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Sample records for benzodiazepine receptor ligands

  1. Imaging of a glioma using peripheral benzodiazepine receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Starosta-Rubinstein, S.; Ciliax, B.J.; Penney, J.B.; McKeever, P.; Young, A.B.

    1987-02-01

    Two types of benzodiazepine receptors have been demonstrated in mammalian tissues, one which is localized on neuronal elements in brain and the other, on glial cells and in peripheral tissues such as kidney. In vivo administration of /sup 3/H-labeled PK 11195 (1-(2-chlorophenyl-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide) or (/sup 3/H)flunitrazepam with 5 mg of clonazepam per kg to rats with intracranial C6 gliomas resulted in high levels of tritiated-drug binding to the tumor as shown by quantitative autoradiography. Pharmacological studies indicated that the bound drugs labeled the peripheral benzodiazepine binding site. Binding to the peripheral benzodiazepine site was confirmed primarily to malignant cells with little binding to adjacent normal brain tissue or to necrotic tissue. Tumor cell binding was completely inhibited by preadministration of the peripheral benzodiazepine blocking agent PK 11195 at 5 mg/kg. The centrally selective benzodiazepine ligand clonazepam had no effect on PK 11195 binding to the tumor cells. When binding to other tumor cell lines grown in nude mice and nude athymic rats was evaluated, little or no peripheral benzodiazepine binding was detected on human pheochromocytoma (RN1) and neuroblastoma (SK-N-MC, SK-N-SH) tumor cells, respectively. However, high densities of peripheral benzodiazepine binding sites were observed on tumors derived from a human glioma cell line (ATCC HTB 14, U-87 MG). The presence of high concentrations of specific peripheral benzodiazepine receptors on glial tumors suggests that human primary central nervous system tumors could be imaged and diagnosed using peripheral benzodiazepine ligands labeled with positron- or gamma-emitting isotopes.

  2. Imaging of a glioma using peripheral benzodiazepine receptor ligands

    International Nuclear Information System (INIS)

    Two types of benzodiazepine receptors have been demonstrated in mammalian tissues, one which is localized on neuronal elements in brain and the other, on glial cells and in peripheral tissues such as kidney. In vivo administration of 3H-labeled PK 11195 [1-(2-chlorophenyl-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide] or [3H]flunitrazepam with 5 mg of clonazepam per kg to rats with intracranial C6 gliomas resulted in high levels of tritiated-drug binding to the tumor as shown by quantitative autoradiography. Pharmacological studies indicated that the bound drugs labeled the peripheral benzodiazepine binding site. Binding to the peripheral benzodiazepine site was confirmed primarily to malignant cells with little binding to adjacent normal brain tissue or to necrotic tissue. Tumor cell binding was completely inhibited by preadministration of the peripheral benzodiazepine blocking agent PK 11195 at 5 mg/kg. The centrally selective benzodiazepine ligand clonazepam had no effect on PK 11195 binding to the tumor cells. When binding to other tumor cell lines grown in nude mice and nude athymic rats was evaluated, little or no peripheral benzodiazepine binding was detected on human pheochromocytoma (RN1) and neuroblastoma (SK-N-MC, SK-N-SH) tumor cells, respectively. However, high densities of peripheral benzodiazepine binding sites were observed on tumors derived from a human glioma cell line (ATCC HTB 14, U-87 MG). The presence of high concentrations of specific peripheral benzodiazepine receptors on glial tumors suggests that human primary central nervous system tumors could be imaged and diagnosed using peripheral benzodiazepine ligands labeled with positron- or gamma-emitting isotopes

  3. Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Gidlöf, Ritha; Nielsen, Elsebet Østergaard;

    2011-01-01

    Based on a pharmacophore model of the benzodiazepine-binding site of GABA(A) receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K(i...

  4. Study on measurement of free ligand concentration in blood and quantitative analysis of brain benzodiazepine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Kenji; Goromaru, Tsuyoshi; Inoue, Osamu; Itoh, Takashi; Yamasaki, Toshiro.

    1988-11-01

    We developed the method to determine rapidly the free ligand concentration in the blood as an input function for the purpose of quantitative analysis of binding potential (B/sub max//K/sub d/) of brain benzodiazepine receptor in vivo. It was found that the unmetabolized radioligand in the blood after intravenous administration of /sup 3/H-Ro 15 - 1788 could be extracted by chloroform, whereas the radioactive metabolites could not be extracted. And the plasma protein binding of /sup 3/H-Ro 15 - 1788 was determined using an ultrafiltration method. The biodistribution of /sup 3/H-Ro 15 - 1788 in the cerebral cortex, cerebellum and pons-medulla after intravenous administration of the radiotracer in the control and forced-swimmed mice was examined. And the time course of the free ligand concentration in the blood was determined as described above. Further, the binding potential of benzodiazepine receptor in the mouse brain was analyzed using a simple mathematical model. It was suggested that the binding potential of benzodiazepine receptor in the mouse brain was significantly decreased by forced-swimming. In conclusion, it was found that these methods would be useful for quantitative analysis of clinical data in the human brain using /sup 11/C-Ro 15 - 1788 and positron emission tomography (PET).

  5. Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand

    OpenAIRE

    Kita, Atsuko; Kohayakawa, Hitoshi; Kinoshita, Tomoko; Ochi, Yoshiaki; Nakamichi, Keiko; Kurumiya, Satoshi; Furukawa, Kiyoshi; Oka, Makoto

    2004-01-01

    We investigated the ability of N-benzyl-N-ethyl-2-(7,8-dihydro-7-methyl-8-oxo-2-phenyl-9H-purin-9-yl)acetamide (AC-5216), a novel mitochondrial benzodiazepine receptor (MBR) ligand, to produce anti-anxiety and antidepressant-like effects in various animal models.AC-5216 showed high affinity for MBRs prepared from rat whole brain (Ki 0.297 nM), rat glioma cells (IC50 3.04 nM) and human glioma cells (IC50 2.73 nM), but only negligible affinity for the other main receptors including central benz...

  6. Effect of peripheral benzodiazepine receptor ligands on lipopolysaccharide-induced tumor necrosis factor activity in thioglycolate-treated mice.

    OpenAIRE

    Matsumoto, T.; Ogata, M.; Koga, K.; Shigematsu, A

    1994-01-01

    To investigate the effect of peripheral and central benzodiazepine receptor ligands on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) activity in mouse macrophages, three types of ligands, 4'-chlorodiazepam (pure peripheral), midazolam (mixed), and clonazepam (pure central), were compared. Midazolam and 4'-chlorodiazepam significantly suppressed LPS (1-microgram/ml)-induced TNF activity in thioglycolate-elicited mouse macrophages. In every concentration examined (0.001 to 100 mi...

  7. Chronic brief restraint decreases in vivo binding of benzodiazepine receptor ligand to mouse brain.

    Science.gov (United States)

    Mosaddeghi, M; Burke, T F; Moerschbaecher, J M

    1993-01-01

    This study examines the effects of chronic brief restraint on in vivo benzodiazepine (BZD) receptor binding in mouse brain. Three groups of mice were used. Mice in group 1 were neither restrained nor injected (ACUTE control). Mice in group 2 were restrained for 5-6 s by grabbing the back skin and holding the subject upside-down at a 45 degrees angle as if to be injected (CHRONIC SHAM control) for 7 d. Mice in group 3 (CHRONIC SALINE) received daily single intraperitoneal (ip) injections of saline (5 mL/kg) for 7 d. On d 8 BZD receptors were labeled in vivo by administration of 3 microCi [3H]flumazenil (ip). The levels of ligand bound in vivo to cerebral cortex (CX), cerebellum (CB), brain stem (BS), striatum (ST), hippocampus (HP), and hypothalamus (HY) were determined. Results indicated that the level of binding was significantly (p stress produces a decrease in BZD receptor binding sites. PMID:8385464

  8. Effects of hippocampal injections of a novel ligand selective for the alpha 5 beta 2 gamma 2 subunits of the GABA/benzodiazepine receptor on Pavlovian conditioning.

    Science.gov (United States)

    Bailey, David J; Tetzlaff, Julie E; Cook, James M; He, Xiaohui; Helmstetter, Fred J

    2002-07-01

    Benzodiazepine pharmacology has led to greater insight into the neural mechanisms underlying learning and anxiety. The synthesis of new compounds capable of modulating responses produced by these receptors has been made possible by the development of an isoform model of the GABA(A)/benzodiazepine receptor complex. In the current experiment, rats were pretreated with several concentrations of the novel ligand RY024 (an alpha 5 beta 2 gamma 2 -selective benzodiazepine receptor inverse agonist) in the hippocampus and were trained in a Pavlovian fear conditioning paradigm. RY024 independently produced fear-related behavior prior to training and, at the highest concentration, decreased the strength of conditioning observed 24 h after training. These data provide further evidence for the involvement of hippocampal GABA(A)/benzodiazepine receptors in learning and anxiety.

  9. Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABAA receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Nielsen, Elsebet Østergaard; Liljefors, Tommy;

    2008-01-01

    A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed...

  10. Mitochondrial benzodiazepine receptors regulate steroid biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Mukhin, A.G.; Papadopoulos, V.; Costa, E.; Krueger, K.E. (Georgetown Univ. School of Medicine, Washington, DC (USA))

    1989-12-01

    Recent observations on the steroid synthetic capability within the brain open the possibility that benzodiazepines may influence steroid synthesis in nervous tissue through interactions with peripheral-type benzodiazepine recognition sites, which are highly expressed in steroidogenic cells and associated with the outer mitochondrial membrane. To examine this possibility nine molecules that exhibit a greater than 10,000-fold difference in their affinities for peripheral-type benzodiazepine binding sites were tested for their effects on a well-established steroidogenic model system, the Y-1 mouse adrenal tumor cell line. 4{prime}-Chlorodiazepam, PK 11195, and PK 14067 stimulated steroid production by 2-fold in Y-1 cells, whereas diazepam, flunitrazepam, zolpidem, and PK 14068 displayed a lower (1.2- to 1.5-fold) maximal stimulation. In contrast, clonazepam and flumazenil did not stimulate steroid synthesis. The potencies of these compounds to inhibit {sup 3}H-labeled PK 11195 binding to peripheral-type benzodiazepine recognition sites correlated with their potencies to stimulate steroid production. Similar findings were observed in bovine and rat adrenocortical cell preparations. These results suggest that ligands of the peripheral-type benzodiazepine recognition site acting on this mitochondrial receptor can enhance steroid production. This action may contribute specificity to the pharmacological profile of drugs preferentially acting on the benzodiazepine recognition site associated with the outer membrane of certain mitochondrial populations.

  11. Mitochondrial benzodiazepine receptors regulate steroid biosynthesis

    International Nuclear Information System (INIS)

    Recent observations on the steroid synthetic capability within the brain open the possibility that benzodiazepines may influence steroid synthesis in nervous tissue through interactions with peripheral-type benzodiazepine recognition sites, which are highly expressed in steroidogenic cells and associated with the outer mitochondrial membrane. To examine this possibility nine molecules that exhibit a greater than 10,000-fold difference in their affinities for peripheral-type benzodiazepine binding sites were tested for their effects on a well-established steroidogenic model system, the Y-1 mouse adrenal tumor cell line. 4'-Chlorodiazepam, PK 11195, and PK 14067 stimulated steroid production by 2-fold in Y-1 cells, whereas diazepam, flunitrazepam, zolpidem, and PK 14068 displayed a lower (1.2- to 1.5-fold) maximal stimulation. In contrast, clonazepam and flumazenil did not stimulate steroid synthesis. The potencies of these compounds to inhibit 3H-labeled PK 11195 binding to peripheral-type benzodiazepine recognition sites correlated with their potencies to stimulate steroid production. Similar findings were observed in bovine and rat adrenocortical cell preparations. These results suggest that ligands of the peripheral-type benzodiazepine recognition site acting on this mitochondrial receptor can enhance steroid production. This action may contribute specificity to the pharmacological profile of drugs preferentially acting on the benzodiazepine recognition site associated with the outer membrane of certain mitochondrial populations

  12. Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines

    OpenAIRE

    Spurny, R.; Ramerstorfer, J.; Price, K; Brams, M.; M. Ernst; Nury, H.; Verheij, M.; Legrand, P.; Bertrand, D.; Bertrand, S.; Dougherty, D A; de Esch, I. J. P.; Corringer, P.-J.; Sieghart, W.; Lummis, S. C. R.

    2012-01-01

    GABA_A receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA_A receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their conc...

  13. Characterization of astrocytic and neuronal benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Bender, A.S.

    1988-01-01

    Primary cultures of astrocytes and neurons express benzodiazepine receptors. Neuronal benzodiazepine receptors were of high-affinity, K{sub D} values were 7.5-43 nM and the densities of receptors (B{sub max}) were 924-4131 fmol/mg protein. Astrocytes posses a high-affinity benzodiazepine receptor, K{sub D} values were 6.6-13 nM. The B{sub max} values were 6,033-12,000 fmol/mg protein. The pharmacological profile of the neuronal benzodiazepine receptor was that of the central-type benzodiazepine receptor, where clonazepam has a high-affinity and Ro 5-4864 (4{prime}-chlorodiazepam) has a low-affinity. Whereas astrocytic benzoidazepine receptor was characteristic of the so called peripheral-type benzodiazepine receptors, which shows a high-affinity towards Ro 5-4863, and a low-affinity towards clonazepam. The astrocytic benzodiazepine receptors was functionally correlated with voltage dependent calcium channels, since dihydropyridines and benzodiazepines interacted with ({sup 3}H) diazepam and ({sup 3}H) nitrendipine receptors with the same rank order of potency, showing a statistically significant correlation. No such correlation was observed in neurons.

  14. Benzodiazepine receptor antagonists for hepatic encephalopathy

    DEFF Research Database (Denmark)

    Als-Nielsen, B; Gluud, L L; Gluud, C

    2004-01-01

    Hepatic encephalopathy may be associated with accumulation of substances that bind to a receptor-complex in the brain resulting in neural inhibition. Benzodiazepine receptor antagonists may have a beneficial effect on patients with hepatic encephalopathy.......Hepatic encephalopathy may be associated with accumulation of substances that bind to a receptor-complex in the brain resulting in neural inhibition. Benzodiazepine receptor antagonists may have a beneficial effect on patients with hepatic encephalopathy....

  15. Evaluation of a radiolabelled peripheral benzodiazepine receptor ligand in the central nervous system inflammation of experimental autoimmune encephalomyelitis: a possible probe for imaging multiple sclerosis

    International Nuclear Information System (INIS)

    Peripheral benzodiazepine receptors (PBRs) are upregulated on macrophages and activated microglia, and radioligands for the PBRs can be used to detect in vivo neuroinflammatory changes in a variety of neurological insults, including multiple sclerosis. Substituted 2-phenyl imidazopyridine-3-acetamides with high affinity and selectivity for PBRs have been prepared that are suitable for radiolabelling with a number of positron emission tomography and single-photon emission computed tomography (SPECT) isotopes. In this investigation, the newly developed high-affinity PBR ligand 6-chloro-2-(4'-iodophenyl)-3-(N,N-diethyl)imidazo [1,2-a]pyridine-3-acetamide, or CLINDE, was radiolabelled with123I and its biodistribution in the central nervous system (CNS) of rats with experimental autoimmune encephalomyelitis (EAE) evaluated. EAE was induced in male Lewis rats by injection of an emulsion of myelin basic protein and incomplete Freund's adjuvant containing Mycobacterium butyricum. Biodistribution studies with123I-CLINDE were undertaken on EAE rats exhibiting different clinical disease severity and compared with results in controls. Disease severity was confirmed by histopathology in the spinal cord of rats. The relationship between inflammatory lesions and PBR ligand binding was investigated using ex vivo autoradiography and immunohistochemistry on rats with various clinical scores. 123I-CLINDE uptake was enhanced in the CNS of all rats exhibiting EAE when compared to controls. Binding reflected the ascending nature of EAE inflammation, with lumbar/sacral cord > thoracic cord > cervical cord > medulla. The amount of ligand binding also reflected the clinical severity of disease. Ex vivo autoradiography and immunohistochemistry revealed a good spatial correspondence between radioligand signal and foci of inflammation and in particular ED-1+ cells representing macrophages and microglia. These results demonstrate the ability of 123I-CLINDE to measure in vivo inflammatory

  16. 3-Alkyl- and 3-amido-isothiazoloquinolin-4-ones as ligands for the benzodiazepine site of GABAA receptors

    DEFF Research Database (Denmark)

    Nilsson, Jakob; Nielsen, Elsebet Østergaard; Liljefors, Tommy;

    2012-01-01

    Based on a pharmacophore model of the benzodiazepine binding site of the GABA(A) receptors, developed with synthetic flavones and potent 3-carbonylquinolin-4-ones, 3-alkyl- and 3-amido-6-methylisothiazoloquinolin-4-ones were designed, prepared and assayed. The suggestion that the interaction betw...

  17. Pharmacology of benzodiazepine receptors: an update.

    OpenAIRE

    Sieghart, W.

    1994-01-01

    Benzodiazepine receptors are allosteric modulatory sites on GABAA receptors. GABAA receptors are probably composed of five protein subunits, at least some of which belong to different subunit classes. So far six alpha-, four beta-, three gamma-, and delta- and two rho = p subunits of GABAA receptors have been identified. A large number of different subunit combinations, each of which will result in a GABAA receptor with distinct electrophysiological and pharmacological properties, are therefo...

  18. Evaluation of a radiolabelled peripheral benzodiazepine receptor ligand in the central nervous system inflammation of experimental autoimmune encephalomyelitis: a possible probe for imaging multiple sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Mattner, F.; Katsifis, A.; Ballantyne, P. [ANSTO, Radiopharmaceuticals Division, Lucas Heights (Australia); Staykova, M.; Willenborg, D.O. [Australian National University Medical School, The Canberra Hospital, Neurosciences Research Unit, Woden, Canberra (Australia)

    2005-04-01

    Peripheral benzodiazepine receptors (PBRs) are upregulated on macrophages and activated microglia, and radioligands for the PBRs can be used to detect in vivo neuroinflammatory changes in a variety of neurological insults, including multiple sclerosis. Substituted 2-phenyl imidazopyridine-3-acetamides with high affinity and selectivity for PBRs have been prepared that are suitable for radiolabelling with a number of positron emission tomography and single-photon emission computed tomography (SPECT) isotopes. In this investigation, the newly developed high-affinity PBR ligand 6-chloro-2-(4'-iodophenyl)-3-(N,N-diethyl)imidazo[1,2-a]pyridine-3-acetamide, or CLINDE, was radiolabelled with{sup 123}I and its biodistribution in the central nervous system (CNS) of rats with experimental autoimmune encephalomyelitis (EAE) evaluated. EAE was induced in male Lewis rats by injection of an emulsion of myelin basic protein and incomplete Freund's adjuvant containing Mycobacterium butyricum. Biodistribution studies with{sup 123}I-CLINDE were undertaken on EAE rats exhibiting different clinical disease severity and compared with results in controls. Disease severity was confirmed by histopathology in the spinal cord of rats. The relationship between inflammatory lesions and PBR ligand binding was investigated using ex vivo autoradiography and immunohistochemistry on rats with various clinical scores. {sup 123}I-CLINDE uptake was enhanced in the CNS of all rats exhibiting EAE when compared to controls. Binding reflected the ascending nature of EAE inflammation, with lumbar/sacral cord > thoracic cord > cervical cord > medulla. The amount of ligand binding also reflected the clinical severity of disease. Ex vivo autoradiography and immunohistochemistry revealed a good spatial correspondence between radioligand signal and foci of inflammation and in particular ED-1{sup +} cells representing macrophages and microglia. These results demonstrate the ability of {sup 123}I

  19. Platelet peripheral benzodiazepine receptors in repeated stress

    Energy Technology Data Exchange (ETDEWEB)

    Dar, D.E.; Bidder, M.; Gavish, M. (Technion-Israel Institute of Technology, Haifa (Israel)); Weizman, A.; Karp, L.; Tyano, S. (Tel Aviv Univ. (Israel)); Grinshpoon, A.; Bleich, A.

    1991-01-01

    ({sup 3}H)PK 11195 binding to platelet membranes and plasma stress hormones were studied in soldiers at the beginning of a parachute training course, following 6 days of preparatory exercises, and after the fourth actual parachute jump. A slight reduction (15%; NS) in the number of peripheral benzodiazepine receptors (PBR) was detected at the end of the exercise period, prior to the first jump. Reduced density of PBR was observed immediately after the repeated actual jumps. Equilibrium dissociation constants were not affected by the stressful situation. Plasma cortisol and prolactin levels remained unaltered during the entire study period.

  20. Repeated swim stress alters brain benzodiazepine receptors measured in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Weizman, R.; Weizman, A.; Kook, K.A.; Vocci, F.; Deutsch, S.I.; Paul, S.M.

    1989-06-01

    The effects of repeated swim stress on brain benzodiazepine receptors were examined in the mouse using both an in vivo and in vitro binding method. Specific in vivo binding of (/sup 3/H)Ro15-1788 to benzodiazepine receptors was decreased in the hippocampus, cerebral cortex, hypothalamus, midbrain and striatum after repeated swim stress (7 consecutive days of daily swim stress) when compared to nonstressed mice. In vivo benzodiazepine receptor binding was unaltered after repeated swim stress in the cerebellum and pons medulla. The stress-induced reduction in in vivo benzodiazepine receptor binding did not appear to be due to altered cerebral blood flow or to an alteration in benzodiazepine metabolism or biodistribution because there was no difference in (14C)iodoantipyrine distribution or whole brain concentrations of clonazepam after repeated swim stress. Saturation binding experiments revealed a change in both apparent maximal binding capacity and affinity after repeated swim stress. Moreover, a reduction in clonazepam's anticonvulsant potency was also observed after repeated swim stress (an increase in the ED50 dose for protection against pentylenetetrazol-induced seizures), although there was no difference in pentylenetetrazol-induced seizure threshold between the two groups. In contrast to the results obtained in vivo, no change in benzodiazepine receptor binding kinetics was observed using the in vitro binding method. These data suggest that environmental stress can alter the binding parameters of the benzodiazepine receptor and that the in vivo and in vitro binding methods can yield substantially different results.

  1. Repeated swim stress alters brain benzodiazepine receptors measured in vivo

    International Nuclear Information System (INIS)

    The effects of repeated swim stress on brain benzodiazepine receptors were examined in the mouse using both an in vivo and in vitro binding method. Specific in vivo binding of [3H]Ro15-1788 to benzodiazepine receptors was decreased in the hippocampus, cerebral cortex, hypothalamus, midbrain and striatum after repeated swim stress (7 consecutive days of daily swim stress) when compared to nonstressed mice. In vivo benzodiazepine receptor binding was unaltered after repeated swim stress in the cerebellum and pons medulla. The stress-induced reduction in in vivo benzodiazepine receptor binding did not appear to be due to altered cerebral blood flow or to an alteration in benzodiazepine metabolism or biodistribution because there was no difference in [14C]iodoantipyrine distribution or whole brain concentrations of clonazepam after repeated swim stress. Saturation binding experiments revealed a change in both apparent maximal binding capacity and affinity after repeated swim stress. Moreover, a reduction in clonazepam's anticonvulsant potency was also observed after repeated swim stress [an increase in the ED50 dose for protection against pentylenetetrazol-induced seizures], although there was no difference in pentylenetetrazol-induced seizure threshold between the two groups. In contrast to the results obtained in vivo, no change in benzodiazepine receptor binding kinetics was observed using the in vitro binding method. These data suggest that environmental stress can alter the binding parameters of the benzodiazepine receptor and that the in vivo and in vitro binding methods can yield substantially different results

  2. Platelet peripheral benzodiazepine receptors are decreased in Parkinson's disease

    International Nuclear Information System (INIS)

    Peripheral benzodiazepine (BDZ) receptors are located in a variety of tissues, including platelets, in the nuclear and/or mitochondrial membranes. The authors studied the density of peripheral BDZ receptors in platelets of 10 de novo Parkinson's disease (PD) patients, 18 PD patients treated with a levodopa/carbidopa combination, and in 15 healthy subjects matched for sex and age. The binding assay was conducted using [3H]PK 11195, a specific ligand for peripheral BDZ receptors. A significant decrease in the density of [3H]PK 11195 binding sites has been observed in PD patients with respect to controls but not between de novo and treated PD patients. No correlation has been found between the decrease in density of [3H]PK 11195 binding sites in platelets and either the duration or severity of PD. Peripheral BDZ receptors are implicated in the regulation of mitochondrial respiratory function. Thus, their decrease in PD might parallel the abnormalities in mitochondrial function recently found in this neurologic disease

  3. Benzodiazepine receptor antagonists for acute and chronic hepatic encephalopathy

    DEFF Research Database (Denmark)

    Als-Nielsen, B; Kjaergard, L L; Gluud, C

    2001-01-01

    The pathogenesis of hepatic encephalopathy is unknown. It has been suggested that liver failure leads to the accumulation of substances that bind to a receptor-complex in the brain resulting in neural inhibition which may progress to coma. Several trials have assessed benzodiazepine receptor...... antagonists for hepatic encephalopathy, but the results are conflicting....

  4. Benzodiazepines

    Science.gov (United States)

    ... longer duration of action are utilized to treat insomnia in patients with daytime anxiety. These benzodiazepines ... of abuse Abuse is frequently associated with adolescents and young adults who take the drug orally or crush it ...

  5. Affinity of 3-acyl substituted 4-quinolones at the benzodiazepine site of GABAA receptors

    DEFF Research Database (Denmark)

    Lager, Erik; Nilsson, Jakob; Nielsen, Elsebet Østergaard;

    2008-01-01

    The finding that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate and N-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxamide derivatives may be high-affinity ligands at the benzodiazepine binding site of the GABA(A) receptor, prompted a study of 3-acyl-1,4-dihydro-4-oxoquinoline (3-acyl-4-quinolones). In...

  6. Benzodiazepine receptor and neurotransmitter studies in the brain of suicides

    International Nuclear Information System (INIS)

    The characteristics of benzodiazepine binding sites were studied on frozen sections of hippocampus of 7 suicides and 5 controls subjects, using biochemical and autoradiographic techniques. 3H flunitrazepam was used as ligand, clonazepam and CL 218,872 as displacing agents. Some neurotransmitters or their derivatives were evaluated quantitatively in parallel in the hippocampal tissue by liquid chromatography. The authors observed mainly an increase in the Ki of CL 218,872 subtype I binding sites in suicides, and an increase in % of type I binding sites. Among neurotransmitters, only norepinephrine differed significantly between controls and suicides. 36 references, 3 figures, 1 table

  7. Benzodiazepine receptor and neurotransmitter studies in the brain of suicides

    Energy Technology Data Exchange (ETDEWEB)

    Manchon, M.; Kopp, N.; Rouzioux, J.J.; Lecestre, D.; Deluermoz, S.; Miachon, S.

    1987-12-14

    The characteristics of benzodiazepine binding sites were studied on frozen sections of hippocampus of 7 suicides and 5 controls subjects, using biochemical and autoradiographic techniques. /sup 3/H flunitrazepam was used as ligand, clonazepam and CL 218,872 as displacing agents. Some neurotransmitters or their derivatives were evaluated quantitatively in parallel in the hippocampal tissue by liquid chromatography. The authors observed mainly an increase in the Ki of CL 218,872 subtype I binding sites in suicides, and an increase in % of type I binding sites. Among neurotransmitters, only norepinephrine differed significantly between controls and suicides. 36 references, 3 figures, 1 table.

  8. AMPA receptor ligands

    DEFF Research Database (Denmark)

    Strømgaard, Kristian; Mellor, Ian

    2004-01-01

    polyamines are known to modulate the function of these receptors in vivo. In this study, recent developments in the medicinal chemistry of polyamine-based ligands are given, particularly focusing on the use of solid-phase synthesis (SPS) as a tool for the facile generation of libraries of polyamine toxin...

  9. Benzodiazepine effect of {sup 125}I-iomazenil-benzodiazepine receptor binding and serum corticosterone level in a rat model

    Energy Technology Data Exchange (ETDEWEB)

    Fukumitsu, Nobuyoshi [Proton Medical Research Center, University of Tsukuba, Ibaragi, 305-8575 (Japan)]. E-mail: gzl13162@nifty.ne.jp; Ogi, Shigeyuki [Department of Radiology, Jikei University School of Medicine, Tokyo, 105-8461 (Japan); Uchiyama, Mayuki [Department of Radiology, Jikei University School of Medicine, Tokyo, 105-8461 (Japan); Mori, Yutaka [Department of Radiology, Jikei University School of Medicine, Tokyo, 105-8461 (Japan)

    2005-01-01

    To test the change in free or unoccupied benzodiazepine receptor (BZR) density in response to diazepam, we investigated {sup 125}I-iomazenil ({sup 125}I-IMZ) binding and serum corticosterone levels in a rat model. Wistar male rats, which received psychological stress using a communication box for 5 days, were divided into two groups according to the amount of administered diazepam: no diazepam [D (0)] group and 10 mg/kg per day [D (10)] group of 12 rats each. The standardized uptake value (SUV) of {sup 125}I-IMZ of the D (10) group were significantly lower (P<.05) than those of the D (0) group in the frontal, parietal and temporal cortices, globus pallidus, hippocampus, amygdala and hypothalamus. The serum corticosterone level ratio in the D (10) group was significantly lower than that in the D (0) group (P<.05). From the change in serum corticosterone levels, diazepam attenuated the psychological stress produced by the physical stress to animals in adjacent compartments. From the reduced binding of {sup 125}I-IMZ, it is clear that diazepam competed with endogenous ligand for the free BZR sites, and the frontal, parietal and temporal cortices, globus pallidus, hippocampus, amygdala and hypothalamus are important areas in which {sup 125}I-IMZ binding is strongly affected by administration of diazepam.

  10. Ligand-Receptor Interactions

    CERN Document Server

    Bongrand, Pierre

    2008-01-01

    The formation and dissociation of specific noncovalent interactions between a variety of macromolecules play a crucial role in the function of biological systems. During the last few years, three main lines of research led to a dramatic improvement of our understanding of these important phenomena. First, combination of genetic engineering and X ray cristallography made available a simultaneous knowledg of the precise structure and affinity of series or related ligand-receptor systems differing by a few well-defined atoms. Second, improvement of computer power and simulation techniques allowed extended exploration of the interaction of realistic macromolecules. Third, simultaneous development of a variety of techniques based on atomic force microscopy, hydrodynamic flow, biomembrane probes, optical tweezers, magnetic fields or flexible transducers yielded direct experimental information of the behavior of single ligand receptor bonds. At the same time, investigation of well defined cellular models raised the ...

  11. Modulation of radioligand binding to the GABA(A)-benzodiazepine receptor complex by a new component from Cyperus rotundus.

    Science.gov (United States)

    Ha, Jeoung-Hee; Lee, Kwang-Youn; Choi, Hyoung-Chul; Cho, Jungsook; Kang, Byung-Soo; Lim, Jae-Chul; Lee, Dong-Ung

    2002-01-01

    Four sesquiterpenes, beta-selinene, isocurcumenol, nootkatone and aristolone and one triterpene, oleanolic acid were isolated from the ethylacetate fraction of the rhizomes of Cyperus rotundus and tested for their ability to modulate gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor function by radioligand binding assays using rat cerebrocortical membranes. Among these compounds, only isocurcumenol, one of the newly identified constituents of this plant, was found to inhibit [3H]Ro15-1788 binding and enhance [3H]flunitrazepam binding in the presence of GABA. These results suggest that isocurcumenol may serve as a benzodiazepine receptor agonist and allosterically modulate GABAergic neurotransmission via enhancement of endogenous receptor ligand binding. PMID:11824542

  12. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    International Nuclear Information System (INIS)

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, 3H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a 3H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of 3H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A4, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each

  13. A Review of the Updated Pharmacophore for the Alpha 5 GABA(A Benzodiazepine Receptor Model

    Directory of Open Access Journals (Sweden)

    Terry Clayton

    2015-01-01

    Full Text Available An updated model of the GABA(A benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1 which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM, SH-053-2′F-R-CH3 (2, has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A receptors.

  14. Biodistribution and dosimetry of [I-123]iodo-PK 11195 : a potential agent for SPET imaging of the peripheral benzodiazepine receptor

    NARCIS (Netherlands)

    Versijpt, J; Dumont, F; Thierens, H; Jansen, H; De Vos, F; Slegers, G; Santens, P; Dierckx, RA; Korf, J

    2000-01-01

    The highest concentrations of the peripheral benzodiazepine receptor (PBR) are found in the kidneys and heart. In addition, the PER has been reported to reflect neuro-inflammatory damage by co-localisation with activated microglia. PK 11195 is a high-affinity ligand for the PER. The aim of this stud

  15. Physiology and physiopathology of central type Benzodiazepine receptors: Study in the monkey and in human brain using positron emission tomography

    International Nuclear Information System (INIS)

    A new non-invasive technique that allows to study in a living subject central type benzodiazepine receptors is developed. A combined approach is applied using a specific positron-emitting radiotracer for the in vivo labelling of the receptors and positron emission tomography allowing, by external detection, a quantitative determination of tissue radioactivity. The radioligand used for the in vivo labelling of benzodiazepine receptors is the antagonist RO 15-1788 labelled with carbon 11. The various stages of the study are described: in vivo characterization in the monkey of central type benzodiazepine receptors; characterization of central type benzodiazepine receptors in human brain using selective molecules for the BZ1 benzodiazepine subclass; demonstration of the heterogeneity of central type benzodiazepine receptors in the brain; study of pathological alteration of benzodiazepine receptors in experimental epilepsy

  16. Benzodiazepine receptor binding in vivo with (/sup 3/)-Ro 15-1788

    Energy Technology Data Exchange (ETDEWEB)

    Goeders, N.E.; Kuhar, M.J.

    1985-07-29

    In vivo benzodiazepine receptor binding has generally been studied by ex vivo techniques. In this investigation, the authors identify the conditions where (/sup 3/H)-Ro 15-1788 labels benzodiazepine receptors by true in vivo binding, i.e. where workable specific to nonspecific ratios are obtained in intact tissues without homogenization or washing. (/sup 3/H)-Flunitrazepam and (/sup 3/H)-clonazepam did not exhibit useful in vivo receptor binding. 39 references, 5 figures, 1 table.

  17. Benzodiazepine receptor binding in vivo with [3]-Ro 15-1788

    International Nuclear Information System (INIS)

    In vivo benzodiazepine receptor binding has generally been studied by ex vivo techniques. In this investigation, the authors identify the conditions where [3H]-Ro 15-1788 labels benzodiazepine receptors by true in vivo binding, i.e. where workable specific to nonspecific ratios are obtained in intact tissues without homogenization or washing. [3H]-Flunitrazepam and [3H]-clonazepam did not exhibit useful in vivo receptor binding. 39 references, 5 figures, 1 table

  18. Autoradiographic localization of benzodiazepine receptors in the rat kidney

    Energy Technology Data Exchange (ETDEWEB)

    Beaumont, K.; Healy, D.P.; Fanestil, D.D.

    1984-11-01

    The localization of benzodiazepine (BZD) receptors in the rat kidney was studied by autoradiography after in vitro labeling of kidney slices with flunitrazepam. The affinity, density, and rank order of displacement of (/sup 3/H)-flunitrazepam by several BZDs (RO 5-4864 > diazepam > clonazepam) demonstrated that binding was to BZD receptors of the peripheral type. In autoradiograms obtained with tritium-sensitive film, a high density of silver grains was obtained in the outer medulla, with lower densities in the cortex. Binding was absent from the inner medulla (papilla). In higher resolution autoradiograms obtained with an emulsion-coated cover slip procedure, silver grains were seen to be concentrated over a tubular element in both outer medulla and cortex, identifiable by morphology and distribution as the thick ascending limb of the loop of Henle and the distal convoluted tubule. The identity of the labeled tubules was confirmed by immunofluorescent localization in adjacent slices of Tamm-Horsfall protein, a specific marker for these segments of tubules. Investigation of the effects of peripherally specific BZDs such as RO 5-4864 on distal tubule function is indicated.

  19. Autoradiographic localization of benzodiazepine receptors in the rat kidney

    International Nuclear Information System (INIS)

    The localization of benzodiazepine (BZD) receptors in the rat kidney was studied by autoradiography after in vitro labeling of kidney slices with flunitrazepam. The affinity, density, and rank order of displacement of [3H]-flunitrazepam by several BZDs (RO 5-4864 > diazepam > clonazepam) demonstrated that binding was to BZD receptors of the peripheral type. In autoradiograms obtained with tritium-sensitive film, a high density of silver grains was obtained in the outer medulla, with lower densities in the cortex. Binding was absent from the inner medulla (papilla). In higher resolution autoradiograms obtained with an emulsion-coated cover slip procedure, silver grains were seen to be concentrated over a tubular element in both outer medulla and cortex, identifiable by morphology and distribution as the thick ascending limb of the loop of Henle and the distal convoluted tubule. The identity of the labeled tubules was confirmed by immunofluorescent localization in adjacent slices of Tamm-Horsfall protein, a specific marker for these segments of tubules. Investigation of the effects of peripherally specific BZDs such as RO 5-4864 on distal tubule function is indicated

  20. The expression of peripheral benzodiazepine receptors in human skin: the relationship with epidermal cell differentiation.

    Science.gov (United States)

    Stoebner, P E; Carayon, P; Penarier, G; Fréchin, N; Barnéon, G; Casellas, P; Cano, J P; Meynadier, J; Meunier, L

    1999-06-01

    The peripheral benzodiazepine receptor (PBR) is a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands. PBR is part of a heteromeric receptor complex involved in the formation of mitochondrial permeability transition pores and in the early events of apoptosis. PBR may function as an oxygen-dependent signal generator; recent data indicate that these receptors may preserve the mitochondria of haematopoietic cell lines from damage caused by oxygen radicals. To identify PBRs in human skin, we used a specific monoclonal antibody directed against the C-terminus fragment of the human receptor. PBR immunoreactivity was found in keratinocytes, Langerhans cells, hair follicles and dermal vascular endothelial cells. Interestingly, confocal microscopic examination of skin sections revealed that PBR expression was strongly upregulated in the superficial differentiated layers of the epidermis. Ultrastructurally, PBRs were distributed throughout the cytoplasm but were selectively expressed on the mitochondrial membranes of epidermal cells. The elevated level of PBRs in the spinous layer was not associated with an increased number of mitochondria nor with an increased amount of mRNA as assessed by in situ hybridization on microautoradiographed skin sections. The present work provides, for the first time, evidence of PBR immunoreactivity in human skin. This mitochondrial receptor may modulate apoptosis in the epidermis; its increased expression in differentiated epidermal layers may represent a novel mechanism of natural skin protection against free radical damage generated by ultraviolet exposure. PMID:10354064

  1. Extraction and purification from Ceratonia siliqua of compounds acting on central and peripheral benzodiazepine receptors.

    Science.gov (United States)

    Avallone, R; Cosenza, F; Farina, F; Baraldi, C; Baraldi, M

    2002-08-01

    The presence of molecules with high affinity for central and peripheral benzodiazepine receptors was determined in the pod and leaves of Ceratonia siliqua (carob). The amount of the substances able to selectively bind the central benzodiazepine receptor recovered from carob pods and leaves was respectively 12.17 and 18.7 ng diazepam equivalent/g. The amount of compounds active on peripheral benzodiazepine receptor in both pods and leaves was higher in comparison with the central one, being 49.83 and 40.00 PK 11195 equivalent/g, respectively. In particular the compounds acting on peripheral benzodiazepine receptors were found to be extremely concentrated in the young leaves (2572.57 ng PK 11195 equivalent/g). The presence of substances with central benzodiazepine activity in carob extracts seems of great importance in view of the possibility to use carob extract as potential natural products with anxiolytic-sedative effects. Moreover, the prevalence in leaves of substances acting on peripheral benzodiazepine receptor suggests the possible utilisation of leave extracts as chemopreventive agents.

  2. Early ontogeny of the central benzodiazepine receptor in human embryos and fetuses

    Energy Technology Data Exchange (ETDEWEB)

    Hebebrand, J.; Hofmann, D.; Reichelt, R.; Schnarr, S.; Knapp, M.; Propping, P.; Foedisch, H.J.

    1988-01-01

    The early ontogeny of the central benzodiazepine receptor (BZR) was investigated in human embryos and fetuses between 7 and 26 weeks of gestation. Brain tissue was gained from terminated pregnancies or spontaneous abortions. Binding studies, which were performed with /sup 3/H-flunitrazepam (FNZ), revealed that specific benzodiazepine binding is already detectable at an embryonal age of 7 weeks post conception. Binding at this early stage can be displaced potently by clonazepam and the inverse agonist ..beta..-CCE. Additionally, /sup 3/H-FNZ binding is enhanced by GABA. Thus, benzodiazepine binding is of the central type. Receptor density increases steeply in whole brain between weeks 8 and 11 of gestation. In frontal cortex receptor density increases gradually between weeks 12 and 26 of gestation. No specific fetal disease entity (including trisomy 21) was consistently associated with exceptionally high or low B/sub max/-values.

  3. Early ontogeny of the central benzodiazepine receptor in human embryos and fetuses

    International Nuclear Information System (INIS)

    The early ontogeny of the central benzodiazepine receptor (BZR) was investigated in human embryos and fetuses between 7 and 26 weeks of gestation. Brain tissue was gained from terminated pregnancies or spontaneous abortions. Binding studies, which were performed with 3H-flunitrazepam (FNZ), revealed that specific benzodiazepine binding is already detectable at an embryonal age of 7 weeks post conception. Binding at this early stage can be displaced potently by clonazepam and the inverse agonist β-CCE. Additionally, 3H-FNZ binding is enhanced by GABA. Thus, benzodiazepine binding is of the central type. Receptor density increases steeply in whole brain between weeks 8 and 11 of gestation. In frontal cortex receptor density increases gradually between weeks 12 and 26 of gestation. No specific fetal disease entity (including trisomy 21) was consistently associated with exceptionally high or low B/sub max/-values

  4. Radioiodinated ligands for dopamine receptors

    International Nuclear Information System (INIS)

    The dopamine receptor system is important for normal brain function; it is also the apparent action site for various neuroleptic drugs for the treatment of schizophrenia and other metal disorders. In the past few years radioiodinated ligands for single photon emission tomography (SPECT) have been successfully developed and tested in humans: [123I]TISCH for D1 dopamine receptors; [123I]IBZM, epidepride, IBF and FIDA2, four iodobenzamide derivatives, for D2/D3 dopamine receptors. In addition, [123I]β-CIT (RTI-55) and IPT, cocaine derivatives, for the dopamine reuptake site are potentially useful for diagnosis of loss of dopamine neurons. The first iodinated ligand, (R)trans-7-OH-PIPAT, for D3 dopamine receptors, was synthesized and characterized with cloned cell lines (Spodoptera frugiperda, Sf9) expressing the D2 and D3 dopamine receptors and with rat basal forebrain membrane preparations. Most of the known iodobenzamides displayed similar potency in binding to both D2 and D3 dopamine receptors expressed in the cell lines. Initial studies appear to suggest that by fine tuning the structures it may be possible to develop agents specific for D2 and D3 dopamine receptors. It is important to investigate D2/D3 selectivity for this series of potent ligands

  5. Comparison of blood flow and distribution of benzodiazepine receptors in focal epilepsy: Preliminary results of a SPECT study. Vergleich von Blutfluss und Benzodiazepin-Rezeptorverteilung bei fokaler Epilepsie: Vorlaeufige Ergebnisse einer SPECT-Studie

    Energy Technology Data Exchange (ETDEWEB)

    Bartenstein, P.; Schober, O.; Lottes, G.; Boettger, I. (Muenster Univ. (Germany, F.R.). Klinik und Poliklinik fuer Nuklearmedizin); Ludolph, A. (Muenster Univ. (Germany, F.R.). Klinik und Poliklinik fuer Neurologie); Beer, H.F. (Paul Scherrer Inst., Wuerenlingen (Switzerland))

    1989-10-01

    {sup 99m}Tc-HMPAO-SPECT and SPECT with the {sup 123}I-labelled benzodiazepine (Bz) receptor ligand Ro 16-0154 were performed in 10 patients suffering from partial epilepsy, without cerebral lesion in MRT or CT.2 h p.i. of Ro 16-0154 the distribution of activity correlated with the known distribution of Bz-receptors in the human brain. Perfusion and receptor-binding were found decreased in 7 patients of each study in the suspicious brain-area. {sup 123}I-labelled Ro 16-0154 is suitable for Bz-receptor mapping by SPECT. The decrease of Bz-receptor binding in epileptic foci, as described in PET-studies, was also detected by SPECT in 7 of 10 patients. (orig.).

  6. Polypharmacology of dopamine receptor ligands.

    Science.gov (United States)

    Butini, S; Nikolic, K; Kassel, S; Brückmann, H; Filipic, S; Agbaba, D; Gemma, S; Brogi, S; Brindisi, M; Campiani, G; Stark, H

    2016-07-01

    Most neurological diseases have a multifactorial nature and the number of molecular mechanisms discovered as underpinning these diseases is continuously evolving. The old concept of developing selective agents for a single target does not fit with the medical need of most neurological diseases. The development of designed multiple ligands holds great promises and appears as the next step in drug development for the treatment of these multifactorial diseases. Dopamine and its five receptor subtypes are intimately involved in numerous neurological disorders. Dopamine receptor ligands display a high degree of cross interactions with many other targets including G-protein coupled receptors, transporters, enzymes and ion channels. For brain disorders like Parkinsońs disease, schizophrenia and depression the dopaminergic system, being intertwined with many other signaling systems, plays a key role in pathogenesis and therapy. The concept of designed multiple ligands and polypharmacology, which perfectly meets the therapeutic needs for these brain disorders, is herein discussed as a general ligand-based concept while focusing on dopaminergic agents and receptor subtypes in particular. PMID:27234980

  7. Benzodiazepine receptor quantification in vivo in humans using [11C]flumazenil and PET

    DEFF Research Database (Denmark)

    Lassen, N A; Bartenstein, P A; Lammertsma, A A;

    1995-01-01

    Carbon-11-labeled flumazenil combined with positron emission tomography (PET) was used to measure the concentration (Bmax) of the benzodiazepine (Bz) receptor in the brain and its equilibrium dissociation constant (KD) for flumazenil in five normal subjects. The steady-state approach was used inj...

  8. Quantitative autoradiography of muscarinic and benzodiazepine receptors in the forebrain of the turtle, Pseudemys scripta

    International Nuclear Information System (INIS)

    The distribution of muscarinic and benzodiazepine receptors was investigated in the turtle forebrain by the technique of in vitro receptor autoradiography. Muscarinic binding sites were labeled with 1 nM 3H-quinuclidinyl benzilate (3H-QNB), and benzodiazepine sites were demonstrated with the aid of 1 nM 3H-flunitrazepam (3H-FLU). Autoradiograms generated on 3H-Ultrofilm apposed to tissue slices revealed regionally specific distributions of muscarinic and benzodiazepine binding sites that are comparable with those for mammalian brain. Dense benzodiazepine binding was found in the anterior olfactory nucleus, the lateral and dorsal cortices, and the dorsal ventricular ridge (DVR), a structure with no clear mammalian homologue. Muscarinic binding sites were most dense in the striatum, accumbens, DVR, lateral geniculate, and the anterior olfactory nucleus. Cortical binding sites were studied in greater detail by quantitative analysis of autoradiograms generated by using emulsion-coated coverslips. Laminar gradients of binding were observed that were specific for each radioligand; 3H-QNB sites were most dense in the inner molecular layer in all cortical regions, whereas 3H-FLU binding was generally most concentrated in the outer molecular layer and was least dense through all layers in the dorsomedial cortex. Because pyramidal cells are arranged in register in turtle cortex, the laminar patterns of receptor binding may reflect different receptor density gradients along pyramidal cell dendrites

  9. Benzodiazepine receptor quantification in Huntington's disease with [123I]iomazenil and SPECT

    OpenAIRE

    Pinborg, L; Videbak, C; Hasselbalch, S; Sorensen, S.; Wagner, A; Paulson, O; Knudsen, G

    2001-01-01

    OBJECTIVES—Increasing evidence suggests that metabolic changes predate neuronal death in Huntington's disease and emission tomography methods (PET and SPECT) have shown changes in glucose consumption and receptor function in early and possibly even presymptomatic disease. Because the GABAA-benzodiazepine receptor complex (BZR) is expressed on virtually all cerebral neurons BZR density images may be used to detect neuronal death. In this study the regional cerebral [123...

  10. Benzodiazepine receptor-mediated behavioral effects of nitrous oxide in the rat social interaction test.

    Science.gov (United States)

    Quock, R M; Wetzel, P J; Maillefer, R H; Hodges, B L; Curtis, B A; Czech, D A

    1993-09-01

    The present study was conducted to ascertain whether an anxiolytic effect of nitrous oxide was demonstrable in rats using the social interaction test and whether this drug effect might be mediated by benzodiazepine receptors. Compared to behavior of vehicle-pretreated, room air-exposed rats, rat pairs exposed to nitrous oxide showed a generally inverted U-shaped dose-response curve with the maximum increase in social interaction encounters occurring at 25% and significant increase in time of active social interaction at 15-35%; higher concentrations produced a sedative effect that reduced social interaction. Treatment with 5.0 mg/kg of the anxiolytic benzodiazepine chlordiazepoxide also increased social interaction. Pretreatment with 10 mg/kg of the benzodiazepine receptor blocker flumazenil, which alone had no effect, significantly antagonized the social interaction-increasing effects of both nitrous oxide and chlordiazepoxide. In summary, these findings suggest that nitrous oxide produces a flumazenil-sensitive effect comparable to that of chlordiazepoxide and implicate central benzodiazepine mechanisms in mediation of the anxiolytic effect of nitrous oxide.

  11. Abnormal benzodiazepine and zinc modulation of GABAA receptors in an acquired absence epilepsy model.

    Science.gov (United States)

    Wu, Jie; Ellsworth, Kevin; Ellsworth, Marc; Schroeder, Katherine M; Smith, Kris; Fisher, Robert S

    2004-07-01

    Brain cholesterol synthesis inhibition (CSI) at a young age in rats has been shown to be a faithful model of acquired absence epilepsy, a devastating condition for which few therapies or models exist. We employed the CSI model to study cellular mechanisms of acquired absence epilepsy in Long-Evans Hooded rats. Patch-clamp, whole-cell recordings were compared from neurons acutely dissociated from the nucleus reticularis of thalamus (nRt) treated and untreated with a cholesterol synthesis inhibitor, U18666A. In U18666A-treated animals, 91% of rats developed EEG spike-waves (SWs). Patchclamp results revealed that although there was no remarkable change in GABAA receptor affinity, both a loss of ability of benzodiazepines to enhance GABAA-receptor responses and an increase of Zn2+ inhibition of GABAA-receptor responses of nRt neurons occurred in Long-Evans Hooded rats previously administered U18666A. This change was specific, since no significant changes were found in neurons exposed to the GABA allosteric modulator, pentobarbital. Taken collectively, these findings provide evidence for abnormalities in benzodiazepine and Zn2+ modulation of GABAA receptors in the CSI model, and suggest that decreased gamma2 subunit expression may underlie important aspects of generation of thalamocortical SWs in atypical absence seizures. The present results are also consistent with recent findings that mutation of the gamma2 subunit of the GABAA receptor changes benzodiazepine modulation in families with generalized epilepsy syndromes.

  12. Characteristics of benzodiazepine receptors in rats differing in predisposition to experimental alcoholism

    Energy Technology Data Exchange (ETDEWEB)

    Burov, Yu.V.; Maiskii, A.I.; Yukhananov, R.Yu.

    1986-02-01

    This paper studies the number and affinity of benzodiazepine receptors for diazepam in the cerebral cortex and hippocampus of rats differently predisposed to the development of experimental alcoholism. Ethanol was injected once intraperitoneally, in a dose of 2.5 g/kg. Control animals received the same volume of physiological saline. Bound and free N-methyl-tritium-diazepam were separated by means of GF/B filters. The characteristics of benzodiazepine receptors are shown in rats differing in predisposition to the development of experimental alcoholism and in rats during voluntary chronic alcoholization. It is shown that weakening of functional acitivity of the GABA-benzodiazepam complex in animals predisposed to the development of experimental alcoholism is one of the neurochemical mechanisms of development of the abstinence syndrome.

  13. The GABA-A benzodiazepine receptor complex: Role of pet and spect in neurology and psychiatry

    International Nuclear Information System (INIS)

    Nuclear medicine imaging techniques such as positron emission tomography (PET) and single photon emission tomography (SPECT) for selective depiction of GABA-A-benzodiazepine receptor (GBZR) binding are complementary investigations in the diagnostic process of neurological and psychiatric disorders. This review summarizes the current knowledge about options and limitations of PET and SPECT for in vivo diagnostics in neurology and psychiatry. The growing importance of GBZR-imaging for the understanding of pathophysiology and pharmacological treatment in different psychiatric syndromes is discussed. (orig.)

  14. Possible interaction of fluoroquinolones with the benzodiazepine-GABAA-receptor complex.

    OpenAIRE

    Unseld, E; Ziegler, G.; Gemeinhardt, A; Janssen, U.; Klotz, U

    1990-01-01

    1. The possible involvement of the benzodiazepine (BZD)-GABAA-receptor complex in mediating CNS stimulatory effects of fluoroquinolones was tested in vitro, in a binding inhibition assay and in vivo, in a clinical drug interaction study using electro-encephalogram (EEG) monitoring. 2. The specific binding of [3H]-flunitrazepam to rat synaptic brain membranes was inhibited by various fluoroquinolones in a concentration-dependent manner. 3. Ofloxacin had CNS-stimulating effects as revealed by t...

  15. GABA/benzodiazepine receptor complex in long-sleep and short-sleep mice

    Energy Technology Data Exchange (ETDEWEB)

    Marley, R.J.

    1987-01-01

    LS mice are more sensitive to benzodiazepine-induced anesthesia; however, the two lines do not differ in their hypothermic response to flurazepam. SS mice are more resistant to 3-mercaptopropionic acid-induced seizures and more sensitive to the anticonvulsant effects of benzodiazepines. The various correlates of GABA and benzodiazepine actions probably are the results of different mechanisms of action and/or differential regional control. Bicuculline competition for /sup 3/H-GABA binding sites is greater in SS cerebellar tissue and /sup 3/H-flunitrazepam binding is greater in the mid-brain region of LS mice. GABA enhancement of /sup 3/H-flunitrazepma binding is greater in SS mice. Ethanol also enhances /sup 3/H-flunitrazepam binding and increases the levels of /sup 3/H-flunitrazepam binding above those observed for GABA. Using correlational techniques on data from LS and SS mice and several inbred mouse strains, it was demonstrated that a positive relationship exists between the degree of receptor coupling within the GABA receptor complex and the degree of resistance to seizures.

  16. GABA/benzodiazepine receptor complex in long-sleep and short-sleep mice

    International Nuclear Information System (INIS)

    LS mice are more sensitive to benzodiazepine-induced anesthesia; however, the two lines do not differ in their hypothermic response to flurazepam. SS mice are more resistant to 3-mercaptopropionic acid-induced seizures and more sensitive to the anticonvulsant effects of benzodiazepines. The various correlates of GABA and benzodiazepine actions probably are the results of different mechanisms of action and/or differential regional control. Bicuculline competition for 3H-GABA binding sites is greater in SS cerebellar tissue and 3H-flunitrazepam binding is greater in the mid-brain region of LS mice. GABA enhancement of 3H-flunitrazepma binding is greater in SS mice. Ethanol also enhances 3H-flunitrazepam binding and increases the levels of 3H-flunitrazepam binding above those observed for GABA. Using correlational techniques on data from LS and SS mice and several inbred mouse strains, it was demonstrated that a positive relationship exists between the degree of receptor coupling within the GABA receptor complex and the degree of resistance to seizures

  17. In vivo study of drug interaction with brain benzodiazepine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, O.; Shinotoh, H.; Ito, T.; Suzuki, K.; Hashimoto, K.; Yamasaki, T.

    1985-05-01

    The possibility of direct estimation of in vivo Bz receptor occupancy in brain was evaluated using C-11, or H-3-flumazepil (Ro15-1788). In animal experiments, 1 ..mu..Ci of H-3-Ro15-1788 was injected at 0.5 or 20 hr after i.v. injection of various dosage of clonazepam. Then radioactivity in cerebral cortex, cerebellum and blood at 5 min. after injection of the tracer was compared. Competitive inhibition of in vivo binding was clearly observed when clonazepam was pretreated at 0.5 hr before injection of the tracer. On the other hand, brain radioactivity was increased when clonazepam was administered at 20 hr before injection of the tracer. This increase in binding of H-3-Ro15-1788 might be caused by rebound of Bz receptor function by treatment with Bz agonist, and this rebound may have an important role in physiological function. Clinical investigation concerning drug interaction with brain Bz receptor was performed in normal volunteer and patients with neurological disorders. The distribution of C-11-Ro15-1788 in the brain of patients chronically treated with clonazepam were significantly heterogeneous. However, cerebral blood flow estimated with N-13 NH3 of these patients were normal.

  18. In vivo study of drug interaction with brain benzodiazepine receptor

    International Nuclear Information System (INIS)

    The possibility of direct estimation of in vivo Bz receptor occupancy in brain was evaluated using C-11, or H-3-flumazepil (Ro15-1788). In animal experiments, 1 μCi of H-3-Ro15-1788 was injected at 0.5 or 20 hr after i.v. injection of various dosage of clonazepam. Then radioactivity in cerebral cortex, cerebellum and blood at 5 min. after injection of the tracer was compared. Competitive inhibition of in vivo binding was clearly observed when clonazepam was pretreated at 0.5 hr before injection of the tracer. On the other hand, brain radioactivity was increased when clonazepam was administered at 20 hr before injection of the tracer. This increase in binding of H-3-Ro15-1788 might be caused by rebound of Bz receptor function by treatment with Bz agonist, and this rebound may have an important role in physiological function. Clinical investigation concerning drug interaction with brain Bz receptor was performed in normal volunteer and patients with neurological disorders. The distribution of C-11-Ro15-1788 in the brain of patients chronically treated with clonazepam were significantly heterogeneous. However, cerebral blood flow estimated with N-13 NH3 of these patients were normal

  19. GABA(A) receptors implicated in REM sleep control express a benzodiazepine binding site.

    Science.gov (United States)

    Nguyen, Tin Quang; Liang, Chang-Lin; Marks, Gerald A

    2013-08-21

    It has been reported that non-subtype-selective GABAA receptor antagonists injected into the nucleus pontis oralis (PnO) of rats induced long-lasting increases in REM sleep. Characteristics of these REM sleep increases were identical to those resulting from injection of muscarinic cholinergic agonists. Both actions were blocked by the muscarinic antagonist, atropine. Microdialysis of GABAA receptor antagonists into the PnO resulted in increased acetylcholine levels. These findings were consistent with GABAA receptor antagonists disinhibiting acetylcholine release in the PnO to result in an acetylcholine-mediated REM sleep induction. Direct evidence has been lacking for localization in the PnO of the specific GABAA receptor-subtypes mediating the REM sleep effects. Here, we demonstrated a dose-related, long-lasting increase in REM sleep following injection (60 nl) in the PnO of the inverse benzodiazepine agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline (DMCM, 10(-2)M). REM sleep increases were greater and more consistently produced than with the non-selective antagonist gabazine, and both were blocked by atropine. Fluorescence immunohistochemistry and laser scanning confocal microscopy, colocalized in PnO vesicular acetylcholine transporter, a presynaptic marker of cholinergic boutons, with the γ2 subunit of the GABAA receptor. These data provide support for the direct action of GABA on mechanisms of acetylcholine release in the PnO. The presence of the γ2 subunit at this locus and the REM sleep induction by DMCM are consistent with binding of benzodiazepines by a GABAA receptor-subtype in control of REM sleep.

  20. Further characterization of benzodiazepine receptor differences in long-sleep and short-sleep mice

    International Nuclear Information System (INIS)

    Molecular and conformational characteristics of benzodiazepine (BZ) receptors in cortex and cerebellum from long-sleep and mice were investigated using heat inactivation and beta-carboline competition techniques. To investigate differences in the allosteric coupling between GABA and BZ receptors, the protection of BZ receptors from heat inactivation, by GABA, was also evaluated. The two genotypes do not differ in the affinity or number of BZ receptors in the cortex or cerebellum. They do, however, appear to differ in the molecular structure and/or regulation of the conformational state of the receptor in the cortex, as indicated by a greater sensitivity of LS mice to both heat inactivation and beta-carboline competition of 3H-flunitrazepam (FNZ) binding in this region. Evidence for differences in the nature of coupling between GABA and BZ receptors is provided by the finding in that in both regions, GABA protected BZ receptors from inactivation to a greater degree in LS mice. The relationship between these differences and the multiplicity of expression of BZ receptors is discussed

  1. Further characterization of benzodiazepine receptor differences in long-sleep and short-sleep mice

    Energy Technology Data Exchange (ETDEWEB)

    Marley, R.J.; Stinchcomb, A.; Wehner, J.M.

    1988-01-01

    Molecular and conformational characteristics of benzodiazepine (BZ) receptors in cortex and cerebellum from long-sleep and mice were investigated using heat inactivation and beta-carboline competition techniques. To investigate differences in the allosteric coupling between GABA and BZ receptors, the protection of BZ receptors from heat inactivation, by GABA, was also evaluated. The two genotypes do not differ in the affinity or number of BZ receptors in the cortex or cerebellum. They do, however, appear to differ in the molecular structure and/or regulation of the conformational state of the receptor in the cortex, as indicated by a greater sensitivity of LS mice to both heat inactivation and beta-carboline competition of /sup 3/H-flunitrazepam (FNZ) binding in this region. Evidence for differences in the nature of coupling between GABA and BZ receptors is provided by the finding in that in both regions, GABA protected BZ receptors from inactivation to a greater degree in LS mice. The relationship between these differences and the multiplicity of expression of BZ receptors is discussed.

  2. Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

    Energy Technology Data Exchange (ETDEWEB)

    Supavilai, P.; Karobath, M.

    1985-02-04

    GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with /sup 3/H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 ..mu..M. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BR agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro. 24 references, 3 figures, 5 table.

  3. Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

    International Nuclear Information System (INIS)

    GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with 3H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 μM. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BR agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro. 24 references, 3 figures, 5 table

  4. Midazolam inhibits hippocampal long-term potentiation and learning through dual central and peripheral benzodiazepine receptor activation and neurosteroidogenesis

    OpenAIRE

    Tokuda, Kazuhiro; O’Dell, Kazuko A.; Izumi, Yukitoshi; Charles F. Zorumski

    2010-01-01

    Benzodiazepines (BDZs) enhance γ-aminobutyric acid-A (GABAA) receptor inhibition by direct actions on central BDZ receptors (CBRs). Although some BDZs also bind mitochondrial receptors (translocator protein 18kDa, TSPO) and promote the synthesis of GABA-enhancing neurosteroids, the role of neurosteroids in the clinical effects of BDZs is unknown. In rat hippocampal slices, we compared midazolam, an anesthetic BDZ with clonazepam, an anticonvulsant/anxiolytic BDZ that activates CBRs selectivel...

  5. [3H]Clonazepam, like [3H]flunitrazepam, is a photoaffinity label for the central type of benzodiazepine receptors

    International Nuclear Information System (INIS)

    [3H]Clonazepam, like [3H]flunitrazepam, is irreversibly bound to membrane proteins of brain tissue when exposed to UV light. In polyacrylamide gel electrophoresis followed by fluorography, the same pattern of photolabelled proteins was obtained in cerebellum and in hippocampus when either [3H]clonazepam or [3H]flunitrazepam was used as photoaffinity label. Since [3H]clonazepam does not interact with the peripheral type of benzodiazepine binding site present in the brain, these results confirm previous evidence that the proteins photolabelled with [3H]flunitrazepam are associated with the central type of benzodiazepine receptor. (Auth.)

  6. Methodology for benzodiazepine receptor binding assays at physiological temperature. Rapid change in equilibrium with falling temperature

    Energy Technology Data Exchange (ETDEWEB)

    Dawson, R.M.

    1986-12-01

    Benzodiazepine receptors of rat cerebellum were assayed with (/sup 3/H)-labeled flunitrazepam at 37/sup 0/C, and assays were terminated by filtration in a cold room according to one of three protocols: keeping each sample at 37 degrees C until ready for filtration, taking the batch of samples (30) into the cold room and filtering sequentially in the order 1-30, and taking the batch of 30 samples into the cold room and filtering sequentially in the order 30-1. the results for each protocol were substantially different from each other, indicating that rapid disruption of equilibrium occurred as the samples cooled in the cold room while waiting to be filtered. Positive or negative cooperativity of binding was apparent, and misleading effects of gamma-aminobutyric acid on the affinity of diazepam were observed, unless each sample was kept at 37/sup 0/C until just prior to filtration.

  7. Benzodiazepine receptor distribution and cerebral blood flow in early blindness. A PET study

    International Nuclear Information System (INIS)

    We studied benzodiazepine receptor (BZR) distribution, which is thought to be affected by neuronal density in the cerebral cortex, and CBF using [11C]flumazenil and [15O]water PET in early blind (EB) and in blindfold sighted control (SC) subjects. PET images were co-registered to the subject's MRI. Using SPM96, MRI images were normalized in the Talairach and Tournoux coordinate system, and accordingly MRI-registered PET images were spatially normalized. Statistical parametric maps were computed on a voxel-by-voxel basis, using the general linear model. CBF for EB was significantly larger in the Brodmann area 17 and 18, especially anterior area, than that for SC, while there was no significant difference in BZR distribution. Our BZR data suggest that the amount of neurons do not change due to early visual deprivation in the visual cortex, in spite of high CBF in visual cortex of EB subjects. (author)

  8. Systematic review of modulators of benzodiazepine receptors in irritable bowel syndrome:Is there hope?

    Institute of Scientific and Technical Information of China (English)

    Pooneh Salari; Mohammad Abdollahi

    2011-01-01

    Several drugs are used in the treatment of irritable bowel syndrome (IBS) but all have side effects and variable efficacy.Considering the role of the gut-brain axis,immune,neural,and endocrine pathways in the patho-genesis of IBS and possible beneficial effects of ben-zodiazepines (BZD) in this axis,the present systematic review focuses on the efficacy of BZD receptor modulators in human IBS.For the years 1966 to February 2011,all literature was searched for any articles on the use of BZD receptor modulators and IBS.After thorough evaluation and omission of duplicate data,10 out of 69 articles were included.BZD receptor modulators can be helpful,especially in the diarrhea-dominant form of IBS,by affecting the inflammatory,neural,and psychologic pathways,however,controversies still exist.Recently,a new BZD receptor modulator,dextofisopam was synthesized and studied in human subjects,but the studies are limited to phase II b clinical trials.None of the existing trials considered the neuroimmunomodulatory effectof BZDs in IBS,but bearing in mind the concentration-dependent effect of BZDs on cytokines and cell proliferation,future studies using pharmacodynamic and pharmacokinetic approaches are highly recommended.

  9. In vivo molecular imaging of the GABA/benzodiazepine receptor complex in the aged rat brain.

    Science.gov (United States)

    Hoekzema, Elseline; Rojas, Santiago; Herance, Raúl; Pareto, Deborah; Abad, Sergio; Jiménez, Xavier; Figueiras, Francisca P; Popota, Foteini; Ruiz, Alba; Flotats, Núria; Fernández, Francisco J; Rocha, Milagros; Rovira, Mariana; Víctor, Víctor M; Gispert, Juan D

    2012-07-01

    The GABA-ergic system, known to regulate neural tissue genesis during cortical development, has been postulated to play a role in cerebral aging processes. Using in vivo molecular imaging and voxel-wise quantification, we aimed to assess the effects of aging on the benzodiazepine (BDZ) recognition site of the GABA(A) receptor. To visualize BDZ site availability, [(11)C]-flumazenil microPET acquisitions were conducted in young and old rats. The data were analyzed and region of interest analyses were applied to validate the voxel-wise approach. We observed decreased [(11)C]-flumazenil binding in the aged rat brains in comparison with the young control group. More specifically, clusters of reduced radioligand uptake were detected in the bilateral hippocampus, cerebellum, midbrain, and bilateral frontal and parieto-occipital cortex. Our results support the pertinence of voxel-wise quantification in the analysis of microPET data. Moreover, these findings indicate that the aging process involves declines in neural BDZ recognition site availability, proposed to reflect alterations in GABA(A) receptor subunit polypeptide expression.

  10. Ligands for Ionotropic Glutamate Receptors

    Science.gov (United States)

    Swanson, Geoffrey T.; Sakai, Ryuichi

    Marine-derived small molecules and peptides have played a central role in elaborating pharmacological specificities and neuronal functions of mammalian ionotropic glutamate receptors (iGluRs), the primary mediators of excitatory syn-aptic transmission in the central nervous system (CNS). As well, the pathological sequelae elicited by one class of compounds (the kainoids) constitute a widely-used animal model for human mesial temporal lobe epilepsy (mTLE). New and existing molecules could prove useful as lead compounds for the development of therapeutics for neuropathologies that have aberrant glutamatergic signaling as a central component. In this chapter we discuss natural source origins and pharmacological activities of those marine compounds that target ionotropic glutamate receptors.

  11. Nitrosamines as nicotinic receptor ligands.

    Science.gov (United States)

    Schuller, Hildegard M

    2007-05-30

    Nitrosamines are carcinogens formed in the mammalian organism from amine precursors contained in food, beverages, cosmetics and drugs. The potent carcinogen, NNK, and the weaker carcinogen, NNN, are nitrosamines formed from nicotine. Metabolites of the nitrosamines react with DNA to form adducts responsible for genotoxic effects. We have identified NNK as a high affinity agonist for the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) whereas NNN bound with high affinity to epibatidine-sensitive nAChRs. Diethylnitrosamine (DEN) bound to both receptors but with lower affinity. High levels of the alpha7nAChR were expressed in human small cell lung cancer (SCLC) cell lines and in hamster pulmonary neuroendocrine cells (PNECs), which serve as a model for the cell of origin of human SCLC. Exposure of SCLC or PNECs to NNK or nicotine increased expression of the alpha7nAChR and caused influx of Ca(2+), activation of PKC, Raf-1, ERK1/2, and c-myc, resulting in the stimulation of cell proliferation. Signaling via the alpha7nAChR was enhanced when cells were maintained in an environment of 10-15% CO(2) similar to that in the diseased lung. Hamsters with hyperoxia-induced pulmonary fibrosis developed neuroendocrine lung carcinomas similar to human SCLC when treated with NNK, DEN, or nicotine. The development of the NNK-induced tumors was prevented by green tea or theophylline. The beta-adrenergic receptor agonist, isoproterenol or theophylline blocked NNK-induced cell proliferation in vitro. NNK and nicotine-induced hyperactivity of the alpha7nAChR/RAF/ERK1/2 pathway thus appears to play a crucial role in the development of SCLC in smokers and could be targeted for cancer prevention. PMID:17459420

  12. Stimulation of Hepatic Apolipoprotein A-I Production by Novel Thieno-Triazolodiazepines: Roles of the Classical Benzodiazepine Receptor, PAF Receptor, and Bromodomain Binding

    OpenAIRE

    Herman J. Kempen; Daniel Bellus; Oleg Fedorov; Silke Nicklisch; Panagis Filippakopoulos; Sarah Picaud; Stefan Knapp

    2013-01-01

    Expression and secretion of apolipoprotein A-I (apoA-I) by cultured liver cells can be markedly stimulated by triazolodiazepines (TZDs). It has been shown previously that the thieno-TZD Ro 11-1464 increases plasma levels of apoA-I and in vivomacrophage reverse cholesterol transport in mice. However, these effects were only seen at high doses, at which the compound could act on central benzodiazepine (BZD) receptors or platelet activating factor (PAF) receptors, interfering with its potential ...

  13. Interactions of pyrethroid insecticides with GABA sub A and peripheral-type benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Devaud, L.L.

    1988-01-01

    Pyrethroid insecticides are potent proconvulsants in the rat. All pyrethroids evincing proconvulsant activity elicited a similar 25-30% maximal reduction of seizure threshold. The Type II pyrethroids were the most potent proconvulsants with 1R{alpha}S, cis cypermethrin having an ED{sub 50} value of 6.3 nmol/kg. The proconvulsant activity of both Type I and Type II pyrenthroids was blocked by pretreatment with PK 11195, the peripheral-type benzodiazepine receptor (PTBR) antagonist. In contrast, phenytoin did not antagonize the proconvulsant activity of either deltamethrin or permethrin. Pyrethroids displaced the specific binding of ({sup 3}H)Ro5-4864 to rat brain membranes with a significant correlation between the log EC{sub 50} values for their activities as proconvulsants and the log IC{sub 50} values for their inhibition of ({sup 3}H)Ro5-4864 binding. Both Ro5-4864 and pyrethroid insecticides were found to influence specific ({sup 35}S)TBPS binding in a GABA-dependent manner. PK 11195 and the Type II pyrethroid, deltamethrin antagonized the Ro5-4864-induced modulation of ({sup 35}S)TBPS binding. Pyrethroid insecticides, Ro5-4864 and veratridine influenced GABA-gated {sup 36}Chloride influx. Moreover, the Type II pyrethroids elicited an increase in {sup 36}chloride influx in the absence of GABA-stimulation. Both of these actions were antagonized by PK 11195 and tetrodotoxin.

  14. Maternal Characteristics of Women Exposed to Hypnotic Benzodiazepine Receptor Agonist during Pregnancy

    Directory of Open Access Journals (Sweden)

    Bjarke Askaa

    2014-01-01

    Full Text Available Background. There is little knowledge regarding the characteristics of women treated with hypnotic benzodiazepine receptor agonists (HBRAs during pregnancy. In this large Danish cohort study, we characterize women exposed to HBRA during pregnancy. We determined changes in prevalence of HBRA use from 1997 to 2010 and exposure to HBRAs in relation to pregnancy. Methods. We performed a retrospective cohort study including 911,017 pregnant women in the period from 1997 to 2010. Information was retrieved from The Danish Birth Registry and The Registry of Medicinal Product Statistics to identify pregnant women redeeming a prescription of HBRAs. Results. We identified 2,552 women exposed to HBRAs during pregnancy, increasing from 0.18% in 1997 to 0.23% in 2010. Compared to unexposed women, exposed women were characterized by being older, with higher BMI, in their third or fourth parity, of lower income and education level, more frequently smokers, and more likely to be comedicated with antipsychotic, anxiolytic, or antidepressant drugs (P<0.0001. Conclusion. Women using HBRAs during their pregnancy differ from unexposed women in socioeconomic factors and were more likely to receive comedication. The consumption of HBRAs was reduced during pregnancy compared to before conception.

  15. Benzodiazepine receptor imaging with iomazenil SPECT in aphasic patients with cerebral infarction

    Energy Technology Data Exchange (ETDEWEB)

    Koshi, Yasuhiko; Kitamura, Shin; Ohyama, Masashi [Nippon Medical School, Tokyo (Japan)] (and others)

    1999-08-01

    To investigate the relationship between prognosis of aphasia and neuronal damage in the cerebral cortex, we evaluated the distribution of central-type benzodiazepine receptor (BZR) binding in post-stroke aphasics with [{sup 123}I]iomazenil and SPECT. We performed iomazenil SPECT in six aphasic patients (aged from 45 to 75 years; all right-handed) with unilateral left cerebral infarction. Three patients showed signs of Broca's aphasia and the other three Wernicke's aphasia. Cerebral blood flow (CBF) imaging was performed with [{sup 123}I]iodoamphetamine (IMP). The regions of interest (ROIs) on both images were set in the cerebral cortex, cerebellar cortex and language relevant area in both hemispheres. Three patients were classified in the mild prognosis group and the other three in the moderate prognosis group. The left language-relevant area was more closely concerned with the difference in aphasic symptoms than the right one in both BZR and CBF distribution, but the ipsilateral to the contralateral ratio (I/C ratio) in the language-relevant areas in the BZR distribution was significantly lower in the moderate prognosis group than in the mild prognosis group, although no difference was seen for these values between the two groups in the CBF distribution. These results suggest that BZR imaging, which makes possible an increase in neuronal cell viability in the cerebral cortex, is useful not only for clarifying the aphasic symptoms but also for evaluating the prognosis of aphasia in patients with cerebral infarction. (author)

  16. Potential role of cortical 5-HT(2A) receptors in the anxiolytic action of cyamemazine in benzodiazepine withdrawal.

    Science.gov (United States)

    Benyamina, Amine; Naassila, Mickaël; Bourin, Michel

    2012-07-30

    The antipsychotic cyamemazine is a potent serotonin 5-HT(2A) receptor (5-HT(2AR)) antagonist. A positron emission tomography (PET) study in human patients showed that therapeutic doses of cyamemazine produced near saturation of 5-HT(2AR) occupancy in the frontal cortex, whereas dopamine D(2) occupancy remained below the level for motor side effects observed with typical antipsychotics. Recently, numerous studies have revealed the involvement of 5-HT(2AR) in the pathophysiology of anxiety and a double-blind, randomized clinical trial showed similar efficacy of cyamemazine and bromazepam in reducing the anxiety associated with benzodiazepine withdrawal. Therefore, we reviewed the above articles about 5-HT(2AR) and anxiety in order to understand better the anxiolytic mechanisms of cyamemazine in benzodiazepine withdrawal. The 5-HT(2AR) is the most abundant serotonin receptor subtype in the cortex. Non-pharmacological studies with antisense oligodeoxynucleotides and genetically modified mice clearly showed that cortical 5-HT(2AR) signaling positively modulates anxiety-like behavior. With a few exceptions, most other studies reviewed here further support this view. Therefore, the anxiolytic efficacy of cyamemazine in benzodiazepine withdrawal can be due to a 5-HT(2AR) antagonistic activity at the cortical level.

  17. Glycomimetic ligands for the human asialoglycoprotein receptor.

    Science.gov (United States)

    Mamidyala, Sreeman K; Dutta, Sanjay; Chrunyk, Boris A; Préville, Cathy; Wang, Hong; Withka, Jane M; McColl, Alexander; Subashi, Timothy A; Hawrylik, Steven J; Griffor, Matthew C; Kim, Sung; Pfefferkorn, Jeffrey A; Price, David A; Menhaji-Klotz, Elnaz; Mascitti, Vincent; Finn, M G

    2012-02-01

    The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.

  18. Pharmacological properties of AC-3933, a novel benzodiazepine receptor partial inverse agonist.

    Science.gov (United States)

    Hashimoto, T; Kiyoshi, T; Kohayakawa, H; Iwamura, Y; Yoshida, N

    2014-01-01

    We investigated in this study the pharmacological properties of AC-3933 (5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-1,6-naphthyridin-2(1H)-one), a novel benzodiazepine receptor (BzR) partial inverse agonist. AC-3933 potently inhibited [3H]-flumazenil binding to rat whole brain membrane with a Ki value of 5.15 ± 0.39 nM and a GABA ratio of 0.84 ± 0.03. AC-3933 exhibited almost no affinity for the other receptors, transporters and ion channels used in this study. In addition, AC-3933, in the presence of GABA (1 μM), gradually but significantly increased [³⁵S] tert-butylbicyclophosphorothionate binding to rat cortical membrane to 117.1% of the control (maximum increase ratio) at 3000 nM. However, this increase reached a plateau at 30 nM with hardly any change at a concentration range of 100-3000 nM (from 115.2% to 117.1%). AC-3933 (0.1-10 μM) significantly enhanced KCl-evoked acetylcholine (ACh) release from rat hippocampal slices in a concentration-dependent manner. Moreover, in vivo brain microdialysis showed that intragastric administration of AC-3933 at the dose of 10 mg/kg significantly increased extracellular ACh levels in the hippocampus of freely moving rats (area under the curve (AUC₀₋₂ h) of ACh level; 288.3% of baseline). These results indicate that AC-3933, a potent and selective BzR inverse agonist with low intrinsic activity, might be useful in the treatment of cognitive disorders associated with degeneration of the cholinergic system.

  19. Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

    Energy Technology Data Exchange (ETDEWEB)

    Lentes, K.U.; Venter, J.C.

    1986-05-01

    Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 ..mu..M clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 ..mu..g trypsin/mg membrane protein yielded H/sub 2/O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain.

  20. Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

    International Nuclear Information System (INIS)

    Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 μM clonazepam) with 10 nM 3H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 μg trypsin/mg membrane protein yielded H2O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain

  1. Ligand binding was acquired during evolution of nuclear receptors

    OpenAIRE

    Escriva, Hector; Safi, Rachid; Hänni, Catherine; Langlois, Marie-Claire; Saumitou-Laprade, Pierre; Stehelin, Dominique; Capron, André; Pierce, Raymond; Laudet, Vincent

    1997-01-01

    The nuclear receptor (NR) superfamily comprises, in addition to ligand-activated transcription factors, members for which no ligand has been identified to date. We demonstrate that orphan receptors are randomly distributed in the evolutionary tree and that there is no relationship between the position of a given liganded receptor in the tree and the chemical nature of its ligand. NRs are specific to metazoans, as revealed by a screen of NR-related sequences in early- and non-metazoan organism...

  2. High density of benzodiazepine binding sites in the substantia innominata of the rat

    Energy Technology Data Exchange (ETDEWEB)

    Sarter, M.; Schneider, H.H.

    1988-07-01

    In order to study the neuronal basis of the pharmacological interactions between benzodiazepine receptor ligands and cortical cholinergic turnover, we examined the regional distribution of specific benzodiazepine binding sites using in vitro autoradiography. In the basal forebrain, the substantia innominata contained a high density of (/sup 3/H)lormetazepam (LMZ) binding sites (Bmax = 277 fmol/mg tissue; Kd = 0.55 nM). The label could be displaced by diazepam (IC50 = 100 nM), the benzodiazepine receptor antagonist beta-carboline ZK 93426 (45 nM) and the partial inverse agonist beta-carboline FG 7142 (540 nM). It is hypothesized that the amnesic effects of benzodiazepine receptor agonists are exerted through benzodiazepine receptors which are situated on cholinergic neurons in the substantia innominata and are involved in a tonic inhibition of cortical acetylcholine release. The benzodiazepine receptor antagonist ZK 93426 may exert its nootropic effects via benzodiazepine receptors in the substantia innominata and, consequently, by disinhibiting cortical acetylcholine release.

  3. Decrease in benzodiazepine receptor binding in a patient with Angelman syndrome detected by iodine-123 iomazenil and single-photon emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Odano, Ikuo [Dept. of Radiology, Niigata Univ. School of Medicine, Niigata (Japan); Anezaki, Toshiharu [Dept. of Neurology, Brain Research Inst., Niigata Univ., Niigata (Japan); Ohkubo, Masaki [Dept. of Radiology, Niigata Univ. School of Medicine, Niigata (Japan); Yonekura, Yoshiharu [Nihon Medi-Physics Co. Ltd., Hyogo (Japan); Onishi, Yoshihiro [Biomedical Imaging Research Center, Fukui Medical School, Fukui (Japan); Inuzuka, Takashi [Dept. of Neurology, Brain Research Inst., Niigata Univ., Niigata (Japan); Takahashi, Makoto [Dept. of Radiology, Niigata Univ. School of Medicine, Niigata (Japan); Tsuji, Shoji [Dept. of Neurology, Brain Research Inst., Niigata Univ., Niigata (Japan)

    1996-05-01

    A receptor mapping technique using iodine-123 iomazenil and single-photon emission tomography (SPET) was employed to examine benzodiazepine receptor binding in a patient with Angelman syndrome (AS). AS is characterized by developmental delay, seizures, inappropriate laughter and ataxic movement. In this entity there is a cytogenic deletion of the proximal long arm of chromosome 15q11-q13, where the gene encoding the GABA{sub A} receptor {beta}3 subunit (GABRB3) is located. Since the benzodiazepine receptor is constructed as a receptor-ionophore complex that contains the GABA{sub A} receptor, it is a suitable marker for GABA-ergic synapsis. To determine whether benzodiazepine receptor density, which indirectly indicates changes in GABA{sub A} receptor density, is altered in the brain in patients with AS, we investigated a 27-year-old woman with AS using {sup 123}I-iomazenil and SPET. Receptor density was quantitatively assessed by measuring the binding potential using a simplified method. Regional cerebral blood flow was also measured with N-isopropyl-p-[{sup 123}]iodoamphetamine. We demonstrated that benzodiazepine receptor density is severely decreased in the cerebellum, and mildly decreased in the frontal and temporal cortices and basal ganglia, a result which is considered to indicate decreased GABA{sub A} receptor density in these regions. Although the deletion of GABRB3 was not observed in the present study, we indirectly demonstrated the disturbance of inhibitory neurotransmission mediated by the GABA{sub A} receptor in the investigated patient. {sup 123}I-iomazenil with SPET was useful to map benzodiazepine receptors, which indicate GABA{sub A} receptor distribution and their density. (orig.)

  4. Radiosynthesis and initial evaluation of [18F]-FEPPA for PET imaging of peripheral benzodiazepine receptors

    International Nuclear Information System (INIS)

    Introduction: A novel [18F]-radiolabelled phenoxyanilide, [18F]-FEPPA, has been synthesized and evaluated, in vitro and ex vivo, as a potential positron emission tomography imaging agent for the peripheral benzodiazepine receptor (PBR). Methods: [18F]-FEPPA and two other radiotracers for imaging PBR, namely [11C]-PBR28 and [11C]-PBR28-d3, were synthesised and evaluated in vitro and ex vivo as potential PBR imaging agents. Results: [18F]-FEPPA is efficiently prepared in one step from its tosylate precursor and [18F]-fluoride in high radiochemical yields and at high specific activity. FEPPA displayed a Ki of 0.07 nM for PBR in rat mitochondrial membrane preparations and a suitable lipophilicity for brain penetration (log P of 2.99 at pH 7.4). Upon intravenous injection into rats, [18F]-FEPPA showed moderate brain uptake [standard uptake value (SUV) of 0.6 at 5 min] and a slow washout (SUV of 0.35 after 60 min). Highest uptake of radioactivity was seen in the hypothalamus and olfactory bulb, regions previously reported to be enriched in PBR in rat brain. Analysis of plasma and brain extracts demonstrated that [18F]-FEPPA was rapidly metabolized, but no lipophilic metabolites were observed in either preparation and only 5% radioactive metabolites were present in brain tissue extracts. Blocking studies to determine the extent of specific binding of [18F]-FEPPA in rat brain were problematic due to large perturbations in circulating radiotracer and the lack of a reference region. Conclusions: Further evaluation of the potential of [18F]-FEPPA will require the employment of rigorous kinetic models and/or appropriate animal models

  5. Radiosynthesis and in vivo evaluation of N-[{sup 11}C]methylated imidazopyridineacetamides as PET tracers for peripheral benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Sekimata, Katsuhiko [Department of Brain Sciences and Molecular Imaging, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8522 (Japan); Hatano, Kentaro [Department of Brain Sciences and Molecular Imaging, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8522 (Japan)], E-mail: hatanok@nils.go.jp; Ogawa, Mikako [Photon Medical Research Center, Hamamatsu University School of Medicine, Shizuoka 431-3192 Japan (Japan); Abe, Junichiro [Department of Brain Sciences and Molecular Imaging, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8522 (Japan); Magata, Yasuhiro [Photon Medical Research Center, Hamamatsu University School of Medicine, Shizuoka 431-3192 Japan (Japan); Biggio, Giovanni; Serra, Mariangela [Department of Experimental Biology, University of Cagliari, Cagliari 09100 (Italy); Laquintana, Valentino; Denora, Nunzio; Latrofa, Andrea; Trapani, Giuseppe; Liso, Gaetano [Pharmaco-Chemistry Department, University of Bari, Bari 70125 (Italy); Ito, Kengo [Department of Brain Sciences and Molecular Imaging, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8522 (Japan)

    2008-04-15

    Imidazopyridineacetoamide 5-8, a series of novel and potentially selective peripheral benzodiazepine receptor (PBR) ligands with affinities comparable to those of known PBR ligands, was investigated. Radiosyntheses of [{sup 11}C]5, 6, 7 or 8 was accomplished by N-methylation of the corresponding desmethyl precursors with [{sup 11}C]methyl iodide in the presence of NaH in dimethylformamide (DMF), resulting in 25% to 77% radiochemical yield and specific activitiy of 20 to 150 MBq/nmol. Each of the labeled compounds was injected in ddY mice, and the radioactivity and weight of dissected peripheral organs and brain regions were measured. Organ distribution of [{sup 11}C]7 was consistent with the known PBR distribution. Moreover, [{sup 11}C]7 showed the best combination of brain uptake and PBR binding, leading to its high retention in the olfactory bulb and cerebellum, areas where PBR density is high in mouse brain. Coinjection of PK11195 or unlabeled 7 significantly reduced the brain uptake of [{sup 11}C]7. These results suggest that [{sup 11}C]7 could be a useful radioligand for positron emission tomography imaging of PBRs.

  6. Structural basis for ligand recognition of incretin receptors

    DEFF Research Database (Denmark)

    Underwood, Christina Rye; Parthier, Christoph; Reedtz-Runge, Steffen

    2010-01-01

    The glucose-dependent insulinotropic polypeptide (GIP) receptor and the glucagon-like peptide-1 (GLP-1) receptor are homologous G-protein-coupled receptors (GPCRs). Incretin receptor agonists stimulate the synthesis and secretion of insulin from pancreatic β-cells and are therefore promising agents...... for the treatment of type 2 diabetes. It is well established that the N-terminal extracellular domain (ECD) of incretin receptors is important for ligand binding and ligand specificity, whereas the transmembrane domain is involved in receptor activation. Structures of the ligand-bound ECD of incretin receptors have...... appear to be the main driving force for ligand binding to the ECD of incretin receptors. Obviously, the-still missing-structures of full-length incretin receptors are required to construct a complete picture of receptor function at the molecular level. However, the progress made recently in structural...

  7. Integrin receptors and ligand-gated channels.

    Science.gov (United States)

    Morini, Raffaella; Becchetti, Andrea

    2010-01-01

    Plastic expression of different integrin subunits controls the different stages of neural development, whereas in the adult integrins regulate synaptic stability. Evidence of integrin-channel crosstalk exists for ionotropic glutamate receptors. As is often the case in other tissues, integrin engagement regulates channel activity through complex signaling pathways that often include tyrosine phosphorylation cascades. The specific pathways recruited by integrin activation depend on cerebral region and cell type. In turn, ion channels control integrin expression onto the plasma membrane and their ligand binding affinity. The most extensive studies concern the hippocampus and suggest implications for neuronal circuit plasticity. The physiological relevance of these findings depends on whether adhesion molecules, aside from determining tissue stability, contribute to synaptogenesis and the responsiveness of mature synapses, thus contributing to long-term circuit consolidation. Little evidence is available for other ligand-gated channels, with the exception of nicotinic receptors. These exert a variety of functions in neurons and non neural tissue, both in development and in the adult, by regulating cell cycle, synaptogenesis and synaptic circuit refinement. Detailed studies in epidermal keratinocytes have shed some light on the possible mechanisms through which ACh can regulate cell motility, which may be of general relevance for morphogenetic processes. As to the control of mature synapses, most results concern the integrinic control of nicotinic receptors in the neuromuscular junction. Following this lead, a few studies have addressed similar topics in adult cerebral synapses. However, pursuing and interpreting these results in the brain is especially difficult because of the complexity of the nicotinic roles and the widespread contribution of nonsynaptic, paracrine transmission. From a pathological point of view, considering the well-known contribution of both

  8. Antiplasmodial and GABA(A)-benzodiazepine receptor binding activities of five plants used in traditional medicine in Mali, West Africa.

    Science.gov (United States)

    Bah, Sekou; Jäger, Anna K; Adsersen, Anne; Diallo, Drissa; Paulsen, Berit Smestad

    2007-04-01

    Extracts of five medicinal plants: Boscia angustifolia, Cissus quadrangularis, Securidaca longipedunculata, Stylosanthes erecta and Trichilia emetica, used traditionally in Malian traditional medicine were screened for in vitro antiplasmodial activity and GABA(A)-benzodiazepine receptor binding activity. Four extracts showed significant antiplasmodial activities, with the dichloromethane extract of leaf of Securidaca longipedunculata being the most active (IC(50) of 7 microg/ml [95% CI: 5-9]). The dichloromethane extract of leaf of Trichilia emetica, in addition to its antiplasmodial activity (IC(50): 12 microg/ml [95% CI: 12-14]), exhibited a good binding activity to the GABA(A)-benzodiazepine receptor, while water and methanol extracts of the same plant did not show any activity. A strong GABA(A)-receptor complex binding activity was observed in the methanol extract of aerial part of Stylosanthes erecta. The results in this study justify some of the traditional indications of the plants investigated and may thus be candidates for Improved Traditional Medicines in Mali. PMID:17126508

  9. Substrate coated with receptor and labelled ligand for assays

    International Nuclear Information System (INIS)

    Improvements in the procedures for assaying ligands are described. The assay consists of a polystyrene tube on which receptors are present for both the ligand to be assayed and a radioactively labelled form of the ligand. The receptors on the bottom portion of the tube are also coated with labelled ligands, thus eliminating the necessity for separate addition of the labelled ligand and sample during an assay. Examples of ligands to which this method is applicable include polypeptides, nucleotides, nucleosides and proteins. Specific examples are given in which the ligand to be assayed is digoxin, the labelled form of the ligand is 3-0-succinyl digoxyigenin tyrosine (125I) and the receptor is digoxin antibody. (U.K.)

  10. Increased accuracy of ligand sensing by receptor internalization

    CERN Document Server

    Aquino, Gerardo

    2010-01-01

    Many types of cells can sense external ligand concentrations with cell-surface receptors at extremely high accuracy. Interestingly, ligand-bound receptors are often internalized, a process also known as receptor-mediated endocytosis. While internalization is involved in a vast number of important functions for the life of a cell, it was recently also suggested to increase the accuracy of sensing ligand as the overcounting of the same ligand molecules is reduced. Here we show, by extending simple ligand-receptor models to out-of-equilibrium thermodynamics, that internalization increases the accuracy with which cells can measure ligand concentrations in the external environment. Comparison with experimental rates of real receptors demonstrates that our model has indeed biological significance.

  11. {sup 125}I-iomazenil - benzodiazepine receptor binding and serum corticosterone level during psychological stress in a rat model

    Energy Technology Data Exchange (ETDEWEB)

    Fukumitsu, Nobuyoshi E-mail: GZL13162@nifty.ne.jp; Ogi, Shigeyuki; Uchiyama, Mayuki; Mori, Yutaka

    2004-02-01

    To test the hypothesis that benzodiazepine receptor density decreases in response to stress, we correlated {sup 125}I-iomazenil ({sup 125}I-IMZ) binding with serum corticosterone levels in a rat model. Wistar male rats were divided into four groups; control group (CON, 10 rats), no physical or psychological stress; and one-, three-, and five-day stress groups of 12 rats each (1-DAY, 3-DAY, and 5-DAY, respectively), receiving psychological stress for the given number of days. Psychological stress were given to rats with a communication box. The standardized uptake value (SUV) of {sup 125}I-iomazenil of the 3-DAY and 5-DAY showed that {sup 125}I-iomazenil - benzodiazepine receptor binding was significantly reduced in the cortices, accumbens nuclei, amygdala and caudate putamen (p<0.05). Serum corticosterone level ratio appeared to be slightly elevated in 3-DAY and 5-DAY, although this elevation was not significant. These data suggest that {sup 125}I-IMZ is a useful radioligand to reflect received stress and its binding in the cortices, accumbens nuclei, amygdala and caudate putamen is strongly affected by psychological stress.

  12. [Benzodiazepine and nonbenzodiazepine hypnotics].

    Science.gov (United States)

    Nakamura, Masaki; Inoue, Yuichi

    2015-06-01

    The prevalence of insomnia shows an age-associated increase. Especially, persons with age over 60 years frequently suffer from arousal during sleep and early-morning awakening. The reason of this phenomenon can be explained by age-related change in sleepwake regulation, comorbid diseases and psycho-social status. Benzodiazepine derivatives and benzodiazepine agonists have been widely used for treatment of insomnia. These GABA-A receptor agonist hypnotics have sedative effect, possibly causing various adverse events, i.e. falls and hip fracture, anterograde amnesia, next morning hangover especially in the elderly. When making a choice of treatment drugs for the elderly, low dose benzodiazepine hypnotics with relatively high Ω1-selectivity, and newer hypnotics including melatonic receptor agonist or orexin receptor antagonist can become important candidates considering their comorbid diseases or drug interaction with other medications. PMID:26065134

  13. Death receptors and ligands in cervical carcinogenesis : an immunohistochemical study

    NARCIS (Netherlands)

    Reesink-Peters, N; Hougardy, B M T; van den Heuvel, F A J; Ten Hoor, K A; Hollema, H; Boezen, H M; de Vries, E G E; de Jong, S; van der Zee, A G J

    2005-01-01

    OBJECTIVE: Increasing imbalance between proliferation and apoptosis is important in cervical carcinogenesis. The death ligands FasL and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induce apoptosis by binding to their cognate cell-surface death receptors Fas or death receptor (DR)

  14. Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

    Science.gov (United States)

    Huang, Xi-Ping; Karpiak, Joel; Kroeze, Wesley K; Zhu, Hu; Chen, Xin; Moy, Sheryl S; Saddoris, Kara A; Nikolova, Viktoriya D; Farrell, Martilias S; Wang, Sheng; Mangano, Thomas J; Deshpande, Deepak A; Jiang, Alice; Penn, Raymond B; Jin, Jian; Koller, Beverly H; Kenakin, Terry; Shoichet, Brian K; Roth, Bryan L

    2015-11-26

    At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs. PMID:26550826

  15. Influences of housing conditions and ethanol intake on binding characteristics of D2, 5-HT1A, and benzodiazepine receptors of rats.

    Science.gov (United States)

    Rilke, O; May, T; Oehler, J; Wolffgramm, J

    1995-09-01

    The effects of different housing conditions and ethanol treatment (6 vol % in the drinking water) on the in vitro binding characteristics of striatal dopaminergic D2 ([3H]spiperone), hippocampal serotonergic 5-HT1A ([3H]8-OH-DPAT), and cortical benzodiazepine ([3H]flunitrazepam) receptors have been examined. Social deprivation due to contact caging, short- (1 day) and long-term isolation (5 weeks) yielded a significant decrease of striatal D2 receptor density with the greatest decrease after long-term isolation (-21% Bmax) without changes of Kd in comparison to group animals. The effect of ethanol on striatal D2 receptor density depended on the housing conditions. Whereas ethanol treatment reduced receptor density of group animals (down to 88%), chronic exposure to ethanol under long-term isolation elicited no significant alteration of D2 receptor density compared with group animals. Different housing and ethanol treatment had no effect on 5-HT1A receptor affinity and density. Alterations of benzodiazepine receptor density were not found, but social deprivation as well as ethanol treatment of group animals caused an increased affinity of [3H]flunitrazepam (reduced Kd value). These results indicate that different housing conditions of adult rats evoked significant alterations in D2 and benzodiazepine receptor binding assays, which were modified by ethanol treatment in the case of striatal D2 receptor density.

  16. PK11195 binding to the peripheral benzodiazepine receptor as a marker of microglia activation in multiple sclerosis and experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Vowinckel, E; Reutens, D; Becher, B;

    1997-01-01

    Activated glial cells are implicated in regulating and effecting the immune response that occurs within the CNS as part of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). The peripheral benzodiazepine receptor (PBR) is expressed in glial cells. We exa...

  17. Quantitative autoradiographic determination of binding sites for a peripheral benzodiazepine ligand ((/sup 3/H)PK 11195) in human iris

    Energy Technology Data Exchange (ETDEWEB)

    Valtier, D.; Malgouris, C.; Uzan, A.

    1987-01-01

    Specific binding sites of peripheral-type benzodiazepines were investigated in human iris/ciliary body (8 eyes). Examination of color-coded prints and densitometric quantification of autoradiograms were performed on slides (20 ..mu..m) labelled with (/sup 3/H)PK 11195 (1 nM) at 25 deg C. Nonspecific binding was determined with PK 11211 (5 ..mu..M) or Ro 5-4864 (5 ..mu..M). Binding sites were present on all the slides, with equivalent density in the 3 regions of the preparation (ciliary body, iris and pupil margin). The numbers of binding sites in ciliary body, iris, and pupil margin, respectively were: 42.7 +- 0.2, 30.1 +- 0.5 and 37.4 +- 0.4 femtomol/mg protein. Labelling on the pupil margin seemed to coincide with the iris sphincter muscle. The presence of peripheral benzodiazepine binding sites in iris muscular tissue, and particularly in the pupil margin, suggests that the iris preparation may be a valuable tool to detect putative physiological effects of peripheral benzodiazepines on muscular motility.

  18. Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

    OpenAIRE

    Brown, M. J.; Bristow, D. R.

    1996-01-01

    1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treat...

  19. (/sup 3/H)Clonazepam, like (/sup 3/H)flunitrazepam, is a photoaffinity label for the central type of benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Sieghart, W. (Vienna Univ. (Austria)); Moehler, H. (Hoffmann-La Roche (F.) and Co., Basel (Switzerland))

    1982-06-16

    (/sup 3/H)Clonazepam, like (/sup 3/H)flunitrazepam, is irreversibly bound to membrane proteins of brain tissue when exposed to UV light. In polyacrylamide gel electrophoresis followed by fluorography, the same pattern of photolabelled proteins was obtained in cerebellum and in hippocampus when either (/sup 3/H)clonazepam or (/sup 3/H)flunitrazepam was used as photoaffinity label. Since (/sup 3/H)clonazepam does not interact with the peripheral type of benzodiazepine binding site present in the brain, these results confirm previous evidence that the proteins photolabelled with (/sup 3/H)flunitrazepam are associated with the central type of benzodiazepine receptor.

  20. A response calculus for immobilized T cell receptor ligands

    DEFF Research Database (Denmark)

    Andersen, P S; Menné, C; Mariuzza, R A;

    2001-01-01

    determine the level of T cell activation. When fitted to T cell responses against purified ligands immobilized on plastic surfaces, the 2D-affinity model adequately simulated changes in cellular activation as a result of varying ligand affinity and ligand density. These observations further demonstrated......To address the molecular mechanism of T cell receptor (TCR) signaling, we have formulated a model for T cell activation, termed the 2D-affinity model, in which the density of TCR on the T cell surface, the density of ligand on the presenting surface, and their corresponding two-dimensional affinity...... the importance of receptor cross-linking density in determining TCR signaling. Moreover, it was found that the functional two-dimensional affinity of TCR ligands was affected by the chemical composition of the ligand-presenting surface. This makes it possible that cell-bound TCR ligands, despite their low...

  1. Midazolam ameliorates the behavior deficits of a rat posttraumatic stress disorder model through dual 18 kDa translocator protein and central benzodiazepine receptor and neurosteroidogenesis.

    Directory of Open Access Journals (Sweden)

    Yu-Liang Miao

    Full Text Available Post-traumatic stress disorder (PTSD is a debilitating anxiety disorder that may develop after an individual has experienced or witnessed a severe traumatic event. It has been shown that the 18 kDa translocator protein (TSPO may be correlated with PTSD and that the TSPO ligand improved the behavioral deficits in a mouse model of PTSD. Midazolam, a ligand for TSPO and central benzodiazepine receptor (CBR, induces anxiolytic- and anti-depressant-like effects in animal models. The present study aimed to determine whether midazolam ameliorates PTSD behavior in rats as assessed by the single prolonged stress (SPS model. The SPS rats received daily Sertraline (Ser (15 mg/kg, i.p. [corrected] and midazolam (0.125, 0.25, 0.5, and 1 mg/kg, i.p. [corrected] during the exposure to SPS and behavioral assessments, which included the open field (OF test, the contextual fear paradigm (CFP, and the elevated plus-maze (EPM. The results showed that, like Ser (15 mg/kg, i.p. [corrected], midazolam (0.25 and 0.5 mg/kg, i.p. [corrected] significantly reversed the behavioral deficiencies of the SPS rats, including PTSD-associated freezing and anxiety-like behavior but not the effects on spontaneous locomotor activity. In addition, the anti-PTSD effects of midazolam (0.5 mg/kg, i.p. [corrected] were antagonized by the TSPO antagonist PK11195 (3 mg/kg, i.p., the CBR antagonist flumazenil (15 mg/kg, i.p. [corrected] and the inhibitor of steroidogenic enzymes finasteride (30 mg/kg, i.p. [corrected], which by themselves had no effect on PTSD-associated freezing and anxiety-like behavior. In summary, this study demonstrated that midazolam improves the behavioral deficits in the SPS model through dual TSPO and CBR and neurosteroidogenesis.

  2. The 18 kDa translocator protein (peripheral benzodiazepine receptor expression in the bone of normal, osteoprotegerin or low calcium diet treated mice.

    Directory of Open Access Journals (Sweden)

    Winnie Wai-Ying Kam

    Full Text Available The presence of the translocator protein (TSPO, previously named as the mitochondrial or peripheral benzodiazepine receptor, in bone cells was studied in vitro and in situ using RT-qPCR, and receptor autoradiography using the selective TSPO ligand PK11195.In vitro, the TSPO is highly expressed in osteoblastic and osteoclastic cells.In situ, constitutive expression of TSPO is found in bone marrow and trabecular bone, e.g., spongiosa. Mice with a reduction of bone turnover induced by a 4-day treatment of osteoprotegerin reduces [(3H]PK11195 binding in the spongiosa (320±128 Bq x mg(-1, 499±106 Bq x mg(-1 in saline-treated controls. In contrast, mice with an increase in bone turnover caused by a 4-day low calcium diet increases [(3H]PK11195 binding in the spongiosa (615±90 Bq x mg(-1. Further, our study includes technical feasibility data on [(18F]fluoride microPET imaging of rodent bone with altered turnover. Despite [(18F]fluoride having high uptake, the in vivo signal differences were small. Using a phantom model, we describe the spillover effect and partial volume loss that affect the quantitative microPET imaging of the small bone structures in experimental mouse models. In summary, we demonstrate the expression of TSPO in small rodent bone tissues, including osteoblasts and osteoclasts. A trend increase in TSPO expression was observed in the spongiosa from low to high bone turnover conditions. However, despite the potential utility of TSPO expression as an in vivo biomarker of bone turnover in experimental rodent models, our small animal PET imaging data using [(18F]fluoride show that even under the condition of a good biological signal-to-noise ratio and high tracer uptake, the currently achievable instrument sensitivity and spatial resolution is unlikely to be sufficient to detect subtle differences in small structures, such as mouse bone.

  3. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    Energy Technology Data Exchange (ETDEWEB)

    Taguchi, J.; Kuriyama, K. (Kyoto Prefectural Univ. of Medicine (Japan))

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  4. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    International Nuclear Information System (INIS)

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited [3H]flunitrazepam binding to benzodiazepine receptor, but not [3H]muscimol binding to GABAA receptor as well as t-[3H]butylbicycloorthobenzoate [( 3H] TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively [3H] flunitrazepam binding. On the other hand, the binding of beta-[3H]CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated [3H]muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-[3H]CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for [3H]flunitrazepam, [3H]muscimol and [3H]TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested

  5. Sliding tethered ligands add topological interactions to the toolbox of ligand-receptor design

    Science.gov (United States)

    Bauer, Martin; Kékicheff, Patrick; Iss, Jean; Fajolles, Christophe; Charitat, Thierry; Daillant, Jean; Marques, Carlos M.

    2015-09-01

    Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand-receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand-receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering.

  6. Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    indicate that channel opening is accompanied by conformational rearrangements in both beta-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on alpha1 glycine receptors to compare changes mediated by the agonist, glycine...... in the inner beta-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded...... differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors....

  7. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABA(A) receptors expressed in Xenopus laevis oocytes.

    Science.gov (United States)

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik Sindal; Jensen, Anders A

    2015-01-01

    The 1,5-benzodiazepine clobazam is indicated for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in patients 2 years of age or older in the United States, and for treatment of anxiety and various forms of epilepsy elsewhere. Clobazam has been reported to exhibit different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences. The functional properties of the two 1,5-benzodiazepines were characterized at the human γ-aminobutyric acid type A receptor (GABA(A)R) subtypes α1β2γ(2S), α2β2γ(2S), α3β2γ(2S), α5β2γ(2S) and α6β2δ expressed in Xenopus laevis oocytes by use of two-electrode voltage-clamp electrophysiology and compared to those exhibited by the 1,4-benzodiazepine clonazepam. All three compounds potentiated GABA EC20-evoked responses through the α(1,2,3,5)β2γ(2S) GABA(A)Rs in a reversible and concentration-dependent manner, with each displaying similar EC50 values at the four subtypes. Furthermore, the degrees of potentiation of the GABA EC20 currents through the four receptors mediated by saturating modulator concentrations did not differ substantially for any of the three benzodiazepines. The three compounds were substantially less potent (200-3900 fold) as positive allosteric modulators at the α6β2δ GABA(A)R than at the α(1,2,3,5)β2γ(2S) receptors. Interestingly, however, clobazam and especially N-desmethylclobazam were highly efficacious potentiators of α6β2δ receptor signaling. Although this activity component is unlikely to contribute to the in vivo effects of clobazam/N-desmethylclobazam, the 1,5-benzodiazepine could constitute an interesting lead for novel modulators targeting this low-affinity binding site in GABAARs. In conclusion, the non-selective modulation

  8. SPECT imaging of GABA{sub A}/benzodiazepine receptors and cerebral perfusion in mild cognitive impairment

    Energy Technology Data Exchange (ETDEWEB)

    Pappata, Sabina; Varrone, Andrea; Vicidomini, Caterina; Sansone, Valeria; Comerci, Marco; Panico, Maria Rosaria; Quarantelli, Mario [CNR, Institute of Biostructure and Bioimaging, Naples (Italy); Milan, Graziella; De Falco, Caterina; Lore, Elisa; Postiglione, Alfredo [University ' ' Federico II' ' , Department of Clinical and Experimental Medicine, Naples (Italy); Iavarone, Alessandro [Neurologic and Stroke Unit, CTO Hospital, Naples (Italy); Salvatore, Marco [CNR, Institute of Biostructure and Bioimaging, Naples (Italy); University ' ' Federico II' ' , Department of Biomorphological and Functional Sciences, Naples (Italy)

    2010-06-15

    The involvement of neocortical and limbic GABA{sub A}/benzodiazepine (BZD) receptors in Alzheimer's disease (AD) is controversial and mainly reported in advanced stages. The status of these receptors in the very early stages of AD is unclear and has not been explored in vivo. Our aims were to investigate in vivo the integrity of cerebral cortical GABA{sub A}/BZD receptors in subjects with amnestic mild cognitive impairment (MCI) and to compare possible receptor changes to those in cerebral perfusion. [{sup 123}I]Iomazenil and [{sup 99m}Tc]HMPAO SPECT images were acquired in 16 patients with amnestic MCI and in 14 normal elderly control subjects (only [{sup 123}I]iomazenil imaging in 5, only [{sup 99m}Tc]HMPAO imaging in 4, and both [{sup 123}I]iomazenil and [{sup 99m}Tc]HMPAO imaging in 5). Region of interest (ROI) analysis and voxel-based analysis were performed with cerebellar normalization. Neither ROI analysis nor voxel-based analysis showed significant [{sup 123}I]iomazenil binding changes in MCI patients compared to control subjects, either as a whole group or when considering only those patients with MCI that converted to AD within 2 years of clinical follow-up. In contrast, the ROI analysis revealed significant hypoperfusion of the precuneus and posterior cingulate cortex in the whole group of MCI patients and in MCI converters as compared to control subjects. Voxel-based analysis showed similar results. These results indicate that in the very early stages of AD, neocortical and limbic neurons/synapses expressing GABA{sub A}/BZD receptors are essentially preserved. They suggest that in MCI patients functional changes precede neuronal/synaptic loss in neocortical posterior regions and that [{sup 99m}Tc]HMPAO rCBF imaging is more sensitive than [{sup 123}I]iomazenil GABA{sub A}/BZD receptor imaging in detecting prodromal AD. (orig.)

  9. Modulation of cholinephosphotransferase activity in breast cancer cell lines by Ro5-4864, a peripheral benzodiazepine receptor agonist

    International Nuclear Information System (INIS)

    Changes in phospholipid and fatty acid profile are hallmarks of cancer progression. Increase in peripheral benzodiazepine receptor expression has been implicated in breast cancer. The benzodiazepine, Ro5-4864, increases cell proliferation in some breast cancer cell lines. Biosynthesis of phosphatidylcholine (PC) has been identified as a marker for cells proliferating at high rates. Cholinephosphotransferase (CPT) is the terminal enzyme for the de novo biosynthesis of PC. We have addressed here whether Ro5-4864 facilitates some cancer causing mechanisms in breast cancer. We report that cell proliferation increases exponentially in aggressive breast cancer cell lines 11-9-1-4 and BT-549 when treated with nanomolar concentrations of Ro5-4864. This increase is seen within 24 h of treatment, consistent with the cell doubling time in these cells. Ro5-4864 also upregulates c-fos expression in breast cancer cell lines 11-9-1-4 and BT-549, while expression in non-tumorigenic cell line MCF-12A was either basal or slightly downregulated. We further examined the expression of the CPT gene in breast cancer (11-9-1-4, BT-549) and non-tumorigenic cell lines (MCF-12A, MCF-12F). We found that the CPT gene is overexpressed in breast cancer cell lines compared to the non-tumorigenic cell lines. Furthermore, the activity of CPT in forming PC is increased in the breast cancer cell lines cultured for 24 h. Additionally, we examined the CPT activity in the presence of nanomolar concentrations of Ro5-4864. Biosynthesis of PC was increased in breast cancer cell lines upon treatment. We therefore propose that Ro5-4864 facilitates PC formation, a process important in membrane biogenesis for proliferating cells

  10. Quantification of human brain benzodiazepine receptors using [{sup 18}F]fluoroethylflumazenil: a first report in volunteers and epileptic patients

    Energy Technology Data Exchange (ETDEWEB)

    Leveque, Philippe [Unite de Tomographie par Positrons, Universite Catholique de Louvain, Louvain-la-Neuve (Belgium); Unite de Chimie Pharmaceutique et de Radiopharmacie, CMFA/REMA, Universite Catholique de Louvain, 73-40 Avenue Mounier, 1200, Bruxelles (Belgium); Sanabria-Bohorquez, Sandra [Imaging Research, Merck Research Laboratories, West Point, Philadelphia (United States); Bol, Anne; Volder, Anne de; Labar, Daniel [Unite de Tomographie par Positrons, Universite Catholique de Louvain, Louvain-la-Neuve (Belgium); Rijckevorsel, K. van [Service de Neurologie, Cliniques Universitaires Saint-Luc, Bruxelles (Belgium); Gallez, Bernard [Unite de Chimie Pharmaceutique et de Radiopharmacie, CMFA/REMA, Universite Catholique de Louvain, 73-40 Avenue Mounier, 1200, Bruxelles (Belgium); Unite de Resonance Magnetique Biomedicale, Universite Catholique de Louvain, Bruxelles (Belgium)

    2003-12-01

    Fluorine-18 fluoroethylflumazenil ([{sup 18}F]FEF) is a tracer for central benzodiazepine (BZ) receptors which is proposed as an alternative to carbon-11 flumazenil for in vivo imaging using positron emission tomography (PET) in humans. In this study, [{sup 18}F]FEF kinetic data were acquired using a 60-min two-injection protocol on three normal subjects and two patients suffering from mesiotemporal epilepsy as demonstrated by abnormal magnetic resonance imaging and [{sup 18}F]fluorodeoxyglucose positron emission tomography. First, a tracer bolus injection was performed and [{sup 18}F]FEF rapidly distributed in the brain according to the known BZ receptor distribution. Thirty minutes later a displacement injection of 0.01 mg/kg of unlabelled flumazenil was performed. Activity was rapidly displaced from all BZ receptor regions demonstrating the specific binding of [{sup 18}F]FEF. No displacement was observed in the pons. Plasma input function was obtained from arterial blood sampling, and metabolite analysis was performed by high-performance liquid chromatography. Metabolite quantification revealed a fast decrease in tracer plasma concentration, such that at 5 min post injection about 70% of the total radioactivity in plasma corresponded to [{sup 18}F]FEF, reaching 24% at 30 min post injection. The interactions between [{sup 18}F]FEF and BZ receptors were described using linear compartmental models with plasma input and reference tissue approaches. Binding potential values were in agreement with the known distribution of BZ receptors in human brain. Finally, in two patients with mesiotemporal sclerosis, reduced uptake of [{sup 18}F]FEF was clearly observed in the implicated left hippocampus. (orig.)

  11. Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor.

    Directory of Open Access Journals (Sweden)

    Xavier Charest-Morin

    Full Text Available The bradykinin (BK B1 receptor (B1R is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B1R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP (enhanced green FP [EGFP] or mCherry prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg9-BK, [Leu8]des-Arg9-BK, respectively. The design of the spacer-ligand joint peptide was validated by a competition assay for [3H]Lys-des-Arg9-BK binding to the human B1R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B1R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy. Both assays indicated that the best design was FP-(Asn-Glyn-Lys-des-Arg9-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B1R antagonist. EGFP-(Asn-Gly15-Lys-des-Arg9-BK competed for the binding of [3H]Lys-des-Arg9-BK to human recombinant B1R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg9-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B1R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology.

  12. Single dose efficacy evaluation of two partial benzodiazepine receptor agonists in photosensitive epilepsy patients: A placebo-controlled pilot study.

    Science.gov (United States)

    Kasteleijn-Nolst Trenité, Dorothée G A; Groenwold, Rolf H H; Schmidt, Bernd; Löscher, Wolfgang

    2016-05-01

    Benzodiazepines (BZDs) are highly effective to suppress various types of seizures; however, their clinical use is limited due to adverse effects and tolerance and dependence liability. Drugs that act only as partial agonists at the BZD recognition site (initially termed "BZD receptor") of the GABAA receptor chloride ionophore complex or exhibit a GABAA receptor subtype-selectivity are thought to have advantages vs. full agonists such as diazepam and most other clinically used BZDs in that such compounds have less adverse effects and reduced or absent tolerance and dependence liability. One of such compounds, abecarnil, has been clinically evaluated as a novel anxiolytic drug, but, despite its potent preclinical anti-seizure activity, it has not yet been evaluated in patients with epilepsy. In the present proof-of-concept study, we performed a within-subject placebo-controlled, single oral dose study of abecarnil in patients with photosensitive epilepsy. Flumazenil, which is generally considered a BZD receptor antagonist, but has slight partial agonistic properties, was used for comparison. In total, 12 patients were enrolled in this study. Abecarnil, 5 or 10mg, completely abolished the photo-paroxysmal EEG response, while flumazenil, 30, 60 or 100mg, was less effective. The anti-epileptic effect of abecarnil was significantly different from both placebo and flumazenil. Sedative adverse effects were observed after abecarnil but not flumazenil. The study substantiates previous pre-clinical experiments that abecarnil exerts pronounced anti-seizure activity. Epilepsy is often associated with anxiety, so that the anxiolytic activity of abecarnil would be an added advantage when using this compound in epilepsy patients. PMID:26921854

  13. The imidazoline receptors and ligands in pain modulation

    Directory of Open Access Journals (Sweden)

    Nurcan Bektas

    2015-01-01

    Full Text Available Pain is an unpleasant experience and effects daily routine negatively. Although there are various drugs, many of them are not entirely successful in relieving pain, since pain modulation is a complex process involving numerous mediators and receptors. Therefore, it is a rational approach to identify the factors involved in the complex process and develop new agents that act on these pain producing mechanisms. In this respect, the involvement of the imidazoline receptors in pain modulation has drawn attention in recent years. In this review, it is aimed to focus on the imidazoline receptors and their ligands which contribute to the pain modulation. It is demonstrated that imidazoline-2 (I2 receptors are steady new drug targets for analgesics. Even if the mechanism of I2receptor is not well known in the modulation of pain, it is known that it plays a role in tonic and chronic pain but not in acute phasic pain. Moreover, the I2receptor ligands increase the analgesic effects of opioids in both acute and chronic pain and prevent the development of opioid tolerance. So, they are valuable for the chronic pain treatment and also therapeutic coadjuvants in the management of chronic pain with opiate drugs due to the attenuation of opioid tolerance and addiction. Thus, the use of the ligands which bind to the imidazoline receptors is an effective strategy for relieving pain. This educational forum exhibits the role of imidazoline receptors and ligands in pain process by utilizing experimental studies.

  14. Biodistribution and dosimetry of [{sup 123}I]iodo-Pk 11195: a potential agent for SPET imaging of the peripheral benzodiazepine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Versijpt, J. [Groningen Univ. Hospital (Netherlands). Dept. of Biological Psychiatry; Div. of Nuclear Medicine, Ghent Univ. Hospital (Belgium); Dumont, F.; Vos, F. de; Slegers, G. [Dept. of Radiopharmacy, Ghent Univ. (Belgium); Thierens, H. [Dept. of Biomedical Physics and Radiation Protection, Ghent Univ. (Belgium); Jansen, H.; Dierckx, R.A. [Div. of Nuclear Medicine, Ghent Univ. Hospital (Belgium); Santens, P. [Dept. of Neurology, Ghent Univ. Hospital (Belgium); Korf, J. [Groningen Univ. Hospital (Netherlands). Dept. of Biological Psychiatry

    2000-09-01

    The highest concentrations of the peripheral benzodiazepine receptor (PBR) are found in the kidneys and heart. In addition, the PBR has been reported to reflect neuro-inflammatory damage by co-localisation with activated microglia. PK 11195 is a high-affinity ligand for the PBR. The aim of this study was to investigate in humans the biodistribution and dosimetry of [{sup 123}I]iodo-PK 11195, a potential single-photon emission tomography tracer for the PBR. Five healthy volunteers were injected with 112 MBq of [{sup 123}I]iodo-PK 11195. Sequential whole-body scans were performed up to 72 h post injection. Multiple blood samples were taken, and urine was collected to measure the fraction voided by the renal system. Decay-corrected regions of interest of the whole-body images were analysed, and geometric mean count rates were used to determine organ activity. Organ absorbed doses and effective dose were calculated using the MIRD method. [{sup 123}I]iodo-PK 11195 was rapidly cleared from the blood, mainly by the hepatobiliary system. Approximately 22% was voided in urine after 48 h. Average organ residence times were 0.74, 0.44 and 0.29 h for the liver, upper large intestine and lower large intestine, respectively. The testes received the highest dose, 109.4 {mu}Gy/MBq. All other organs investigated received doses of less than 50 {mu}Gy/MBq. The effective dose was 40.3 {mu}Sv/MBq. In conclusion, [{sup 123}I]iodo-PK 11195 is a suitable agent for the visualisation of the PBR and indirectly for the imaging of neuro-inflammatory lesions. Taking into account the radiation burden of 7.46 mSv following an administration of 185 MBq, a [{sup 123}I]iodo-PK 11195 investigation has to be considered an ICRP risk category IIb investigation. (orig.)

  15. Biodistribution and dosimetry of [123I]iodo-Pk 11195: a potential agent for SPET imaging of the peripheral benzodiazepine receptor

    International Nuclear Information System (INIS)

    The highest concentrations of the peripheral benzodiazepine receptor (PBR) are found in the kidneys and heart. In addition, the PBR has been reported to reflect neuro-inflammatory damage by co-localisation with activated microglia. PK 11195 is a high-affinity ligand for the PBR. The aim of this study was to investigate in humans the biodistribution and dosimetry of [123I]iodo-PK 11195, a potential single-photon emission tomography tracer for the PBR. Five healthy volunteers were injected with 112 MBq of [123I]iodo-PK 11195. Sequential whole-body scans were performed up to 72 h post injection. Multiple blood samples were taken, and urine was collected to measure the fraction voided by the renal system. Decay-corrected regions of interest of the whole-body images were analysed, and geometric mean count rates were used to determine organ activity. Organ absorbed doses and effective dose were calculated using the MIRD method. [123I]iodo-PK 11195 was rapidly cleared from the blood, mainly by the hepatobiliary system. Approximately 22% was voided in urine after 48 h. Average organ residence times were 0.74, 0.44 and 0.29 h for the liver, upper large intestine and lower large intestine, respectively. The testes received the highest dose, 109.4 μGy/MBq. All other organs investigated received doses of less than 50 μGy/MBq. The effective dose was 40.3 μSv/MBq. In conclusion, [123I]iodo-PK 11195 is a suitable agent for the visualisation of the PBR and indirectly for the imaging of neuro-inflammatory lesions. Taking into account the radiation burden of 7.46 mSv following an administration of 185 MBq, a [123I]iodo-PK 11195 investigation has to be considered an ICRP risk category IIb investigation. (orig.)

  16. Benzodiazepine-induced intestinal motor disturbances in rats: mediation by omega 2 (BZ2) sites on capsaicin-sensitive afferent neurones.

    OpenAIRE

    Bonnafous, C; Scatton, B; BUÉNO, L.

    1994-01-01

    1. The central and peripheral effects of the omega (benzodiazepine) site ligands, clonazepam, alpidem, zolpidem, triazolam, flumazenil, ethyl beta carboline-3-carboxylate (beta-CCE) and N-methyl beta carboline-3-carboxylate (beta-CCM) on intestinal myoelectrical activity were evaluated in conscious rats, chronically fitted with Nichrome electrodes implanted on the duodenum and jejunum. The localization of the omega (benzodiazepine) receptors involved in these effects was evaluated by use of s...

  17. Kinetics of Receptor-Ligand Interactions in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    Mian Long; Shouqin Lü; Ganyun Sun

    2006-01-01

    Receptor-ligand interactions in blood flow are crucial to initiate the biological processes as inflammatory cascade,platelet thrombosis, as well as tumor metastasis. To mediate cell adhesions, the interacting receptors and ligands must be anchored onto two apposing surfaces of two cells or a cell and a substratum, i.e., the two-dimensional (2D) binding, which is different from the binding of a soluble ligand in fluid phase to a receptor, i.e., three-dimensional (3D) binding. While numerous works have been focused on 3D kinetics of receptor-ligand interactions in immune systems, 2D kinetics and its regulations have less been understood, since no theoretical framework and experimental assays have been established until 1993. Not only does the molecular structure dominate 2D binding kinetics, but the shear force in blood flow also regulates cell adhesions mediated by interacting receptors and ligands. Here we provided the overview of current progresses in 2D bindings and regulations. Relevant issues of theoretical frameworks, experimental measurements, kinetic rates and binding affinities, and force regulations,were discussed.

  18. Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling.

    Science.gov (United States)

    de Lima, Camila Bento; Tamura, Eduardo K; Montero-Melendez, Trindad; Palermo-Neto, João; Perretti, Mauro; Markus, Regina P; Farsky, Sandra Helena Poliselli

    2012-01-13

    The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response. PMID:22209795

  19. Experiment K-6-18. Study of muscarinic and gaba (benzodiazepine) receptors in the sensory-motor cortex, hippcampus and spinal code

    Science.gov (United States)

    Daunton, N.; Damelio, F.; Krasnov, I.

    1990-01-01

    Frontal lobe samples of rat brains flown aboard Cosmos 1887 were processed for the study of muscarinic (cholinergic) and GABA (benzodiazepine) receptors and for immunocytochemical localization of the neurotransmitter gamma-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP). Although radioactive labeling of both muscarinic cholinergic and GABA (benzodiazepine) receptors proved to be successful with the techniques employed, distinct receptor localization of individual laminae of the frontal neocortex was not possible since the sampling of the area was different in the various groups of animals. In spite of efforts made for proper orientation and regional identification of laminae, it was found that a densitometric (quantitation of autoradiograms) analysis of the tissue did not contribute to the final interpretation of the effects of weightlessness on these receptors. As to the immunocytochemical studies the use of both markers, GFAP and GABA antiserum, confirmed the suitability of the techniques for use in frozen material. However, similar problems to those encountered in the receptor studies prevented an adequate interpretation of the effects of micro-G exposure on the localization and distribution of GABA and GFAP. This study did, however, confirm the feasibility of investigating neurotransmitters and their receptors in future space flight experiments.

  20. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    Directory of Open Access Journals (Sweden)

    Beaudet A.

    1998-01-01

    Full Text Available This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

  1. Syntheses of oxysterol receptors'(LXRs) ligands

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The LXRs' agonist, 24S,25-epoxycholesterol 1, was synthesized stereoselectively (100% d.e.) in 56% overall yield from methyl hyodeoxycholanate 4 in 9 steps with desmosterol acetate 11 as the key intermediate and the modified Sharpless asymmetric dihydroxylation as the key step. The LXR? subtype selective agonist 5α,6α:24S,25-diepoxycho- lesterol 2 and the novel LXRs' ligand 5β,6β:24S,25-diepo- xycholesterol 3 were also synthesized from 1.

  2. Inside job: ligand-receptor pharmacology beneath the plasma membrane

    Science.gov (United States)

    Babcock, Joseph J; Li, Min

    2013-01-01

    Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments. However, these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone. Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell. These additional modes of interaction have been reported for functionally diverse ligands of GPCRs, ion channels, and transporters. Such intracellular drug-target engagements affect cell surface expression. Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation. Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites, and the potential therapeutic opportunities presented by this phenomenon. PMID:23685953

  3. Inside job: ligand-receptor pharmacology beneath the plasma membrane

    Institute of Scientific and Technical Information of China (English)

    Joseph J BABCOCK; Min LI

    2013-01-01

    Most drugs acting on the cell surface receptors are membrane permeable and thus able to engage their target proteins in different subcellular compartments.However,these drugs' effects on cell surface receptors have historically been studied on the plasma membrane alone.Increasing evidence suggests that small molecules may also modulate their targeted receptors through membrane trafficking or organelle-localized signaling inside the cell.These additional modes of interaction have been reported for functionally diverse ligands of GPCRs,ion channels,and transporters.Such intracellular drug-target engagements affect cell surface expression.Concurrent intracellular and cell surface signaling may also increase the complexity and therapeutic opportunities of small molecule modulation.Here we discuss examples of ligand-receptor interactions that are present in both intra- and extracellular sites,and the potential therapeutic opportunities presented by this phenomenon.

  4. Novel retinoic acid receptor ligands in Xenopus embryos.

    OpenAIRE

    Blumberg, B; Bolado, J; Derguini, F; Craig, A G; Moreno, T A; Chakravarti, D; Heyman, R A; Buck, J.; Evans, R M

    1996-01-01

    Retinoids are a large family of natural and synthetic compounds related to vitamin A that have pleiotropic effects on body physiology, reproduction, immunity, and embryonic development. The diverse activities of retinoids are primarily mediated by two families of nuclear retinoic acid receptors, the RARs and RXRs. Retinoic acids are thought to be the only natural ligands for these receptors and are widely assumed to be the active principle of vitamin A. However, during an unbiased, bioactivit...

  5. Stimulation of Hepatic Apolipoprotein A-I Production by Novel Thieno-Triazolodiazepines: Roles of the Classical Benzodiazepine Receptor, PAF Receptor, and Bromodomain Binding.

    Science.gov (United States)

    Kempen, Herman J; Bellus, Daniel; Fedorov, Oleg; Nicklisch, Silke; Filippakopoulos, Panagis; Picaud, Sarah; Knapp, Stefan

    2013-01-01

    Expression and secretion of apolipoprotein A-I (apoA-I) by cultured liver cells can be markedly stimulated by triazolodiazepines (TZDs). It has been shown previously that the thieno-TZD Ro 11-1464 increases plasma levels of apoA-I and in vivomacrophage reverse cholesterol transport in mice. However, these effects were only seen at high doses, at which the compound could act on central benzodiazepine (BZD) receptors or platelet activating factor (PAF) receptors, interfering with its potential utility. In this work, we describe 2 new thieno-TZDs MDCO-3770 and MDCO-3783, both derived from Ro 11-1464. These compounds display the same high efficacy on apoA-I production, metabolic stability, and lack of cytotoxicity in cultured hepatocytes as Ro 11-1464, but they do not bind to the central BZD receptor and PAF receptor. The quinazoline RVX-208 was less efficacious in stimulating apoA-I production and displayed signs of cytotoxicity. Certain TZDs stimulating apoA-I production are now known to be inhibitors of bromodomain (BRD) extra-terminal (BET) proteins BRDT, BRD2, BRD3, and BRD4, and this inhibition was inferred as a main molecular mechanism for their effect on apoA-I expression. We show here that the thieno-TZD (+)-JQ1, a potent BET inhibitor, strongly stimulated apoA-I production in Hep-G2 cells, but that its enantiomer (-)-JQ1, which has no BET inhibitor activity, also showed considerable effect on apoA-I production. MDCO-3770 and MDCO-3783 also inhibited BRD3 and BRD4 in vitro, with potency somewhat below that of (+)-JQ1. We conclude that the effect of thieno-TZDs on apoA-I expression is not due to inhibition of the BZD or PAF receptors and is not completely explained by transcriptional repression by BET proteins. PMID:25278768

  6. Aryl hydrocarbon receptor ligands in cancer: friend and foe.

    Science.gov (United States)

    Murray, Iain A; Patterson, Andrew D; Perdew, Gary H

    2014-12-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is best known for mediating the toxicity and tumour-promoting properties of the carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin, commonly referred to as ‘dioxin’. AHR influences the major stages of tumorigenesis — initiation, promotion, progression and metastasis — and physiologically relevant AHR ligands are often formed during disease states or during heightened innate and adaptive immune responses. Interestingly, ligand specificity and affinity vary between rodents and humans. Studies of aggressive tumours and tumour cell lines show increased levels of AHR and constitutive localization of this receptor in the nucleus. This suggests that the AHR is chronically activated in tumours, thus facilitating tumour progression. This Review discusses the role of AHR in tumorigenesis and the potential for therapeutic modulation of its activity in tumours. PMID:25568920

  7. Competitive antagonism of AMPA receptors by ligands of different classes

    DEFF Research Database (Denmark)

    Hogner, Anders; Greenwood, Jeremy R; Liljefors, Tommy;

    2003-01-01

    Ionotropic glutamate receptors (iGluRs) constitute a family of ligand-gated ion channels that are essential for mediating fast synaptic transmission in the central nervous system. This study presents a high-resolution X-ray structure of the competitive antagonist (S)-2-amino-3-[5-tert-butyl-3-(ph...

  8. Modeling of ligand binding to dopamine D2 receptor

    Directory of Open Access Journals (Sweden)

    Ostopovici-Halip Liliana

    2014-01-01

    Full Text Available The dopaminic receptors have been for long time the major targets for developing new small molecules with high affinity and selectivity to treat psychiatric disorders, neurodegeneration, drug abuse, and other therapeutic areas. In the absence of a 3D structure for the human D2 dopamine (HDD2 receptor, the efforts for discovery and design of new potential drugs rely on comparative models generation, docking and pharmacophore development studies. To get a better understanding of the HDD2 receptor binding site and the ligand-receptor interactions a homology model of HDD2 receptor based on the X-ray structure of β2-adrenergic receptor has been built and used to dock a set of partial agonists of HDD2 receptor. The main characteristics of the binding mode for the HDD2 partial agonists set are given by the ligand particular folding and a complex network of contacts represented by stacking interactions, salt bridge and hydrogen bond formation. The characterization of the partial agonist binding mode at HDD2 receptor provide the needed information to generate pharmacophore models which represent essential information in the future virtual screening studies in order to identify new potential HDD2 partial agonists.

  9. Radiosynthesis and initial evaluation of [{sup 18}F]-FEPPA for PET imaging of peripheral benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Alan A. [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 (Canada)], E-mail: alan.wilson@camhpet.ca; Garcia, Armando; Parkes, Jun [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); McCormick, Patrick [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Institute of Medical Science, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada); Stephenson, Karin A. [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Houle, Sylvain; Vasdev, Neil [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 (Canada)

    2008-04-15

    Introduction: A novel [{sup 18}F]-radiolabelled phenoxyanilide, [{sup 18}F]-FEPPA, has been synthesized and evaluated, in vitro and ex vivo, as a potential positron emission tomography imaging agent for the peripheral benzodiazepine receptor (PBR). Methods: [{sup 18}F]-FEPPA and two other radiotracers for imaging PBR, namely [{sup 11}C]-PBR28 and [{sup 11}C]-PBR28-d3, were synthesised and evaluated in vitro and ex vivo as potential PBR imaging agents. Results: [{sup 18}F]-FEPPA is efficiently prepared in one step from its tosylate precursor and [{sup 18}F]-fluoride in high radiochemical yields and at high specific activity. FEPPA displayed a K{sub i} of 0.07 nM for PBR in rat mitochondrial membrane preparations and a suitable lipophilicity for brain penetration (log P of 2.99 at pH 7.4). Upon intravenous injection into rats, [{sup 18}F]-FEPPA showed moderate brain uptake [standard uptake value (SUV) of 0.6 at 5 min] and a slow washout (SUV of 0.35 after 60 min). Highest uptake of radioactivity was seen in the hypothalamus and olfactory bulb, regions previously reported to be enriched in PBR in rat brain. Analysis of plasma and brain extracts demonstrated that [{sup 18}F]-FEPPA was rapidly metabolized, but no lipophilic metabolites were observed in either preparation and only 5% radioactive metabolites were present in brain tissue extracts. Blocking studies to determine the extent of specific binding of [{sup 18}F]-FEPPA in rat brain were problematic due to large perturbations in circulating radiotracer and the lack of a reference region. Conclusions: Further evaluation of the potential of [{sup 18}F]-FEPPA will require the employment of rigorous kinetic models and/or appropriate animal models.

  10. Capacity of Diffusion-based Molecular Communication with Ligand Receptors

    CERN Document Server

    Einolghozati, Arash; Fekri, Faramarz

    2012-01-01

    A diffusion-based molecular communication system has two major components: the diffusion in the medium, and the ligand-reception. Information bits, encoded in the time variations of the concentration of molecules, are conveyed to the receiver front through the molecular diffusion in the medium. The receiver, in turn, measures the concentration of the molecules in its vicinity in order to retrieve the information. This is done via ligand-reception process. In this paper, we develop models to study the constraints imposed by the concentration sensing at the receiver side and derive the maximum rate by which a ligand-receiver can receive information. Therefore, the overall capacity of the diffusion channel with the ligand receptors can be obtained by combining the results presented in this paper with our previous work on the achievable information rate of molecular communication over the diffusion channel.

  11. Evolution of ligand specificity in vertebrate corticosteroid receptors

    Directory of Open Access Journals (Sweden)

    Deitcher David L

    2011-01-01

    Full Text Available Abstract Background Corticosteroid receptors include mineralocorticoid (MR and glucocorticoid (GR receptors. Teleost fishes have a single MR and duplicate GRs that show variable sensitivities to mineralocorticoids and glucocorticoids. How these receptors compare functionally to tetrapod MR and GR, and the evolutionary significance of maintaining two GRs, remains unclear. Results We used up to seven steroids (including aldosterone, cortisol and 11-deoxycorticosterone [DOC] to compare the ligand specificity of the ligand binding domains of corticosteroid receptors between a mammal (Mus musculus and the midshipman fish (Porichthys notatus, a teleost model for steroid regulation of neural and behavioral plasticity. Variation in mineralocorticoid sensitivity was considered in a broader phylogenetic context by examining the aldosterone sensitivity of MR and GRs from the distantly related daffodil cichlid (Neolamprologus pulcher, another teleost model for neurobehavioral plasticity. Both teleost species had a single MR and duplicate GRs. All MRs were sensitive to DOC, consistent with the hypothesis that DOC was the initial ligand of the ancestral MR. Variation in GR steroid-specificity corresponds to nine identified amino acid residue substitutions rather than phylogenetic relationships based on receptor sequences. Conclusion The mineralocorticoid sensitivity of duplicate GRs in teleosts is highly labile in the context of their evolutionary phylogeny, a property that likely led to neo-functionalization and maintenance of two GRs.

  12. Flavylium salts as in vitro precursors of potent ligands to brain GABA-A receptors

    DEFF Research Database (Denmark)

    Kueny-Stotz, Marie; Chassaing, Stefan; Brouillard, Raymond;

    2008-01-01

    The synthesis of a series of derivatized flavylium cations was undertaken and the affinity to the benzodiazepine binding site of the GABA-A receptor evaluated. The observed high affinity for some derivatives (sub-muM range) was explained by an in vitro transformation of the flavylium cations into...

  13. DMPD: Endogenous ligands of Toll-like receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15178705 Endogenous ligands of Toll-like receptors. Tsan MF, Gao B. J Leukoc Biol. ...2004 Sep;76(3):514-9. Epub 2004 Jun 3. (.png) (.svg) (.html) (.csml) Show Endogenous ligands of Toll-like re...ceptors. PubmedID 15178705 Title Endogenous ligands of Toll-like receptors. Authors Tsan MF, Gao B. Publicat

  14. Benzodiazepine receptor equilibrium constants for flumazenil and midazolam determined in humans with the single photon emission computer tomography tracer [123I]iomazenil

    DEFF Research Database (Denmark)

    Videbaek, C; Friberg, L; Holm, S;

    1993-01-01

    twice, once without receptor blockade and once with a constant degree of partial blockade of the benzodiazepine receptors by infusion of nonradioactive flumazenil (Lanexat) or midazolam (Dormicum). Single photon emission computer tomography and blood sampling were performed intermittently for 6 h after...... bolus injection of [123I]iomazenil. The tracer in plasma was determined by high-pressure liquid chromatography and also by a simple octanol extraction procedure. The free concentration of flumazenil and midazolam in plasma water averaged 52% and 3.5% of that in whole plasma. The Kd values for the entire...... cortical rim for flumazenil were 7.4, 10.0, 10.3 and 17.7 nmol/l plasma water and, for midazolam, 73, 76, 58 and 30 nmol/l plasma water. The variation exceeds random methodological error and is probably due to interindividual differences in receptor affinity. The Kd level of midazolam is considerably...

  15. Optimizing Ligand Efficiency of Selective Androgen Receptor Modulators (SARMs).

    Science.gov (United States)

    Handlon, Anthony L; Schaller, Lee T; Leesnitzer, Lisa M; Merrihew, Raymond V; Poole, Chuck; Ulrich, John C; Wilson, Joseph W; Cadilla, Rodolfo; Turnbull, Philip

    2016-01-14

    A series of selective androgen receptor modulators (SARMs) containing the 1-(trifluoromethyl)benzyl alcohol core have been optimized for androgen receptor (AR) potency and drug-like properties. We have taken advantage of the lipophilic ligand efficiency (LLE) parameter as a guide to interpret the effect of structural changes on AR activity. Over the course of optimization efforts the LLE increased over 3 log units leading to a SARM 43 with nanomolar potency, good aqueous kinetic solubility (>700 μM), and high oral bioavailability in rats (83%).

  16. Structural basis for AMPA receptor activation and ligand selectivity

    DEFF Research Database (Denmark)

    Hogner, A; Kastrup, Jette Sandholm Jensen; Jin, R;

    2002-01-01

    and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5...

  17. Modulation of Opioid Receptor Ligand Affinity and Efficacy Using Active and Inactive State Receptor Models

    OpenAIRE

    Anand, Jessica P.; Purington, Lauren C.; Pogozheva, Irina D.; Traynor, John R.; Mosberg, Henry I.

    2012-01-01

    Mu opioid receptor (MOR) agonists are widely used for the treatment of pain; however chronic use results in the development of tolerance and dependence. It has been demonstrated that co-administration of a MOR agonist with a delta opioid receptor (DOR) antagonist maintains the analgesia associated with MOR agonists, but with reduced negative side effects. Using our newly refined opioid receptor models for structure-based ligand design, we have synthesized several pentapeptides with tailored a...

  18. Region-selective effects of neuroinflammation and antioxidant treatment on peripheral benzodiazepine receptors and NMDA receptors in the rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Biegon, A.; Alvarado, M.; Budinger, T.F.; Grossman, R.; Hensley, K.; West, M.S.; Kotake, Y.; Ono, M.; Floyd, R.A.

    2001-12-10

    Following induction of acute neuroinflammation by intracisternal injection of endotoxin (lipopolysaccharide) in rats, quantitative autoradiography was used to assess the regional level of microglial activation and glutamate (NMDA) receptor binding. The possible protective action of the antioxidant phenyl-tert-butyl nitrone in this model was tested by administering the drug in the drinking water for 6 days starting 24 hours after endotoxin injection. Animals were killed 7 days post-injection and consecutive cryostat brain sections labeled with [3H]PK11195 as a marker of activated microglia and [125I]iodoMK801 as a marker of the open-channel, activated state of NMDA receptors. Lipopolysaccharide increased [3H]PK11195 binding in the brain, with the largest increases (2-3 fold) in temporal and entorhinal cortex, hippocampus, and substantia innominata. A significant (>50 percent) decrease in [125I]iodoMK801 binding was found in the same brain regions. Phenyl-tert-butyl nitrone treatment resulted in a partial inhibition ({approx}25 percent decrease) of the lipopolysaccharide-induced increase in [3H]PK11195 binding but completely reversed the lipopolysaccharide-induced decrease in [125I]iodoMK80 binding in the entorhinal cortex, hippocampus, and substantia innominata. Loss of NMDA receptor function in cortical and hippocampal regions may contribute to the cognitive deficits observed in diseases with a neuroinflammatory component, such as meningitis or Alzheimer's disease.

  19. Steroid receptors and their ligands: Effects on male gamete functions

    Energy Technology Data Exchange (ETDEWEB)

    Aquila, Saveria; De Amicis, Francesca, E-mail: francesca.deamicis@unical.it

    2014-11-01

    In recent years a new picture of human sperm biology is emerging. It is now widely recognized that sperm contain nuclear encoded mRNA, mitochondrial encoded RNA and different transcription factors including steroid receptors, while in the past sperm were considered incapable of transcription and translation. One of the main targets of steroid hormones and their receptors is reproductive function. Expression studies on Progesterone Receptor, estrogen receptor, androgen receptor and their specific ligands, demonstrate the presence of these systems in mature spermatozoa as surface but also as nuclear conventional receptors, suggesting that both systemic and local steroid hormones, through sperm receptors, may influence male reproduction. However, the relationship between the signaling events modulated by steroid hormones and sperm fertilization potential as well as the possible involvement of the specific receptors are still controversial issues. The main line of this review highlights the current research in human sperm biology examining new molecular systems of response to the hormones as well as specific regulatory pathways controlling sperm cell fate and biological functions. Most significant studies regarding the identification of steroid receptors are reported and the mechanistic insights relative to signaling pathways, together with the change in sperm metabolism energy influenced by steroid hormones are discussed.The reviewed evidences suggest important effects of Progesterone, Estrogen and Testosterone and their receptors on spermatozoa and implicate the involvement of both systemic and local steroid action in the regulation of male fertility potential. - Highlights: • One of the main targets of steroid hormones and their receptors is reproductive function. • Pg/PR co-work to stimulate enzymatic activities to sustain a capacitation process. • E2/ERs regulate sperm motility, capacitation and acrosome reaction and act as survival factors. • Androgens

  20. Feedback, receptor clustering, and receptor restriction to single cells yield large Turing spaces for ligand-receptor-based Turing models

    Science.gov (United States)

    Kurics, Tamás; Menshykau, Denis; Iber, Dagmar

    2014-08-01

    Turing mechanisms can yield a large variety of patterns from noisy, homogenous initial conditions and have been proposed as patterning mechanism for many developmental processes. However, the molecular components that give rise to Turing patterns have remained elusive, and the small size of the parameter space that permits Turing patterns to emerge makes it difficult to explain how Turing patterns could evolve. We have recently shown that Turing patterns can be obtained with a single ligand if the ligand-receptor interaction is taken into account. Here we show that the general properties of ligand-receptor systems result in very large Turing spaces. Thus, the restriction of receptors to single cells, negative feedbacks, regulatory interactions among different ligand-receptor systems, and the clustering of receptors on the cell surface all greatly enlarge the Turing space. We further show that the feedbacks that occur in the FGF10-SHH network that controls lung branching morphogenesis are sufficient to result in large Turing spaces. We conclude that the cellular restriction of receptors provides a mechanism to sufficiently increase the size of the Turing space to make the evolution of Turing patterns likely. Additional feedbacks may then have further enlarged the Turing space. Given their robustness and flexibility, we propose that receptor-ligand-based Turing mechanisms present a general mechanism for patterning in biology.

  1. Research Progress in Physical Dependence of Benzodiazepine-type Drugs and Receptor Mechanism%苯二氮(艹卓)类药物的躯体依赖及受体机制研究进展

    Institute of Scientific and Technical Information of China (English)

    王丽华

    2011-01-01

    Prolonged use of benzodiazepines can lead to physical dependence. The diverse behavioral effects of benzodiazepines may reflect the actions on different subtypes of GABAA receptors. Benzodiazepine action appears to be determined by the presence of particular ct subunits. But a complex picture is emerging with respect to abuse of benzodiazepines and the roles of different GABAA receptor subtypes. Recent researches suggest an interaction with all GABAA receptor subtypes is required for physical dependence of benzodiazepines. This article reviews physical dependence of benzodiazepine and mediating GABAA receptor subunits.%苯二氮(艹卓)类药物的长期使用会使患者产生躯体依赖.不同的苯二氮(艹卓)类药物的行为效应可能由不同的GABAA受体亚单位介导.苯二氮(艹卓)类药物主要作用于特定的α亚单位.然而,苯二氮艹 卓类药物的滥用和不同的GABAA受体亚单位所起的作用之间却是复杂的.研究表明,苯二氮(艹卓)类药物躯体依赖的发生需要所有GABAA受体亚单位的相互作用.现重点介绍国内外有关苯二氮(艹卓)类药物的躯体依赖的产生,GABAA受体亚单位介导的苯二氮(艹卓)类药物的躯体依赖等研究情况.

  2. GABA receptor imaging

    International Nuclear Information System (INIS)

    GABA is primary an inhibitory neurotransmitter that is localized in inhibitory interneurons. GABA is released from presynaptic terminals and functions by binding to GABA receptors. There are two types of GABA receptors, GABAA-receptor that allows chloride to pass through a ligand gated ion channel and GABAB-receptor that uses G-proteins for signaling. The GABAA-receptor has a GABA binding site as well as a benzodiazepine binding sites, which modulate GABAA-receptor function. Benzodiazepine GABAA receptor imaging can be accomplished by radiolabeling derivates that activates benzodiazepine binding sites. There has been much research on flumazenil (FMZ) labeled with 11C-FMZ, a benzodiazepine derivate that is a selective, reversible antagonist to GABAA receptors. Recently, 18F-fluoroflumazenil (FFMZ) has been developed to overcome 11C's short half-life. 18F-FFMZ shows high selective affinity and good pharmacodynamics, and is a promising PET agent with better central benzodiazepine receptor imaging capabilities. In an epileptic focus, because the GABA/benzodiazepine receptor amount is decreased, using '11C-FMZ PET instead of 18F-FDG, PET, restrict the foci better and may also help find lesions better than high resolution MR. GABAA receptors are widely distributed in the cerebral cortex, and can be used as an viable neuronal marker. Therefore it can be used as a neuronal cell viability marker in cerebral ischemia. Also, GABA-receptors decrease in areas where neuronal plasticity develops, therefore, GABA imaging can be used to evaluate plasticity. Besides these usages, GABA receptors are related with psychological diseases, especially depression and schizophrenia as well as cerebral palsy, a motor-related disorder, so further in-depth studies are needed for these areas

  3. GABA receptor imaging

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jong Doo [Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2007-04-15

    GABA is primary an inhibitory neurotransmitter that is localized in inhibitory interneurons. GABA is released from presynaptic terminals and functions by binding to GABA receptors. There are two types of GABA receptors, GABA{sub A}-receptor that allows chloride to pass through a ligand gated ion channel and GABA{sub B}-receptor that uses G-proteins for signaling. The GABA{sub A}-receptor has a GABA binding site as well as a benzodiazepine binding sites, which modulate GABA{sub A}-receptor function. Benzodiazepine GABAA receptor imaging can be accomplished by radiolabeling derivates that activates benzodiazepine binding sites. There has been much research on flumazenil (FMZ) labeled with {sup 11}C-FMZ, a benzodiazepine derivate that is a selective, reversible antagonist to GABAA receptors. Recently, {sup 18}F-fluoroflumazenil (FFMZ) has been developed to overcome {sup 11}C's short half-life. {sup 18}F-FFMZ shows high selective affinity and good pharmacodynamics, and is a promising PET agent with better central benzodiazepine receptor imaging capabilities. In an epileptic focus, because the GABA/benzodiazepine receptor amount is decreased, using '1{sup 1}C-FMZ PET instead of {sup 18}F-FDG, PET, restrict the foci better and may also help find lesions better than high resolution MR. GABA{sub A} receptors are widely distributed in the cerebral cortex, and can be used as an viable neuronal marker. Therefore it can be used as a neuronal cell viability marker in cerebral ischemia. Also, GABA-receptors decrease in areas where neuronal plasticity develops, therefore, GABA imaging can be used to evaluate plasticity. Besides these usages, GABA receptors are related with psychological diseases, especially depression and schizophrenia as well as cerebral palsy, a motor-related disorder, so further in-depth studies are needed for these areas.

  4. Halogenated benzamides as ligands for cerebral dopamine receptors

    International Nuclear Information System (INIS)

    In the past several years the authors' has synthesized a series of high affinity iodine-123 and fluorine-18 labeled substituted benzamide ligands for SPECT and PET visualization of the dopamine D-2 receptors in brain regions with low receptor density outside the striatum. Radioiodination and radiofluorination in high yield and high specific activity was achieved by using the tributyltin precursor and nucleophilic displacement of the saturation analysis revealed that the optimal striatum-to-cerebellum uptake ratio in the rat brain is highly correlated with the product of Kw and KD. The authors have used [125I] and [123I] epidepride to detect extra striatal dopamine D2 receptors in vitro by saturation analysis and in vivo with high resolution SPECT imaging

  5. PET and SPECT in medically non-refractory complex partial seizures. Temporal asymmetries of glucose consumption, Benzodiazepine receptor density

    Energy Technology Data Exchange (ETDEWEB)

    Matheja, P.; Kuwert, T.; Wolf, K.; Schober, O. [Muenster Univ. (Germany). Kliniken und Polikliniken fuer Nuklearmedizin; Stodieck, S.R.G.; Diehl, B.; Ringelstein, E.B. [Muenster Univ. (Germany). Klinik fuer Neurologie; Schuierer, G. [Muenster Univ. (Germany). Inst. fuer Klinische Radiologie

    1998-12-31

    Aim: In contrast to medically refractory complex partial seizures (CPS), only limited knowledge exists on cerebral perfusion and metabolism in medically non-refractory CPS. The aim of this study was to investigate the frequency of temporal asymmetries in regional cerebral glucose consumption (rCMRGlc), regional cerebral blood flow (rCBF), and regional cerebral benzodiazepine receptor density (BRD) in this group of patients. Methods: The study included 49 patients with medically non-refractory cryptogenic CPS (age: 36.0{+-}16.1 years). rCMRGlc was studied with F-18-FDG-PET (FDG), rCBF with Tc-99m-ECD-SPECT (ECD), and BRD with I-123-iomazenil-SPECT (IMZ). All studies were performed interictally and within four weeks in each patient. Duration of epilepsy ranged from 0.1 to 42 years (median 4.0 years). SPECT was performed with the triple-headed SPECT camera Multispect 3, PET with the PET camera ECAT EXACT 47. Using linear profiles, glucose consumption, as well as uptake of ECD and IMZ, were measured in four temporal regions of interest (ROIs), and asymmetry indices were calculated (ASY). The results were compared to 95% confidence intervals determined in control subjects. Results: Thirty-five of the 49 (71%) patients had at least one significantly elevated ASY; temporal rCMRGlc was asymmetrical in 41% of the patients, temporal BRD in 29%, and temporal rCBF in 24%. One patient had an asymmetry of all three variables, two of temporal rCMRGlc and BRD, three of temporal rCMRGlc and rCBF, and another four of rCBF and BRD. Fourteen patients had an isolated temporal asymmetry in rCMRGlc, seven in BRD, and four in rCBF. A discrepancy in lateralization between the three modalities was not observed. Conclusion: The majority of patients with medically non-refractory CPS have focal abnormalities of blood flow and metabolism in their temporal lobe. In this group of patients, FDG-PET demonstrates abnormalities with the highest frequency of the three modalities studied, followed by

  6. Benzodiazepine receptor and cerebral blood flow in early Alzheimer's disease. SPECT study using 123I-Iomazenil and 123I-IMP

    International Nuclear Information System (INIS)

    This study was designed to investigate benzodiazepine receptors (BZR) and cerebral blood flow (CBF) in patients with early Alzheimer's disease. Imaging of BZR and measurement of CBF were performed by SPECT using 123I-Iomazenil (IMZ) and 123I-IMP respectively, in seven patients with early Alzheimer's disease and five patients with unilateral left cerebral infarction as controls. The values for the normal cerebral hemisphere (ratio to the contralateral cerebellum) in patients with cerebral infarction were adopted as control values. In patients with Alzheimer's disease, the CBF (ratio to cerebellum) decreased significantly in the frontal cortex and the parietal cortex compared with the control values. There was no significant difference in late IMZ SPECT counts (ratio to cerebellum) and washout (the ratio of late-to-early IMZ SPECT counts) between patients with Alzheimer's disease and the controls. However, the late IMZ SPECT counts and washout decreased in one patient with moderate dementia. There was a significant correlation between the severity of dementia and the late IMZ SPECT counts in the temporal cortex and the parietal cortex. These results suggest that benzodiazepine binding sites are relatively well preserved in patients with early Alzheimer's disease, and reduction of the CBF is caused by neuronal dysfunction rather than by neuronal loss. IMZ SPECT study is useful and necessary for clarifying the pathophysiological state in Alzheimer's disease. (author)

  7. Somatostatin receptor ligands and resistance to treatment in pituitary adenomas.

    Science.gov (United States)

    Cuevas-Ramos, Daniel; Fleseriu, Maria

    2014-06-01

    Somatostatin (SST), an inhibitory polypeptide with two biologically active forms SST14 and SST28, inhibits GH, prolactin (PRL), TSH, and ACTH secretion in the anterior pituitary gland. SST also has an antiproliferative effect inducing cell cycle arrest and apoptosis. Such actions are mediated through five G-protein-coupled somatostatin receptors (SSTR): SSTR1-SSTR5. In GH-secreting adenomas, SSTR2 expression predominates, and somatostatin receptor ligands (SRLs; octreotide and lanreotide) directed to SSTR2 are presently the mainstays of medical therapy. However, about half of patients show incomplete biochemical remission, but the definition of resistance per se remains controversial. We summarize here the determinants of SRL resistance in acromegaly patients, including clinical, imaging features as well as molecular (mutations, SSTR variants, and polymorphisms), and histopathological (granulation pattern, and proteins and receptor expression) predictors. The role of SSTR5 may explain the partial responsiveness to SRLs in patients with adequate SSTR2 density in the cell membrane. In patients with ACTH-secreting pituitary adenomas, i.e. Cushing's disease (CD), SSTR5 is the most abundant receptor expressed and tumors show low SSTR2 density due to hypercortisolism-induced SSTR2 down-regulation. Clinical studies with pasireotide, a multireceptor-targeted SRL with increased SSTR5 activity, lead to approval of pasireotide for treatment of patients with CD. Other SRL delivery modes (oral octreotide), multireceptor-targeted SRL (somatoprim) or chimeric compounds targeting dopamine D2 receptors and SSTR2 (dopastatin), are briefly discussed. PMID:24647046

  8. Studies on 'carrier-free' radiohalogenation of receptor-binding 1,4-benzodiazepines with 18F, 75Br and 123I

    International Nuclear Information System (INIS)

    The aim of this study was to label suitable benzodiazepine derivatives with the short-lived radionuclides F-18, Br-75 and I-123 in order to map the receptor areas of these compounds in-vivo by emission tomography. Selective labelling of the diazepines in the 7th position was achieved starting from the nitro compound and then carrying out reduction, diazotization and dediazotization via triazene decomposition. Greatest radio-chemical yields were achieved with the bromium derivative. This compound was tested with success in clinical trials. A detailed discussion is presented on the choice of diazapines, the reaction conditions and the analytical methods used. In view of the short half-lives of the halogens, the synthesis could be completed within an hour. (PW)

  9. Identification of Putative Receptors for the Novel Adipokine CTRP3 Using Ligand-Receptor Capture Technology

    Science.gov (United States)

    Li, Ying; Ozment, Tammy; Wright, Gary L.

    2016-01-01

    C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects. Our initial work identified CTRP3’s promise as an effective treatment for Nonalcoholic fatty liver disease (NAFLD). Specifically, we demonstrated that mice fed a high fat diet failed to develop NAFLD when treated with CTRP3. The purpose of this current project is to identify putative receptors which mediate the hepatic actions of CTRP3. Methods We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line. Results Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the insulin receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells. Conclusion The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3. Relevance The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD. PMID:27727322

  10. The human peripheral benzodiazepine receptor gene: Cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia

    Energy Technology Data Exchange (ETDEWEB)

    Lin, D.; Miller, W.L. (Univ. of California, San Francisco, CA (United States)); Chang, Y.J.; Strauss, J.F. III (Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States))

    1993-12-01

    The mitochondrial benzodiazepine receptor (mBzR) appears to be a key factor in the flow of cholesterol into mitochondia to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, the authors have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5[prime] untranslated segment. The 5[prime] flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of [open quotes]housekeeping[close quotes] genes. The encoded PBR mRNA is alternately spliced into two forms: [open quotes]authentic[close quotes] PBR mRNA retains all four exons, while a short form termed PBR-S lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression of PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [[sup 3]H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease. 36 refs., 6 figs.

  11. Imaging brain inflammation with [(11)C]PK11195 by PET and induction of the peripheral-type benzodiazepine receptor after transient focal ischemia in rats.

    Science.gov (United States)

    Rojas, Santiago; Martín, Abraham; Arranz, Maria J; Pareto, Deborah; Purroy, Jesús; Verdaguer, Esther; Llop, Jordi; Gómez, Vanessa; Gispert, Joan D; Millán, Olga; Chamorro, Angel; Planas, Anna M

    2007-12-01

    [(11)C]PK11195 is used in positron emission tomography (PET) studies for imaging brain inflammation in vivo as it binds to the peripheral-type benzodiazepine receptor (PBR) expressed by reactive glia and macrophages. However, features of the cellular reaction required to induce a positive [(11)C]PK11195 signal are not well characterized. We performed [(11)C]PK11195 PET and autoradiography in rats after transient focal cerebral ischemia. We determined [(3)H]PK11195 binding and PBR expression in brain tissue and examined the lesion with several markers. [(11)C]PK11195 standard uptake value increased at day 4 and grew further at day 7 within the ischemic core. Accordingly, ex vivo [(3)H]PK11195 binding increased at day 4, and increases further at day 7. The PET signal also augmented in peripheral regions, but to a lesser extent than in the core. Binding in the region surrounding infarction was supported by [(11)C]PK11195 autoradiography at day 7 showing that the radioactive signal extended beyond the infarcted core. Enhanced binding was preceded by increases in PBR mRNA expression in the ipsilateral hemisphere, and a 18-kDa band corresponding to PBR protein was detected. Peripheral-type benzodiazepine receptor immunohistochemistry showed subsets of ameboid microglia/macrophages within the infarcted core showing a distinctive strong PBR expression from day 4. These cells were often located surrounding microhemorrhages. Reactive astrocytes forming a rim surrounding infarction at day 7 also showed some PBR immunostaining. These results show cellular heterogeneity in the level of PBR expression, supporting that PBR is not a simple marker of inflammation, and that the extent of [(11)C]PK11195 binding depends on intrinsic features of the inflammatory cells.

  12. Using [(11)C]Ro15 4513 PET to characterise GABA-benzodiazepine receptors in opiate addiction: Similarities and differences with alcoholism.

    Science.gov (United States)

    Lingford-Hughes, Anne; Myers, James; Watson, Ben; Reid, Alastair G; Kalk, Nicola; Feeney, Adrian; Hammers, Alexander; Riaño-Barros, Daniela A; McGinnity, Colm J; Taylor, Lindsay G; Rosso, Lula; Brooks, David J; Turkheimer, Federico; Nutt, David J

    2016-05-15

    The importance of the GABA-benzodiazepine receptor complex and its subtypes are increasingly recognised in addiction. Using the α1/α5 benzodiazepine receptor PET radioligand [(11)C]Ro15 4513, we previously showed reduced binding in the nucleus accumbens and hippocampus in abstinent alcohol dependence. We proposed that reduced [(11)C]Ro15 4513 binding in the nucleus accumbens was a marker of addiction whilst the reduction in hippocampus and positive relationship with memory was a consequence of chronic alcohol abuse. To examine this further we assessed [(11)C]Ro15 4513 binding in another addiction, opiate dependence, and used spectral analysis to estimate contributions of α1 and α5 subtypes to [(11)C]Ro15 4513 binding in opiate and previously acquired alcohol-dependent groups. Opiate substitute maintained opiate-dependent men (n=12) underwent an [(11)C]Ro15 4513 PET scan and compared with matched healthy controls (n=13). We found a significant reduction in [(11)C]Ro15 4513 binding in the nucleus accumbens in the opiate-dependent compared with the healthy control group. There was no relationship between [(11)C]Ro15 4513 binding in the hippocampus with memory. We found that reduced [(11)C]Ro15 4513 binding was associated with reduced α5 but not α1 subtypes in the opiate-dependent group. This was also seen in an alcohol-dependent group where an association between memory performance and [(11)C]Ro15 4513 binding was primarily driven by α5 and not α1 subtype. We suggest that reduced α5 levels in the nucleus accumbens are associated with addiction since we have now shown this in dependence to two pharmacologically different substances, alcohol and opiates.

  13. Cherry-picked ligands at histamine receptor subtypes.

    Science.gov (United States)

    Sadek, Bassem; Stark, Holger

    2016-07-01

    Histamine, a biogenic amine, is considered as a principle mediator of multiple physiological effects through binding to its H1, H2, H3, and H4 receptors (H1-H4Rs). Currently, the HRs have gained attention as important targets for the treatment of several diseases and disorders ranging from allergy to Alzheimer's disease and immune deficiency. Accordingly, medicinal chemistry studies exploring histamine-like molecules and their physicochemical properties by binding and interacting with the four HRs has led to the development of a diversity of agonists and antagonists that display selectivity for each HR subtype. An overview on H1-R4Rs and developed ligands representing some key steps in development is provided here combined with a short description of structure-activity relationships for each class. Main chemical diversities, pharmacophores, and pharmacological profiles of most innovative H1-H4R agonists and antagonists are highlighted. Therefore, this overview should support the rational choice for the optimal ligand selection based on affinity, selectivity and efficacy data in biochemical and pharmacological studies. This article is part of the Special Issue entitled 'Histamine Receptors'. PMID:26581501

  14. Biotinylated recombinant human erythropoietins: Bioactivity and utility as receptor ligand

    Energy Technology Data Exchange (ETDEWEB)

    Wojchowski, D.M.; Caslake, L. (Pennsylvania State Univ., University Park (USA))

    1989-08-15

    Recombinant human erythropoietin labeled covalently with biotin at sialic acid moieties has been prepared, and has been shown to possess high biological activity plus utility as a receptor ligand. Initially, the effects on biological activity of covalently attaching biotin to erythropoietin alternatively at carboxylate, amino, or sialic acid groups were compared. Biotinylation of erythropoietin at carboxylate groups using biotin-amidocaproyl hydrazide plus 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide led to substantial biological inactivation, although biotinylated molecules retained detectable activity when prepared at low stoichiometries. Biotinylation at amino groups using sulfosuccinimidyl 6-(biotinamido) hexanoate resulted in a high level of biological inactivation with little, if any, retention of biological activity, regardless of labeling stoichiometries. Biotinylation at sialic acid moieties using periodate and biotinamidocaproyl hydrazide proceeded efficiently (greater than 95% and 80% labeling efficiencies for human urinary and recombinant erythropoietin, respectively) and yielded stably biotinylated erythropoietin molecules possessing comparably high biological activity (ie, 45% of the activity of unmodified hormone). Utility of recombinant biotin-(sialyl)-erythropoietin (in combination with 125I-streptavidin) in the assay of cell surface receptors was demonstrated using two distinct murine erythroleukemia cell lines, Friend 745 and Rauscher Red 1. The densities and affinities of specific hormone binding sites were 116 +/- 4 sites, 3.3 +/- 0.4 nmol/L kd and 164 +/- 5 sites, 2.7 +/- 0.4 nmol/L kd, respectively. It is predicted that the present development of biotin-(sialyl)-erythropoietin as a chemically and biologically stable, bioactive ligand will assist in advancing an understanding of the regulated expression and physicochemistry of the human and murine erythropoietin receptors.

  15. Activation profiles of opioid ligands in HEK cells expressing δ opioid receptors

    Directory of Open Access Journals (Sweden)

    Clark J

    2002-11-01

    Full Text Available Abstract Background The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only δ opioid receptors. Activation profiles of most of these ligands at δ opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production. Results Naltrexone and nalorphine were classified as antagonists at δ opioid receptor. The other ligands studied were agonists at δ opioid receptors and demonstrated IC50 values of 0.1 nM to 2 μM, maximal inhibition of 39–77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol ≥ fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine ≥ met-enkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relative efficacy of several ligands at δ opioid receptors. Conclusions The data produced from this study can lead to elucidation of the complete activation profiles of several opioid ligands, leading to clarification of the mechanisms involved in physiological effects of these ligands at δ opioid receptors. Furthermore, these data can be used as a basis for novel use of existing opioid ligands based on their pharmacology at δ opioid receptors.

  16. Ligands specify estrogen receptor alpha nuclear localization and degradation

    Directory of Open Access Journals (Sweden)

    Caze-Subra Stéphanie

    2010-12-01

    Full Text Available Abstract Background The estrogen receptor alpha (ERα is found predominately in the nucleus, both in hormone stimulated and untreated cells. Intracellular distribution of the ERα changes in the presence of agonists but the impact of different antiestrogens on the fate of ERα is a matter of debate. Results A MCF-7 cell line stably expressing GFP-tagged human ERα (SK19 cell line was created to examine the localization of ligand-bound GFP-ERα. We combined digitonin-based cell fractionation analyses with fluorescence and immuno-electron microscopy to determine the intracellular distribution of ligand-bound ERα and/or GFP-ERα. Using fluorescence- and electron microscopy we demonstrate that both endogenous ERα and GFP-ERα form numerous nuclear focal accumulations upon addition of agonist, 17β-estradiol (E2, and pure antagonists (selective estrogen regulator disruptor; SERD, ICI 182,780 or RU58,668, while in the presence of partial antagonists (selective estrogen regulator modulator; SERM, 4-hydroxytamoxifen (OHT or RU39,411, diffuse nuclear staining persisted. Digitonin based cell fractionation analyses confirmed that endogenous ERα and GFP-ERα predominantly reside in the nuclear fraction. Overall ERα protein levels were reduced after estradiol treatment. In the presence of SERMs ERα was stabilized in the nuclear soluble fraction, while in the presence of SERDs protein levels decreased drastically and the remaining ERα was largely found in a nuclear insoluble fraction. mRNA levels of ESR1 were reduced compared to untreated cells in the presence of all ligands tested, including E2. E2 and SERDs induced ERα degradation occurred in distinct nuclear foci composed of ERα and the proteasome providing a simple explanation for ERα sequestration in the nucleus. Conclusions Our results indicate that chemical structure of ligands directly affect the nuclear fate and protein turnover of the estrogen receptor alpha independently of their impact on

  17. Origin and evolution of the ligand-binding ability of nuclear receptors.

    Science.gov (United States)

    Markov, Gabriel V; Laudet, Vincent

    2011-03-01

    The origin of the ligand-binding ability of nuclear receptors is still a matter of discussion. Current opposing models are the early evolution of an ancestral receptor that would bind a specific ligand with high affinity and the early evolution of an ancestral orphan that was a constitutive transcription factor. Here we review the arguments in favour or against these two hypotheses, and we discuss an alternative possibility that the ancestor was a ligand sensor, which would be able to explain the apparently contradictory data generated in previous models for the evolution of ligand binding in nuclear receptors. PMID:21055443

  18. Identification of VDR Antagonists among Nuclear Receptor Ligands Using Virtual Screening

    Directory of Open Access Journals (Sweden)

    Kelly Teske

    2014-04-01

    Full Text Available Herein, we described the development of two virtual screens to identify new vitamin D receptor (VDR antagonists among nuclear receptor (NR ligands. Therefore, a database of 14330 nuclear receptor ligands and their NR affinities was assembled using the online available “Binding Database.” Two different virtual screens were carried out in conjunction with a reported VDR crystal structure applying a stringent and less stringent pharmacophore model to filter docked NR ligand conformations. The pharmacophore models were based on the spatial orientation of the hydroxyl functionalities of VDR's natural ligands 1,25(OH2D3 and 25(OH2D3. The first virtual screen identified 32 NR ligands with a calculated free energy of VDR binding of more than -6.0 kJ/mol. All but nordihydroguaiaretic acid (NDGA are VDR ligands, which inhibited the interaction between VDR and coactivator peptide SRC2-3 with an IC50 value of 15.8 μM. The second screen identified 162 NR ligands with a calculated free energy of VDR binding of more than -6.0 kJ/mol. More than half of these ligands were developed to bind VDR followed by ERα/β ligands (26%, TRα/β ligands (7%, and LxRα/β ligands (7%. The binding between VDR and ERα ligand H6036 as well as TRα/β ligand triiodothyronine and a homoserine analog thereof was confirmed by fluorescence polarization.

  19. Toll-Like Receptors, Their Ligands, and Atherosclerosis

    Directory of Open Access Journals (Sweden)

    Conrad P. Hodgkinson

    2011-01-01

    Full Text Available Atherosclerosis is a disease characterized by inflammation in the arterial wall. Atherogenesis is dependent on the innate immune response involving activation of Toll-like receptors (TLRs and the expression of inflammatory proteins. TLRs, which recognize various pathogen-associated molecular patterns, are expressed in various cell types within the atherosclerotic plaque. Microbial agents are associated with an increased risk of atherosclerosis and this is, in part, due to activation of TLRs. Recently considerable evidence has been provided suggesting that endogenous proteins promote atherosclerosis by binding to TLRs. In this review, we describe the role of TLRs in atherosclerosis with particular emphasis on those atherogenic endogenous proteins that have been implicated as TLR ligands.

  20. Structural and functional characterization of a novel type of ligand-independent RXR-USP receptor

    OpenAIRE

    Iwema, Thomas; Billas, Isabelle ML; Beck, Yannick; Bonneton, François; Nierengarten, Hélène; Chaumot, Arnaud; Richards, Geoff; Laudet, Vincent; Moras, Dino

    2007-01-01

    Retinoid X receptor (RXR) and Ultraspiracle (USP) play a central role as ubiquitous heterodimerization partners of many nuclear receptors. While it has long been accepted that a wide range of ligands can activate vertebrate/mollusc RXRs, the existence and necessity of specific endogenous ligands activating RXR-USP in vivo is still matter of intense debate. Here we report the existence of a novel type of RXR-USP with a ligand-independent functional conformation. Our studies involved Tribolium ...

  1. Bioluminescent Ligand-Receptor Binding Assays for Protein or Peptide Hormones.

    Science.gov (United States)

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    Bioluminescence has been widely used in biomedical research due to its high sensitivity, low background, and broad linear range. In recent studies, we applied bioluminescence to ligand-receptor binding assays for some protein or peptide hormones based on a newly developed small monomeric Nanoluciferase (NanoLuc) reporter that has the so far brightest bioluminescence. The conventional ligand-receptor binding assays rely on radioligands that have drawbacks, such as radioactive hazards and short shelf lives. In contrast, the novel bioluminescent binding assays use the NanoLuc-based protein or peptide tracers that are safe, stable, and ultrasensitive. Thus, the novel bioluminescent ligand-receptor binding assay would be applied to more and more protein or peptide hormones for ligand-receptor interaction studies in future. In the present article, we provided detailed protocols for setting up the novel bioluminescent ligand-receptor binding assays using two representative protein hormones as examples. PMID:27424896

  2. Development of radioiodinated receptor ligands for cerebral single photon emission tomography

    International Nuclear Information System (INIS)

    In the last decade the use of radiolabeled ligands for the imaging of cerebral receptors by emission computed tomography (ECT) has seen rapid growth. The opportunity to routinely perform cerebral single photon emission tomography (SPET) with iodine-123-labeled ligands depends on the availability of receptor ligands into which iodine can be introduced without decreasing the required high target receptor specificity. The use of iodine-123-labeled receptor-specific ligands also depends on the availability of high purity iodine-123 at reasonable costs and the necessary imaging instrumentation. In this paper, the development and current stage of evaluation of various iodine-123-labeled ligands for SPET imaging of dopaminergic, serotonergic and muscarinic acetylcholinergic receptor classes are discussed

  3. Effects of benzodiazepines and non-benzodiazepine compounds on the GABA-induced response in frog isolated sensory neurones.

    OpenAIRE

    Yakushiji, T; Fukuda, T.; Oyama, Y.; Akaike, N.

    1989-01-01

    1. The effects of benzodiazepines and non-benzodiazepine compounds on the gamma-aminobutyric acid (GABA)-induced chloride current (ICl) were studied in frog isolated sensory neurones by use of a concentration-jump (termed 'concentration-clamp') technique, under single-electrode voltage-clamp conditions. The drugs used were classified into four categories as follows: full benzodiazepine receptor agonists (diazepam, clonazepam, nitrazepam, midazolam, clotiazepam and etizolam), partial agonists ...

  4. Computational approaches to modeling receptor flexibility upon ligand binding: Application to interfacially activated enzymes

    DEFF Research Database (Denmark)

    Wade, R.C.; Sobolev, V.; Ortiz, A.R. .;

    1998-01-01

    Receptors generally undergo conformational change upon ligand binding. We describe how fairly simple techniques may be used in docking and design studies to account for some of the changes in the conformations of proteins on ligand binding. Simulations of protein-ligand interactions that give...... a more complete description of the dynamics important for ligand binding are then discussed. These methods are illustrated for phospholipase A(2) and lipase, enzymes that both undergo interfacial activation....

  5. SELECTIVITY AND SPECIFICITY OF SPHINGOSINE 1-PHOSPHATE RECEPTOR LIGANDS: ‘OFF-TARGETS’ OR COMPLEX PHARMACOLOGY?

    Directory of Open Access Journals (Sweden)

    Nigel John Pyne

    2011-05-01

    Full Text Available A recent perspective published in frontiers of Pharmacology by Salomone and Waeber (2011 discussed the selectivity and specificity of sphingosine 1-phosphate (S1P receptor ligands. This perspective surveyed the use of various S1P receptor ligands and attempted to reconcile a number of inconsistencies in the predicted biological outcomes: these were interpreted as ‘off-target’ effects. Therefore the perspective cautioned against the use of these S1P receptor ligands. Here we highlight the complex pharmacology of S1P receptors, which along with ‘inside-out’ signalling might provide an alternative explanation for ‘off-target’ effects.

  6. The Anticonvulsant Activity of a Flavonoid-Rich Extract from Orange Juice Involves both NMDA and GABA-Benzodiazepine Receptor Complexes

    Directory of Open Access Journals (Sweden)

    Rita Citraro

    2016-09-01

    Full Text Available The usage of dietary supplements and other natural products to treat neurological diseases has been growing over time, and accumulating evidence suggests that flavonoids possess anticonvulsant properties. The aim of this study was to examine the effects of a flavonoid-rich extract from orange juice (OJe in some rodent models of epilepsy and to explore its possible mechanism of action. The genetically audiogenic seizures (AGS-susceptible DBA/2 mouse, the pentylenetetrazole (PTZ-induced seizures in ICR-CD1 mice and the WAG/Rij rat as a genetic model of absence epilepsy with comorbidity of depression were used. Our results demonstrate that OJe was able to exert anticonvulsant effects on AGS-sensible DBA/2 mice and to inhibit PTZ-induced tonic seizures, increasing their latency. Conversely, it did not have anti-absence effects on WAG/Rij rats. Our experimental findings suggest that the anti-convulsant effects of OJe are likely mediated by both an inhibition of NMDA receptors at the glycine-binding site and an agonistic activity on benzodiazepine-binding site at GABAA receptors. This study provides evidences for the antiepileptic activity of OJe, and its results could be used as scientific basis for further researches aimed to develop novel complementary therapy for the treatment of epilepsy in a context of a multitarget pharmacological strategy.

  7. Progesterone in pregnancy; receptor-ligand interaction and signaling pathways.

    Science.gov (United States)

    Szekeres-Bartho, Julia; Halasz, Melinda; Palkovics, Tamas

    2009-12-01

    Progesterone is indispensable in creating a suitable endometrial environment for implantation, and also for the maintenance of pregnancy. Successful pregnancy depends on an appropriate maternal immune response to the fetus. Along with its endocrine effects, progesterone also acts as an "immunosteroid", by contributing to the establishment of a pregnancy protective immune milieu. Progesterone plays a role in uterine homing of NK cells and upregulates HLA-G gene expression, the ligand for NK inhibitory and activating receptors. At high concentrations, progesterone is a potent inducer of Th2-type cytokines as well as of LIF and M-CSF production by T cells. A protein called progesterone-induced blocking factor (PIBF), by inducing a Th2-dominant cytokine production mediates the immunological effects of progesterone. PIBF binds to a novel type of the IL-4 receptor and signals via the Jak/STAT pathway, to induce a number of genes, that not only affect the immune response, but might also play a role in trophoblast invasiveness. PMID:19880194

  8. Pharmacological profiles of the metabotropic glutamate receptor ligands.

    Science.gov (United States)

    Naples, M A; Hampson, D R

    2001-01-01

    Metabotropic glutamate receptors (mGluRs) are a family of G-protein coupled receptors that are expressed in the central and peripheral nervous systems. The purpose of this study was to compare the ligand binding selectivity profiles of the mGluR agonist [(3)H]L-AP4 and the novel radiolabeled phenylglycine antagonist [(3)H]CPPG at all eight rat mGluR subtypes expressed in transfected human embryonic kidney cells. At a concentration of 30 nM [(3)H]L-AP4, no specific binding was detected in membranes expressing the group I receptors mGluR1a or mGluR5a, or in membranes expressing the group II mGluRs, mGluR2 and mGluR3. Among the group III mGluRs, specific [(3)H]L-AP4 binding was detected in cells expressing mGluR4a and mGluR8a but not in cells expressing mGluR6 or mGluR7a. The binding of [(3)H]CPPG showed an exceptional pattern of selectivity amongst the mGluR subtypes; at a concentration of 20 nM [(3)H]CPPG, a high level of specific binding was seen in membranes containing mGluR8a but not in any of the other mGluR subtypes. The affinity constant (K(D)) calculated for [(3)H]CPPG binding to mGluR8a was 183 nM. In competition experiments, the phosphono-substituted phenylglycine congeners including MPPG, (RS)-PPG, and unlabeled CPPG were the most potent inhibitors of [(3)H]CPPG binding while non-phosphonated compounds such as L-glutamate and MCPG were substantially less potent. These results demonstrate that [(3)H]L-AP4 and [(3)H]CPPG can be used as probes to selectively label group III mGluRs and that CPPG and related phenylglycine derivatives are useful for studying differences in the ligand recognition sites of highly homologous mGluRs. PMID:11114395

  9. 5-HT1A and benzodiazepine receptors in the basolateral amygdala modulate anxiety in the social interaction test, but not in the elevated plus-maze.

    Science.gov (United States)

    Gonzalez, L E; Andrews, N; File, S E

    1996-09-01

    In order to investigate the role of the 5-HT1A receptors of the amygdala in modulating anxiety, rats were implanted with bilateral cannulae aimed at the basolateral nucleus of the amygdala complex and infused with either artificial cerebrospinal fluid (aCSF) or the selective 5-HT1A receptor agonist 8-OH-DPAT (50-200 ng) and tested in two animal models of anxiety. In the elevated plus-maze test, no significant effects were detected in this dose range. In contrast, 8-OH-DPAT caused an overall reduction in levels of social investigation, thus indicating anxiogenic actions in the social interaction test. At 50 ng, 8-OH-DPAT had a selective action on anxiety, while at 200 ng there was a concomitant reduction in locomotor activity and, in some animals, signs of the 5-HT1A syndrome. Evidence that the anxiogenic effect of 8-OH-DPAT (50 ng) was due to activation of 5-HT1A receptors came from the finding that (-)-tertatolol, a 5-HT1A receptor antagonist, reversed this effect at a dose (1.5 micrograms) which was silent when given alone. The benzodiazepine receptor agonist, midazolam (1 and 2 micrograms) was bilaterally administered into the basolateral nucleus of the amygdala and evoked clear-cut anxiolytic effects in the social interaction test. These data indicate that the agonist activation of post-synaptic 5-HT1A receptors in the basolateral nucleus of the amygdala may produce anxiogenic effects, while agonist activation of BDZ receptors in the same areas evokes anxiolytic effects. Our results from the social interaction test are similar to those previously reported from tests of anxiety using punished paradigms, but contrast with those found in the elevated plus-maze. Thus, it is concluded that either the two tests have different sensitivities to midazolam and 8-OH-DPAT or more intriguingly, the tests are evoking fundamentally different states of anxiety, with that evoked by the plus-maze being mediated via brain areas or receptors different from those studied here.

  10. Cell surface receptors for signal transduction and ligand transport: a design principles study.

    Directory of Open Access Journals (Sweden)

    Harish Shankaran

    2007-06-01

    Full Text Available Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor-ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation.

  11. Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

    Directory of Open Access Journals (Sweden)

    Graham M West

    Full Text Available Activation of the glucagon-like peptide-1 receptor (GLP-1R in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM. Like other class B G protein-coupled receptors (GPCRs, the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

  12. Elimination of a ligand gating site generates a supersensitive olfactory receptor

    OpenAIRE

    Kanika Sharma; Gaurav Ahuja; Ashiq Hussain; Sabine Balfanz; Arnd Baumann; Korsching, Sigrun I.

    2016-01-01

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unex...

  13. Rescue of ligand binding of a mutant IGF-I receptor by complementation

    DEFF Research Database (Denmark)

    Chakravarty, Arjun Anders; Hinrichsen, Jane; Whittaker, Linda;

    2005-01-01

    from a wild-type receptor monomer and a mutant receptor monomer devoid of binding activity. Receptor hybrids were generated by transient co-transfection of cDNAs encoding wild-type and mutant receptors with unique epitope tags. Hybrid receptors were purified from transfected cells by sequential immuno......-affinity chromatography and their ligand-binding properties were determined. Complementation produced a hybrid with near wild-type affinity. Dissociation studies demonstrated that the hybrid did not exhibit negative cooperativity....

  14. CLE Peptides in Plants: Proteolytic Processing,Structure-Activity Relationship, and Ligand-Receptor Interaction

    Institute of Scientific and Technical Information of China (English)

    Xiaoming Gao; Yongfeng Guo

    2012-01-01

    Ligand-receptor signaling initiated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) family peptides is critical in regulating cell division and differentiation in meristematic tissues in plants.Biologically active CLE peptides are released from precursor proteins via proteolytic processing.The mature form of CLE ligands consists of 12-13 amino acids with several post-translational modifications.This review summarizes recent progress toward understanding the proteolytic activities that cleave precursor proteins to release CLE peptides,the molecular structure and function of mature CLE ligands,and interactions between CLE ligands and corresponding leucine-rich repeat (LRR) receptor-like kinases (RLKs).

  15. Triton X-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABA(A) receptors in Xenopus oocytes

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Ebert, Bjarke; Klaerke, Dan;

    2009-01-01

    effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA(A) receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA(A) alpha(1)beta(3)gamma(2S) receptor currents in a...

  16. The orthosteric GABAA receptor ligand Thio-4-PIOL displays distinctly different functional properties at synaptic and extrasynaptic receptors

    DEFF Research Database (Denmark)

    Hoestgaard-Jensen, K; O'Connor, R M; Dalby, Nils Ole;

    2013-01-01

    Explorations into the heterogeneous population of native GABA type A receptors (GABAA Rs) and the physiological functions governed by the multiple GABAA R subtypes have for decades been hampered by the lack of subtype-selective ligands....

  17. Cell surface receptors for signal transduction and ligand transport - a design principles study

    Energy Technology Data Exchange (ETDEWEB)

    Shankaran, Harish; Resat, Haluk; Wiley, H. S.

    2007-06-01

    Although many different receptors undergo endocytosis, the system-level design principles that govern the evolution of receptor dynamics are far from fully understood. We have constructed a generalized mathematical model to understand how receptor internalization dynamics encodes receptor function and regulation. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptors can be categorized a being: i) avidity-controlled where the response control depends primarily on the extracelluar ligand capture efficiency, ii) consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii) dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled and epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to anhance the accuracy of signaling receptors rather than serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulations.

  18. Complex pharmacology of novel allosteric free fatty acid 3 receptor ligands

    DEFF Research Database (Denmark)

    Hudson, Brian D; Christiansen, Elisabeth; Murdoch, Hannah;

    2014-01-01

    Analysis of the roles of the short chain fatty acid receptor, free fatty acid 3 receptor (FFA3), has been severely limited by the low potency of its endogenous ligands, the crossover of function of these on the closely related free fatty acid 2 receptor, and a dearth of FFA3-selective synthetic l...

  19. 1,2,3-triazolyl amino acids as AMPA receptor ligands

    DEFF Research Database (Denmark)

    Stanley, Nathan J.; Pedersen, Daniel Sejer; Nielsen, Birgitte;

    2010-01-01

    The central nervous system glutamate receptors are an important target for drug discovery. Herein we report initial investigations into the synthesis and glutamate receptor activity of 1,2,3-triazolyl amino acids. Two compounds were found to be selective AMPA receptor ligands, which warrant further...

  20. Ligand Promiscuity of Aryl Hydrocarbon Receptor Agonists and Antagonists Revealed by Site-Directed Mutagenesis

    OpenAIRE

    Soshilov, Anatoly A; DENISON, Michael S.

    2014-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse chemicals. To examine the mechanisms responsible for the promiscuity in AhR ligand binding, we determined the effects of mutations within the AhR ligand-binding domain (LBD) on the activity of diverse AhR ligands. Site-directed mutagenesis identified Ile319 of the mouse AhR and, to a lesser extent, Phe318 as residues involved in ligand-selective modulation of AhR transf...

  1. Rapid and efficient radiosynthesis of [{sup 123}I]I-PK11195, a single photon emission computed tomography tracer for peripheral benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Pimlott, Sally L. [Department of Clinical Physics, West of Scotland Radionuclide Dispensary, Western Infirmary, G11 6NT Glasgow (United Kingdom)], E-mail: s.pimlott@clinmed.gla.ac.uk; Stevenson, Louise [Department of Chemistry, WestCHEM, University of Glasgow, G12 8QQ Glasgow (United Kingdom); Wyper, David J. [Institute of Neurological Sciences, Southern General Hospital, G51 4TF Glasgow (United Kingdom); Sutherland, Andrew [Department of Chemistry, WestCHEM, University of Glasgow, G12 8QQ Glasgow (United Kingdom)

    2008-07-15

    Introduction: [{sup 123}I]I-PK11195 is a high-affinity single photon emission computed tomography radiotracer for peripheral benzodiazepine receptors that has previously been used to measure activated microglia and to assess neuroinflammation in the living human brain. This study investigates the radiosynthesis of [{sup 123}I]I-PK11195 in order to develop a rapid and efficient method that obtains [{sup 123}I]I-PK11195 with a high specific activity for in vivo animal and human imaging studies. Methods: The synthesis of [{sup 123}I]I-PK11195 was evaluated using a solid-state interhalogen exchange method and an electrophilic iododestannylation method, where bromine and trimethylstannyl derivatives were used as precursors, respectively. In the electrophilic iododestannylation method, the oxidants peracetic acid and chloramine-T were both investigated. Results: Electrophilic iododestannylation produced [{sup 123}I]I-PK11195 with a higher isolated radiochemical yield and a higher specific activity than achievable using the halogen exchange method investigated. Using chloramine-T as oxidant provided a rapid and efficient method of choice for the synthesis of [{sup 123}I]I-PK11195. Conclusions: [{sup 123}I]I-PK11195 has been successfully synthesized via a rapid and efficient electrophilic iododestannylation method, producing [{sup 123}I]I-PK11195 with a higher isolated radiochemical yield and a higher specific activity than previously achieved.

  2. Different sensitivities to competitive inhibition of benzodiazepine receptor binding of {sup 11}C-iomazenil and {sup 11}C-flumazenil in rhesus monkey brain

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Osamu; Hosoi, Rie; Kobayashi, Kaoru [Osaka Univ., Suita (Japan). Medical School; Itoh, Takashi; Gee, A.; Suzuki, Kazutoshi

    2001-04-01

    The in vivo binding kinetics of {sup 11}C-iomazenil were compared with those of {sup 11}C-flumazenil binding in rhesus monkey brain. The monkey was anesthetized with ketamine and intravenously injected with either {sup 11}C-iomazenil or {sup 11}C-flumazenil in combination with the coadministration of different doses of non-radioactive flumazenil (0, 5 and 20 {mu}g/kg). The regional distribution of {sup 11}C-iomazenil in the brain was similar to that of {sup 11}C-flumazenil, but the sensitivity of {sup 11}C-iomazenil binding to competitive inhibition by non-radioactive flumazenil was much less than that of {sup 11}C-flumazenil binding. A significant reduction in {sup 11}C-flumazenil binding in the cerebral cortex was observed with 20 {mu}g/kg of flumazenil, whereas a relatively smaller inhibition of {sup 11}C-iomazenil binding in the same region was observed with the same dose of flumazenil. These results suggest that {sup 11}C-flumazenil may be a superior radiotracer for estimating benzodiazepine receptor occupancy in the intact brain. (author)

  3. Multiparameter flow cytometry of a pH sensitive ligand bound to receptors and inside cells

    Energy Technology Data Exchange (ETDEWEB)

    Fay, S.P.; Habbersett, R.; Posner, R.G.; Domalewski, M.D.; Freer, R.J.; Pierson, E.; Whittaker, J.; Haugland, R.P.; Sklar, L.A. (Univ. of New Mexico, Albuquerque (United States) Los Alamos National Lab., NM (United States))

    1993-01-01

    Because fluoresceinated ligands of the neutrophil formyl peptide receptor can be protonated either upon binding to the receptor on the cell surface or in acidified intracellular compartments, the authors synthesized a ligand conjugated to the pH sensitive fluorescent probe SNAFL (CHO-Met-Leu-Phe-Phe-Lys-SNAFL). In the three laser flow cytometer at LANL, protonated dye is excited at 488 nm and emits at 530 nm; unprotonated dye is excited at 568 nm and emits at 650 nm. Detection at the isobestic and isoemissive points at 528 and 600 nm is used to keep track of variations in ligand concentration from sample to sample. The SNAFL-ligand bound to HL-60 cells (which overexpress the formyl peptide receptor) was compared to the free ligand in solution over a pH range from 6.5 to 9.0. The results suggest that the ligand bound to cell surface receptors was protonated in the binding pocket, possibly by virtue of its proximity to His 90, based on sequence data. When the cells were raised from 4[degrees] to 37[degrees], they also observed a time-dependent acidification of the ligand, indicative of ligand-receptor processing beginning 3-4 minutes after internalization.

  4. Monitoring ligand-receptor interactions by photonic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jeney, Sylvia [M E Mueller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, 4056 (Switzerland); Mor, Flavio; Forro, Laszlo [Laboratory of Complex Matter Physics (LPMC), Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland); Koszali, Roland [Institute for Information and Communication Technologies (IICT), University of Applied Sciences of Western Switzerland (HEIG-VD), Rue Galilee 15, CH 1401 Yverdon-les-bains (Switzerland); Moy, Vincent T, E-mail: sylvia.jeney@unibas.ch, E-mail: vmoy@miami.edu [Department of Physiology and Biophysics, University of Miami Miller School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136 (United States)

    2010-06-25

    We introduce a method for the acquisition of single molecule force measurements of ligand-receptor interactions using the photonic force microscope (PFM). Biotin-functionalized beads, manipulated with an optical trap, and a streptavidin-functionalized coverslip were used to measure the effect of different pulling forces on the lifetime of individual streptavidin-biotin complexes. By optimizing the design of the optical trap and selection of the appropriate bead size, pulling forces in excess of 50 pN were achieved. Based on the amplitude of three-dimensional (3D) thermal position fluctuations of the attached bead, we were able to select for a bead-coverslip interaction that was mediated by a single streptavidin-biotin complex. Moreover, the developed experimental system was greatly accelerated by automation of data acquisition and analysis. In force-dependent kinetic measurements carried out between streptavidin and biotin, we observed that the streptavidin-biotin complex exhibited properties of a catch bond, with the lifetime increasing tenfold when the pulling force increased from 10 to 20 pN. We also show that silica beads were more appropriate than polystyrene beads for the force measurements, as tethers, longer than 200 nm, could be extracted from polystyrene beads.

  5. Mutual inactivation of Notch receptors and ligands facilitates developmental patterning.

    Directory of Open Access Journals (Sweden)

    David Sprinzak

    2011-06-01

    Full Text Available Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression perturbations. For lateral inhibition, we find that mutual inactivation speeds up patterning dynamics, relieves the need for cooperative regulatory interactions, and expands the range of parameter values that permit pattern formation, compared to canonical models. Furthermore, mutual inactivation enables a simple lateral inhibition circuit architecture which requires only a single downstream regulatory step. Both model systems show how mutual inactivation can facilitate robust fine-grained patterning processes that would be difficult to implement without it, by encoding a difference-promoting feedback within the signaling system itself. Together, these results provide a framework for analysis of more complex Notch-dependent developmental systems.

  6. [Analysis of the binding capacity of the benzodiazepine site of gabaa receptor in mice C57BL/6 and BALB/C pretreated with anxiolytics].

    Science.gov (United States)

    Iarkova, M A

    2011-01-01

    The level of specific 3H-flunitrazepam binding in synaptosomal membranes of C57BL/6 and BALB/c mice brain underwent to the stress of different types has been studied. Mild stress (Elevated Plus Maze) was shown to induce the decrease of benzodiazepine binding in BALB/c mice only, while the strong one (Exposure to a predator) was revealed to cause this decrease in both strains. Behavioral effects of different non-benzodiazepine drugs possessing anxiolytic properties (Afobazol, Ladasten and Noopept) was accompanied with the normalization of the level of benzodiazepine reception, reduced by the stress of both modalities. PMID:22232906

  7. Effect of size and conformation of the ligand on asialoglycoprotein receptor-mediated ligand internalization and degradation in rat hepatocytes

    International Nuclear Information System (INIS)

    The rates of internalization and degradation of 125-I-labeled desialylated cyanogen bromide fragment I of orosomucoid (AS-CNBr-I) and its reduced and carboxymethylated derivative (AS-RC-CNBr-I) were compared with those of 125I-labeled asialoorosomucoid (ASOR) in rat hepatocytes. At 30 nM the rates of internalization and degradation of 125I-AS-CNBr-I were greater than those of 125I-ASOR. 125I-AS-RC-CNBr-I also had a lower rate of internalization and degradation. In contrast to 125I-ASOR, when degradation was inhibited by 5 μM colchicine there was a significant intracellular accumulation of the smaller ligands. At 40C the hepatocytes were found to bind the fragmented ligands more than 125I-ASOR. Incubation of the cells with bound ligand at 370 indicated that diacytosis of 125I-ASOR was greater than the smaller ligands. Colchincine markedly enhanced diacytosis of 125I-ASOR. On the other hand, there were marked accumulation of the smaller ligands by colchicine. These results suggest that the rates of internalization, degradation and diacytosis of the ligand are affected by the size and conformation of the ligand through different rates of receptor binding and intracellular transport

  8. Challenges predicting ligand-receptor interactions of promiscuous proteins: the nuclear receptor PXR.

    Directory of Open Access Journals (Sweden)

    Sean Ekins

    2009-12-01

    Full Text Available Transcriptional regulation of some genes involved in xenobiotic detoxification and apoptosis is performed via the human pregnane X receptor (PXR which in turn is activated by structurally diverse agonists including steroid hormones. Activation of PXR has the potential to initiate adverse effects, altering drug pharmacokinetics or perturbing physiological processes. Reliable computational prediction of PXR agonists would be valuable for pharmaceutical and toxicological research. There has been limited success with structure-based modeling approaches to predict human PXR activators. Slightly better success has been achieved with ligand-based modeling methods including quantitative structure-activity relationship (QSAR analysis, pharmacophore modeling and machine learning. In this study, we present a comprehensive analysis focused on prediction of 115 steroids for ligand binding activity towards human PXR. Six crystal structures were used as templates for docking and ligand-based modeling approaches (two-, three-, four- and five-dimensional analyses. The best success at external prediction was achieved with 5D-QSAR. Bayesian models with FCFP_6 descriptors were validated after leaving a large percentage of the dataset out and using an external test set. Docking of ligands to the PXR structure co-crystallized with hyperforin had the best statistics for this method. Sulfated steroids (which are activators were consistently predicted as non-activators while, poorly predicted steroids were docked in a reverse mode compared to 5alpha-androstan-3beta-ol. Modeling of human PXR represents a complex challenge by virtue of the large, flexible ligand-binding cavity. This study emphasizes this aspect, illustrating modest success using the largest quantitative data set to date and multiple modeling approaches.

  9. The sigma receptor as a ligand-regulated auxiliary potassium channel subunit.

    Science.gov (United States)

    Aydar, Ebru; Palmer, Christopher P; Klyachko, Vitaly A; Jackson, Meyer B

    2002-04-25

    The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.

  10. Potential applications for sigma receptor ligands in cancer diagnosis and therapy.

    Science.gov (United States)

    van Waarde, Aren; Rybczynska, Anna A; Ramakrishnan, Nisha K; Ishiwata, Kiichi; Elsinga, Philip H; Dierckx, Rudi A J O

    2015-10-01

    Sigma receptors (sigma-1 and sigma-2) represent two independent classes of proteins. Their endogenous ligands may include the hallucinogen N,N-dimethyltryptamine (DMT) and sphingolipid-derived amines which interact with sigma-1 receptors, besides steroid hormones (e.g., progesterone) which bind to both sigma receptor subpopulations. The sigma-1 receptor is a ligand-regulated molecular chaperone with various ion channels and G-protein-coupled membrane receptors as clients. The sigma-2 receptor was identified as the progesterone receptor membrane component 1 (PGRMC1). Although sigma receptors are over-expressed in tumors and up-regulated in rapidly dividing normal tissue, their ligands induce significant cell death only in tumor tissue. Sigma ligands may therefore be used to selectively eradicate tumors. Multiple mechanisms appear to underlie cell killing after administration of sigma ligands, and the signaling pathways are dependent both on the type of ligand and the type of tumor cell. Recent evidence suggests that the sigma-2 receptor is a potential tumor and serum biomarker for human lung cancer and an important target for inhibiting tumor invasion and cancer progression. Current radiochemical efforts are focused on the development of subtype-selective radioligands for positron emission tomography (PET) imaging. Right now, the mostpromising tracers are [18F]fluspidine and [18F]FTC-146 for sigma-1 receptors and [11C]RHM-1 and [18F]ISO-1 for the sigma-2 subtype. Nanoparticles coupled to sigma ligands have shown considerable potential for targeted delivery of antitumor drugs in animal models of cancer, but clinical studies exploring this strategy in cancer patients have not yet been reported. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers. PMID:25173780

  11. Imaging G protein-coupled receptors while quantifying their ligand-binding free-energy landscape.

    Science.gov (United States)

    Alsteens, David; Pfreundschuh, Moritz; Zhang, Cheng; Spoerri, Patrizia M; Coughlin, Shaun R; Kobilka, Brian K; Müller, Daniel J

    2015-09-01

    Imaging native membrane receptors and testing how they interact with ligands is of fundamental interest in the life sciences but has proven remarkably difficult to accomplish. Here, we introduce an approach that uses force-distance curve-based atomic force microscopy to simultaneously image single native G protein-coupled receptors in membranes and quantify their dynamic binding strength to native and synthetic ligands. We measured kinetic and thermodynamic parameters for individual protease-activated receptor-1 (PAR1) molecules in the absence and presence of antagonists, and these measurements enabled us to describe PAR1's ligand-binding free-energy landscape with high accuracy. Our nanoscopic method opens an avenue to directly image and characterize ligand binding of native membrane receptors. PMID:26167642

  12. Ligands, cell-based models, and readouts required for Toll-like receptor action.

    LENUS (Irish Health Repository)

    Dellacasagrande, Jerome

    2012-02-01

    This chapter details the tools that are available to study Toll-like receptor (TLR) biology in vitro. This includes ligands, host cells, and readouts. The use of modified TLRs to circumvent some technical problems is also discussed.

  13. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    Science.gov (United States)

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  14. Computer-aided design of a novel ligand for retinoic acid receptor in cancer chemotherapy

    Science.gov (United States)

    Silva, Carlos H. T. P.; Leopoldino, Andreia M.; Silva, Eloiza H. T.; Espinoza, V. A. A.; Taft, C. A.

    The isotypes of RAR and RXR are retinoic acid and retinoid X acid receptors, respectively, whose ligand-binding domain contains the ligand-dependent activation function, with distinct pharmacological targets for retinoids, involved in the treatment of various cancers and skin diseases. Due to the major challenge which cancer treatment and cure still imposes after many decades to the international scientific community, there is actually considerable interest in new ligands with increased bioactivity. We have focused on the retinoid acid receptor, which is considered an interesting target for drug design. In this work, we carried out density functional geometry optimizations and different docking procedures. We performed screening in a large database (hundreds of thousands of molecules which we optimized at the AM1 level) yielding a set of potential bioactive ligands. A new ligand was selected and optimized at the B3LYP/6-31G* level. A flexible docking program was used to investigate the interactions between the receptor and the new ligand. The result of this work is compared with several crystallographic ligands of RAR. Our theoretically more bioactive new ligand indicates stronger and more hydrogen bonds as well as hydrophobic interactions with the receptor.

  15. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Nie,Y.; Hobbs, J.; Vigues, S.; Olson, W.; Conn, G.; Munger, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

  16. Impact of killer immunoglobulin-like receptor-human leukocyte antigens ligand incompatibility among renal transplantation.

    Science.gov (United States)

    Alam, S; Rangaswamy, D; Prakash, S; Sharma, R K; Khan, M I; Sonawane, A; Agrawal, S

    2015-01-01

    Killer immunoglobulin-like receptor (KIR) gene shows a high degree of polymorphism. Natural killer cell receptor gets activated once they bind to self-human leukocyte antigens (HLAs) with specific ligand. KIR gene and HLA ligand incompatibility due to the presence/absence of KIR in the recipient and the corresponding HLA ligand in the allograft may impact graft survival in solid organ transplantation. This study evaluates the effect of matches between KIR genes and known HLA ligands. KIR genotypes were determined using sequence specific primer polymerase chain reaction. Presence of certain KIR in a recipient, where the donor lacked the corresponding HLA ligand was considered a mismatch. The allograft was considered matched when both KIR receptor and HLA alloantigen reveald compatibility among recipient and donor. The data revealed better survival among individuals with matched inhibitory KIR receptors and their corresponding HLA ligands (KIR2DL2/DL3-HLAC2, KIR3DL1-HLABw4). On the contrary, no adverse effect was seen for matched activating KIR receptors and their corresponding HLA ligands. One of the activating gene KIR2DS4 showed risk (P = 0.0413, odds ratio = 1.91, 95% confidence interval = 1.02-3.57) association with renal allograft rejection. We conclude that the presence of inhibitory KIR gene leads to better survival; whereas activating motifs show no significant role in renal allograft survival.

  17. ASSESSMENT OF COCAINE-LIKE DISCRIMINATIVE STIMULUS EFFECTS OF DOPAMINE D-3 RECEPTOR LIGANDS

    NARCIS (Netherlands)

    ACRI, JB; CARTER, [No Value; ALLING, K; GETERDOUGLASS, B; DIJKSTRA, D; WIKSTROM, H; KATZ, JL; WITKIN, JM

    1995-01-01

    The highly selective dopamine D-3 receptor ligand, (+)-PD 128907 4aR10bR-(+)-trans-3,4,4a,10b-tetrahydro-4-n-propyl- 2H5H[4,3-b]-1,4-oxazin-9-ol), and other dopamine D-3 receptor ligands, (+/-)-7-hydroxy-2-(N,N-di-n-propylamino)tetralin and (+)-7-hydroxy-2-(N,N-di-n-propylamino)tetralin, substituted

  18. Ligand-gated chloride channels are receptors for biogenic amines in C. elegans

    OpenAIRE

    Ringstad, Niels; Abe, Namiko; Horvitz, H. Robert

    2009-01-01

    Biogenic amines such as serotonin and dopamine are intercellular signaling molecules that function widely as neurotransmitters and neuromodulators. We have identified in the nematode Caenorhabditis elegans three ligand-gated chloride channels that are receptors for biogenic amines: LGC-53 is a high-affinity dopamine receptor, LGC-55 is a high-affinity tyramine receptor, and LGC-40 is a low-affinity serotonin receptor that is also gated by choline and acetylcholine. lgc-55 mutants are defectiv...

  19. Effects of 2,3-benzodiazepine AMPA receptor antagonists on dopamine turnover in the striatum of rats with experimental parkinsonism.

    Science.gov (United States)

    Megyeri, Katalin; Marko, Bernadett; Sziray, Nora; Gacsalyi, Istvan; Juranyi, Zsolt; Levay, Gyorgy; Harsing, Laszlo G

    2007-03-15

    Although levodopa is the current "gold standard" for treatment of Parkinson's disease, there has been disputation on whether AMPA receptor antagonists can be used as adjuvant therapy to improve the effects of levodopa. Systemic administration of levodopa, the precursor of dopamine, increases brain dopamine turnover rate and this elevated turnover is believed to be essential for successful treatment of Parkinson's disease. However, long-term treatment of patients with levodopa often leads to development of dyskinesia. Therefore, drugs that feature potentiation of dopamine turnover rate and are able to reduce daily levodopa dosages might be used as adjuvant in the treatment of patients suffering from Parkinson's disease. To investigate such combined treatment, we have examined the effects of two non-competitive AMPA receptor antagonists, GYKI-52466 and GYKI-53405, alone or in combination with levodopa on dopamine turnover rate in 6-hydroxydopamine-lesioned striatum of the rat. We found here that repeated administration of levodopa, added with the peripheral DOPA decarboxylase inhibitor carbidopa, increased dopamine turnover rate after lesioning the striatum with 6-hydroxydopamine. Moreover, combination of levodopa with GYKI-52466 or GYKI-53405 further increased dopamine turnover enhanced by levodopa administration while the AMPA receptor antagonists by themselves failed to influence striatal dopamine turnover. We concluded from the present data that potentiation observed between levodopa and AMPA receptor antagonists may reflect levodopa-sparing effects in clinical treatment indicating the therapeutic potential of such combination in the management of Parkinson's disease.

  20. A ligand channel through the G protein coupled receptor opsin.

    Directory of Open Access Journals (Sweden)

    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

  1. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Directory of Open Access Journals (Sweden)

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  2. Benzodiazepines: Sedation and Agitation.

    Science.gov (United States)

    Gallagher, Catherine

    2016-01-01

    Dental anxiety is common and frequently poses a barrier to necessary dental treatment. The increasing availability of conscious sedation in dental practice has made treatment much more accessible for anxious patients. At present, benzodiazepines are the most commonly used drugs in sedation practice and provide a pleasant experience for most, but not all, patients. An understanding of the mechanism of action of benzodiazepines should inform our practice and deepen our understanding of why and how sedation may fail. CPD/CLINICAL RELEVANCE: As an increasing number of dentists provide sedation for their patients an update on benzodiazepines is timely. PMID:27024905

  3. Directed evolution of estrogen receptor proteins with altered ligand-binding specificities.

    Science.gov (United States)

    Islam, Kazi Mohammed Didarul; Dilcher, Meik; Thurow, Corinna; Vock, Carsten; Krimmelbein, Ilga Kristine; Tietze, Lutz Friedjan; Gonzalez, Victor; Zhao, Huimin; Gatz, Christiane

    2009-01-01

    Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor alpha (hER alpha) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast two-hybrid system that reports the ligand-dependent interaction between hER alpha and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 microM. The optimized receptor mutant (L384F/L387M/Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 microM) and a 10-fold decreased activity to 17beta-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific 'gene switch' as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.

  4. Histamine H4 receptor ligands: future applications and state of art.

    Science.gov (United States)

    Corrêa, Michelle Fidelis; dos Santos Fernandes, João Paulo

    2015-04-01

    Histamine is a chemical transmitter found practically in whole organism and exerts its effects through the interaction with H1 to H4 histaminergic receptors. Specifically, H4 receptors are found mainly in immune cells and blood-forming tissues, thus are involved in inflammatory and immune processes, as well as some actions in central nervous system. Therefore, H4 receptor ligands can have applications in the treatment of chronic inflammatory and immune diseases and may be novel therapeutic option in these conditions. Several H4 receptor ligands have been described from early 2000's until nowadays, being imidazole, indolecarboxamide, 2-aminopyrimidine, quinazoline, and quinoxaline scaffolds the most explored and discussed in this review. Moreover, several studies of molecular modeling using homology models of H4 receptor and QSAR data of the ligands are summarized. The increasing and promising therapeutic applications are leading these compounds to clinical trials, which probably will be part of the next generation of blockbuster drugs. PMID:25228262

  5. The type I interleukin-1 receptor mediates fever in the rat as shown by interleukin-1 receptor subtype selective ligands.

    Science.gov (United States)

    Malinowsky, D; Chai, Z; Bristulf, J; Simoncsits, A; Bartfai, T

    1995-12-01

    The interleukin-1 (IL-1) system possesses two distinct receptors (type I and type II) which, together with the accessory protein, mediate a multitude of responses to IL-1 alpha and IL-1 beta, including fever. So far, no receptor subtype-specific ligands have been described. Since both types of IL-1 receptors occur in the thermoregulatory areas it was unclear which IL-1 receptor type mediates fever. We report here that for a series of deletion mutants of human recombinant IL-1 beta (hrIL-1 beta), the affinity of these ligands for the type I IL-1 receptor correlates with their efficacy to evoke the fever response (hrIL-1 beta > des-SND52-54 > des-QGE48-50 > des-I56). Thus, the results suggest that agonist occupancy of the type I IL-1 receptor is essential for IL-1 beta-mediated fever.

  6. Memory Effects of Benzodiazepines: Memory Stages and Types Versus Binding-Site Subtypes

    Directory of Open Access Journals (Sweden)

    Miroslav M. Savic

    2005-01-01

    Full Text Available Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the α1 and α5 subunits, whereas the effects on procedural memory can be mainly mediated by the α1 subunit. The pervading involvement of the α1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of α5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states.

  7. An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions.

    Science.gov (United States)

    Syedbasha, Mohameedyaseen; Linnik, Janina; Santer, Deanna; O'Shea, Daire; Barakat, Khaled; Joyce, Michael; Khanna, Nina; Tyrrell, D Lorne; Houghton, Michael; Egli, Adrian

    2016-01-01

    A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

  8. Following a TRAIL:Update on a ligand and its five receptors

    Institute of Scientific and Technical Information of China (English)

    Fiona C. KIMBERLEY; Gavin R. SCREATON

    2004-01-01

    Identification of tumour necrosis factor apoptosis inducing ligand (TRAIL), a TNF family ligand, sparked a torrent of research, following an initial observation that it could kill tumour cells, but spare normal cells. Almost a decade after its discovery, and with five known receptors, the true physiological role of TRAIL is still debated and its anti-tumorigenic properties limited by potential toxicity. This review takes a comprehensive look at the story of this enigmatic ligand,addressing its remaining potential as a therapeutic and providing an overview of the TRAIL receptors themselves.

  9. Domain interplay in the urokinase receptor. Requirement for the third domain in high affinity ligand binding and demonstration of ligand contact sites in distinct receptor domains

    DEFF Research Database (Denmark)

    Behrendt, N; Ronne, E; Dano, K

    1996-01-01

    . This result shows that in addition to D1, which has an established function in ligand binding (Behrendt, N., Ploug, M., Patthy, L., Houen, G., Blasi, F., and Dano, K. (1991) J. Biol. Chem. 266, 7842-7847), D3 has an important role in governing a high affinity in the intact receptor. Real-time biomolecular...

  10. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    DEFF Research Database (Denmark)

    Andersen, P S; Geisler, C; Buus, S;

    2001-01-01

    (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency......Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

  11. Benzodiazepine poisoning in elderly

    Directory of Open Access Journals (Sweden)

    Perković-Vukčević Nataša

    2016-01-01

    Full Text Available Background/Aim. Benzodiazepines are among the most frequently ingested drugs in self-poisonings. Elderly may be at greater risk compared with younger individuals due to impaired metabolism and increased sensitivity to benzodiazepines. The aim of this study was to assess toxicity of benzodiazepines in elderly attempted suicide. Methods. A retrospective study of consecutive presentations to hospital after self-poisoning with benzodiazepines was done. Collected data consisted of patient's characteristics (age, gender, benzodiazepine ingested with its blood concentrations at admission, clinical findings including vital signs and Glasgow coma score, routine blood chemistry, complications of poisoning, details of management, length of hospital stay and outcome. According the age, patients are classified as young (15-40-year old, middle aged (41-65-year old and elderly (older than 65. Results. During a 2-year observational period 387 patients were admitted because of pure benzodiazepine poisoning. The most frequently ingested drug was bromazepam, the second was diazepam. The incidence of coma was significantly higher, and the length of hospital stay significantly longer in elderly. Respiratory failure and aspiration pneumonia occurred more frequently in old age. Also, flumazenil was more frequently required in the group of elderly patients. Conclusion. Massive benzodiazepines overdose in elderly may be associated with a significant morbidity, including deep coma with aspiration pneumonia, respiratory failure, and even death. Flumazenil is indicated more often to reduce CNS depression and prevent complications of prolonged unconsciousness, but supportive treatment and proper airway management of comatose patients is the mainstay of the treatment of acute benzodiazepine poisoning.

  12. Adverse effects of benzodiazepines

    OpenAIRE

    Claire Gudex

    1990-01-01

    The growing realisation that the benzodiazepines have potential for causing serious harm has caused concern due to their wide and common use. This has stimulated interest in the costs and benefits of their use. This paper is a review of the adverse effects of benzodiazepines, and concentrates on four areas of particular concern: drug dependence which the consequent withdrawal symptoms; psychological effects while on the drugs; use by the elderly’ and tolerance to the drug effects. Although th...

  13. Revealing a steroid receptor ligand as a unique PPARγagonist

    Institute of Scientific and Technical Information of China (English)

    Shengchen Lin; Ying Han; Yuzhe Shi; Hui Rong; Songyang Zheng; Shikan Jin; Shu-Yong Lin; Sheng-Cai Lin; Yong Li

    2012-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs.We report here the identification of a steroid receptor ligand,RU-486,as an unexpected PPARγ agonist,thereby uncovering a novel signaling route for this steroid drug.Similar to rosiglitazone,RU486 modulates the expression of key PPARγ target genes and promotes adipocyte differentiation,but with a lower adipogenic activity.Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPARγ ligand-binding pocket with distinctive properties and epitopes,providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs.Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPARγligands in the treatment of insulin resistance.

  14. Revealing a steroid receptor ligand as a unique PPAR[gamma] agonist

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Shengchen; Han, Ying; Shi, Yuzhe; Rong, Hui; Zheng, Songyang; Jin, Shikan; Lin, Shu-Yong; Lin, Sheng-Cai; Li, Yong (Pitt); (Xiamen)

    2012-06-28

    Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPAR{gamma} agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPAR{gamma} target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPAR{gamma} ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPAR{gamma} ligands in the treatment of insulin resistance.

  15. Recent developments in A2B adenosine receptor ligands.

    Science.gov (United States)

    Kalla, Rao V; Zablocki, Jeff; Tabrizi, Mojgan Aghazadeh; Baraldi, Pier Giovanni

    2009-01-01

    A selective, high-affinity A(2B) adenosine receptor (AR) antagonist will be useful as a pharmacological tool to help determine the role of the A(2B)AR in inflammatory diseases and angiogenic diseases. Based on early A(2B)AR-selective ligands with nonoptimal pharmaceutical properties, such as 15 (MRS 1754: K(i)(hA(2B)) = 2 nM; K(i)(hA(1)) = 403 nM; K(i)(hA(2A)) = 503 NM, and K(i)(hA(3)) = 570 nM), several groups have discovered second-generation A(2B)AR ligands that are suitable for development. Scientists at CV Therapeutics have discovered the selective, high-affinity A(2B)AR antagonist 22, a 8-(4-pyrazolyl)-xanthine derivative, (CVT-6883, K(i)(hA(2B)) = 22 nM; K(i)(hA(1)) = 1,940 nM; K(i)(hA(2A)) = 3,280; and K(i)(hA(3)) = 1,070 nM). Compound 22 has demonstrated favorable pharmacokinetic (PK) properties (T(1/2) = 4 h and F > 35% rat), and it is a functional antagonist at the A(2B)AR(K (B) = 6 nM). In a mouse model of asthma, compound 22 demonstrated a dose-dependent efficacy supporting the role of the A(2B)AR in asthma. In two Phase I clinical trails, 22 (CVT-6883) was found to be safe, well tolerated, and suitable for once-daily dosing. Baraldi et al. have independently discovered a selective, high-affinity A(2B)AR antagonist, 30 (MRE2029F20), 8-(5-pyrazolyl)-xanthine (K(i)(hA(2B)) = 5.5 nM; K(i)(hA(1)) = 200 nM; K(i)(hA(2A), A(3)) > 1,000, that has been selected for development in conjunction with King Pharmaceuticals. Compound 30 has been demonstrated to be a functional antagonist of the A(2B)AR, and it has been radiolabeled for use in pharmacological studies. A third compound, 58 (LAS-38096), is a 2-aminopyrimidine derivative (discovered by the Almirall group) that has high A(2B)AR affinity and selectivity (K(i)(hA(2B)) = 17 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 2,500; and K(i)(hA(3)) > 1,000 nM), and 58 has been moved into preclinical safety testing. A fourth selective, high-affinity A(2B)AR antagonist, 54 (OSIP339391 K(i))(hA(2B)) = 0.5 nM; K(i))(hA(1

  16. Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level.

    Science.gov (United States)

    Tabor, Alina; Weisenburger, Siegfried; Banerjee, Ashutosh; Purkayastha, Nirupam; Kaindl, Jonas M; Hübner, Harald; Wei, Luxi; Grömer, Teja W; Kornhuber, Johannes; Tschammer, Nuska; Birdsall, Nigel J M; Mashanov, Gregory I; Sandoghdar, Vahid; Gmeiner, Peter

    2016-01-01

    G protein-coupled receptors (GPCRs), including dopamine receptors, represent a group of important pharmacological targets. An increased formation of dopamine receptor D2 homodimers has been suggested to be associated with the pathophysiology of schizophrenia. Selective labeling and ligand-induced modulation of dimerization may therefore allow the investigation of the pathophysiological role of these dimers. Using TIRF microscopy at the single molecule level, transient formation of homodimers of dopamine receptors in the membrane of stably transfected CHO cells has been observed. The equilibrium between dimers and monomers was modulated by the binding of ligands; whereas antagonists showed a ratio that was identical to that of unliganded receptors, agonist-bound D2 receptor-ligand complexes resulted in an increase in dimerization. Addition of bivalent D2 receptor ligands also resulted in a large increase in D2 receptor dimers. A physical interaction between the protomers was confirmed using high resolution cryogenic localization microscopy, with ca. 9 nm between the centers of mass. PMID:27615810

  17. Potential applications for sigma receptor ligands in cancer diagnosis and therapy

    NARCIS (Netherlands)

    van Waarde, Aren; Rybczynska, Anna A.; Kuzhuppilly Ramakrishnan, Nisha; Ishiwata, Kiichi; Elsinga, Philip H.; Dierckx, Rudi A. J. O.

    2015-01-01

    Sigma receptors (sigma-1 and sigma-2) represent two independent classes of proteins. Their endogenous ligands may include the hallucinogen N,N-dimethyltryptamine (DMT) and sphingolipid-derived amines which interact with sigma-1 receptors, besides steroid hormones (e.g., progesterone) which bind to b

  18. Increased accuracy of ligand sensing by receptor diffusion on cell surface

    Science.gov (United States)

    Aquino, Gerardo; Endres, Robert G.

    2010-10-01

    The physical limit with which a cell senses external ligand concentration corresponds to the perfect absorber, where all ligand particles are absorbed and overcounting of same ligand particles does not occur. Here, we analyze how the lateral diffusion of receptors on the cell membrane affects the accuracy of sensing ligand concentration. Specifically, we connect our modeling to neurotransmission in neural synapses where the diffusion of glutamate receptors is already known to refresh synaptic connections. We find that receptor diffusion indeed increases the accuracy of sensing for both the glutamate α -Amino-3-hydroxy-5-Methyl-4-isoxazolePropionic Acid (AMPA) and N -Methyl-D-aspartic Acid (NMDA) receptor, although the NMDA receptor is overall much noisier. We propose that the difference in accuracy of sensing of the two receptors can be linked to their different roles in neurotransmission. Specifically, the high accuracy in sensing glutamate is essential for the AMPA receptor to start membrane depolarization, while the NMDA receptor is believed to work in a second stage as a coincidence detector, involved in long-term potentiation and memory.

  19. Modeling multivalent ligand-receptor interactions with steric constraints on configurations of cell surface receptor aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Monine, Michael [Los Alamos National Laboratory; Posner, Richard [TRANSLATION GENOMICS RESAEARCH INSTITUTE; Savage, Paul [BYU; Faeder, James [UNIV OF PITTSBURGH; Hlavacek, William S [UNM

    2008-01-01

    Signal transduction generally involves multivalent protein-protein interactions, which can produce various protein complexes and post-translational modifications. The reaction networks that characterize these interactions tend to be so large as to challenge conventional simulation procedures. To address this challenge, a kinetic Monte Carlo (KMC) method has been developed that can take advantage of a model specification in terms of reaction rules for molecular interactions. A set of rules implicitly defines the reactions that can occur as a result of the interactions represented by the rules. With the rule-based KMC method, explicit generation of the underlying chemical reaction network implied by rules is avoided. Here, we apply and extend this method to characterize the interactions of a trivalent ligand with a bivalent cell-surface receptor. This system is also studied experimentally. We consider the following kinetic models: an equivalent-site model, an extension of this model, which takes into account steric constraints on the configurations of receptor aggregates, and finally, a model that accounts for cyclic receptor aggregates. Simulation results for the equivalent-site model are consistent with an equilibrium continuum model. Using these models, we investigate the effects of steric constraints and the formation of cyclic aggregates on the kinetics and equilibria of small and large aggregate formation and the percolation phase transition that occurs in this system.

  20. DMPD: Toll-like receptors, Notch ligands, and cytokines drive the chronicity of lunginflammation. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18073395 Toll-like receptors, Notch ligands, and cytokines drive the chronicity of lunginflammation. Raymond...ors, Notch ligands, and cytokines drive the chronicity of lunginflammation. Authors Raymond T, Schaller M, H

  1. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    International Nuclear Information System (INIS)

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the α-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor

  2. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H. (Biopharmaceuticals Div., Bagsvaerd (Denmark))

    1990-08-14

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the {alpha}-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor.

  3. Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors.

    Science.gov (United States)

    Takehara, S; Mikashima, H; Muramoto, Y; Terasawa, M; Setoguchi, M; Tahara, T

    1990-12-01

    The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets. PMID:1965554

  4. Strategy for improved [11C]DAA1106 radiosynthesis and in vivo peripheral benzodiazepine receptor imaging using microPET, evaluation of [11C]DAA1106

    International Nuclear Information System (INIS)

    Introduction: The peripheral benzodiazepine receptor (PBR) has shown considerable potential as a clinical marker of neuroinflammation and tumour progression. [11C]DAA1106 ([11C]N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)-acetamide) is a promising positron emission tomography (PET) radioligand for imaging PBRs. Methods: A four-step synthetic route was devised to prepare DAA1123, the precursor for [11C]DAA1106. Two robust, high yielding methods for radiosynthesis based on [11C]-O-methylation of DAA1123 were developed and implemented on a nuclear interface methylation module, producing [11C]DAA1106 with up to 25% radiochemical yields at end-of-synthesis based on [11C]CH3I trapped. Evaluation of [11C]DAA1106 for in vivo imaging was performed in a rabbit model with microPET, and the presence of PBR receptor in the target organ was further corroborated by immunohistochemistry. Results: The standard solution method produced 2.6-5.2 GBq (n=19) of [11C]DAA1106, whilst the captive solvent method produced 1.6-6.3 GBq (n=10) of [11C]DAA1106. Radiochemical purities obtained were 99% and specific radioactivity at end-of-synthesis was up to 200 GBq/μmol for both methods. Based on radiochemical product, shorter preparation times and simplicity of synthesis, the captive solvent method was chosen for routine productions of [11C]DAA1106. In vivo microPET [11C]DAA1106 scans of rabbit kidney demonstrated high levels of binding in the cortex. The subsequent introduction of nonradioactive DAA1106 (0.2 μmol) produced considerable displacement of the radioactive signal in this region. The presence of PBR in kidney cortex was further corroborated by immunohistochemistry. Conclusions: A robust, high yielding captive solvent method of [11C]DAA1106 production was developed which enabled efficacious in vivo imaging of PBR expressing tissues in an animal model

  5. The Different Ligand-Binding Modes of Relaxin Family Peptide Receptors RXFP1 and RXFP2

    OpenAIRE

    Scott, Daniel J.; Rosengren, K. Johan; Bathgate, Ross A. D.

    2012-01-01

    Relaxin and insulin-like peptide 3 (INSL3) are peptide hormones with a number of important physiological roles in reproduction, regulation of extracellular matrix turnover, and cardiovascular function. Relaxin and INSL3 mediate their actions through the closely related G-protein coupled receptors, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2), respectively. These receptors have large extracellular domains (ECD) that contain high-affinity ligand-binding sites within their 10 leuci...

  6. Characterization of the ligand-dependent transactivation domain of thyroid hormone receptor.

    OpenAIRE

    Barettino, D; Vivanco Ruiz, M M; Stunnenberg, H.G.

    1994-01-01

    Transcriptional activation by nuclear receptors is achieved through autonomous activation functions (AFs), a constitutive N-terminal AF-1 and a C-terminal, ligand-dependent AF-2 that comprises a motif conserved between nuclear receptors. We have performed an extensive mutational analysis of the putative AF-2 domain of chicken thyroid hormone receptor alpha (cT3R alpha). We show that the AF-2 region mediates transactivation as well as transcriptional interference (squelching), not only between...

  7. Melanocortin-1 receptor-mediated signalling pathways activated by NDP-MSH and HBD3 ligands

    OpenAIRE

    Beaumont, Kimberley A.; Smit, Darren J.; Liu, Yan Yan; Chai, Eric; Patel, Mira P.; Millhauser, Glenn L.; Smith, Jennifer J.; Alewood, Paul F.; Sturm, Richard A.

    2012-01-01

    Binding of melanocortin peptide agonists to the melanocortin-1 receptor of melanocytes results in eumelanin production, whereas binding of the agouti signalling protein inverse agonist results in pheomelanin synthesis. Recently, a novel melanocortin-1 receptor ligand was reported. A β-defensin gene mutation was found to beresponsible for black coat colour in domestic dogs. Notably, the human equivalent, β-defensin 3, was found to bind with high affinity to the melanocortin-1 receptor; however...

  8. Bombesin family receptor and ligand gene expression in human colorectal cancer and normal mucosa

    OpenAIRE

    Chave, H S; Gough, A C; Palmer, K.; Preston, S. R.; Primrose, J N

    1999-01-01

    Bombesin-like peptides and their receptors are widely distributed throughout the gut and are potential mitogens for a number of gastrointestinal (GI) cancers. We have analysed the expression of bombesin-like peptides and their receptor subtypes in normal and neoplastic colorectal tissue. Expression was analysed by reverse transcription polymerase chain reaction (RT-PCR) using receptor and ligand subtype-specific primers and then expression localized by in situ hybridization (ISH) with ribopro...

  9. Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands.

    Science.gov (United States)

    Li, Jingjing; Gierach, Izabela; Gillies, Alison R; Warden, Charles D; Wood, David W

    2011-11-15

    The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G(4)S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD. PMID:21893405

  10. Theoretical investigation of interaction between the set of ligands and α7 nicotinic acetylcholine receptor

    Science.gov (United States)

    Glukhova, O. E.; Prytkova, T. R.; Shmygin, D. S.

    2016-03-01

    Nicotinic acetylcholine receptors (nAChRs) are neuron receptor proteins that provide a transmission of nerve impulse through the synapses. They are composed of a pentametric assembly of five homologous subunits (5 α7 subunits for α7nAChR, for example), oriented around the central pore. These receptors might be found in the chemical synapses of central and peripheral nervous system, and also in the neuromuscular synapses. Transmembrane domain of the one of such receptors constitutes ion channel. The conductive properties of ion channel strongly depend on the receptor conformation changes in the response of binding with some molecule, f.e. acetylcholine. Investigation of interaction between ligands and acetylcholine receptor is important for drug design. In this work we investigate theoretically the interaction between the set of different ligands (such as vanillin, thymoquinone, etc.) and the nicotinic acetylcholine receptor (primarily with subunit of the α7nAChR) by different methods and packages (AutodockVina, GROMACS, KVAZAR, HARLEM, VMD). We calculate interaction energy between different ligands in the subunit using molecular dynamics. On the base of obtained calculation results and using molecular docking we found an optimal location of different ligands in the subunit.

  11. Why do receptor-ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

    Science.gov (United States)

    Gao, Zhiwen; Gao, Yanfei

    2016-10-01

    Cell adhesion often exhibits the clustering of the receptor-ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation for the clustering/assembling of the receptor-ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor-ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.

  12. 3,5-T2 is an alternative ligand for the thyroid hormone receptor β1.

    Science.gov (United States)

    Mendoza, A; Navarrete-Ramírez, P; Hernández-Puga, G; Villalobos, P; Holzer, G; Renaud, J P; Laudet, V; Orozco, A

    2013-08-01

    Several liganded nuclear receptors have alternative ligands acting in a tissue-specific fashion and playing important biological roles. We present evidence that 3,5-diiodothyronine (T(2)), a naturally occurring iodothyronine that results from T(3) outer-ring deiodination, is an alternative ligand for thyroid hormone receptor β1 (TRβ1). In tilapia, 2 TRβ isoforms differing by 9 amino acids in the ligand-binding domain were cloned. Binding and transactivation studies showed that T(2) activates the human and the long tilapia TRβ1 isoform, but not the short one. A chimeric human TRβ1 (hTRβ1) that contained the 9-amino-acid insert showed no response to T(2), suggesting that the conformation of the hTRβ1 naturally allows T(2) binding and that other regions of the receptor are implicated in TR activation by T(2). Indeed, further analysis showed that the N terminus is essential for T(2)-mediated transactivation but not for that by T(3) in the long and hTRβ1, suggesting a functional interaction between the N-terminal domain and the insertion in the ligand-binding domain. To establish the functional relevance of T(2)-mediated TRβ1 binding and activation, mRNA expression and its regulation by T(2) and T(3) was evaluated for both isoforms. Our data show that long TRβ1expression is 10(6)-fold higher than that of the short isoform, and T(3) and T(2) differentially regulate the expression of these 2 TRβ1 isoforms in vivo. Taken together, our results prompted a reevaluation of the role and mechanism of action of thyroid hormone metabolites previously believed to be inactive. More generally, we propose that classical liganded receptors are only partially locked to very specific ligands and that alternative ligands may play a role in the tissue-specific action of receptors. PMID:23736295

  13. Gamma-aminobutyric acid and benzodiazepine receptor changes induced by unilateral 6-hydroxydopamine lesions of the medial forebrain bundle

    Energy Technology Data Exchange (ETDEWEB)

    Pan, H.S.; Penney, J.B.; Young, A.B.

    1985-11-01

    Quantitative autoradiography was used to ascertain alterations in (TH)muscimol, (TH)flunitrazepam (FLU), (TH)naloxone, (TH)D-alanine-D-leucine-enkephalin (DADL), and (TH)spiroperidol binding in basal ganglia 1 week, 4 weeks, and 5 months after unilateral 6-hydroxydopamine lesions of the medial forebrain bundle (MFB) in the rat. At 1 and 4 weeks following lesions, (TH)spiroperidol binding increased 33% in striatum. At 5 months, (TH)spiroperidol was only nonsignificantly increased above control. At 1 week, (TH)muscimol binding decreased 39% in ipsilateral globus pallidus (GP), but increased 41% and 11% in entopeduncular nucleus (EPN) and substantia nigra pars reticulata (SNr), respectively. At 4 weeks, (TH)muscimol binding was reduced 19% in striatum and 44% in GP and remained enhanced by 32% in both EPN and SNr. These changes in (TH)muscimol binding persisted at 5 months. (TH)FLU binding was altered in the same direction as (TH)muscimol binding; however, changes were slower in onset and became significant (and remained so) only at 4 weeks after lesions. Decreases in (TH)naloxone and (TH)DADL binding were seen in striatum, GP, EPN, and SNr. Scatchard analyses revealed that only receptor numbers were altered. This study provides biochemical evidence for differential regulation of striatal GABAergic output to GP and EPN/SNr.

  14. Discriminating agonist and antagonist ligands of the nuclear receptors using 3D-pharmacophores.

    Science.gov (United States)

    Lagarde, Nathalie; Delahaye, Solenne; Zagury, Jean-François; Montes, Matthieu

    2016-01-01

    Nuclear receptors (NRs) constitute an important class of therapeutic targets. We evaluated the performance of 3D structure-based and ligand-based pharmacophore models in predicting the pharmacological profile of NRs ligands using the NRLiSt BDB database. We could generate selective pharmacophores for agonist and antagonist ligands and we found that the best performances were obtained by combining the structure-based and the ligand-based approaches. The combination of pharmacophores that were generated allowed to cover most of the chemical space of the NRLiSt BDB datasets. By screening the whole NRLiSt BDB on our 3D pharmacophores, we demonstrated their selectivity towards their dedicated NRs ligands. The 3D pharmacophores herein presented can thus be used as a predictor of the pharmacological activity of NRs ligands.Graphical AbstractUsing a combination of structure-based and ligand-based pharmacophores, agonist and antagonist ligands of the Nuclear Receptors included in the NRLiSt BDB database could be separated.

  15. Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells.

    Science.gov (United States)

    Zheng, Xiangjun; Cheung, Luthur Siu-Lun; Schroeder, Joyce A; Jiang, Linan; Zohar, Yitshak

    2011-10-21

    Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.

  16. A mollusk retinoic acid receptor (RAR) ortholog sheds light on the evolution of ligand binding.

    Science.gov (United States)

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Lima, Daniela; Pierzchalski, Keely; Jones, Jace W; Kane, Maureen; Nishikawa, Jun-Ichi; Hiromori, Youhei; Nakanishi, Tsuyoshi; Santos, Miguel M; Castro, L Filipe C; Bourguet, William; Schubert, Michael; Laudet, Vincent

    2014-11-01

    Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms. PMID:25116705

  17. A novel chemogenomics analysis of G protein-coupled receptors (GPCRs and their ligands: a potential strategy for receptor de-orphanization

    Directory of Open Access Journals (Sweden)

    Emmerich Michael TM

    2010-06-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs represent a family of well-characterized drug targets with significant therapeutic value. Phylogenetic classifications may help to understand the characteristics of individual GPCRs and their subtypes. Previous phylogenetic classifications were all based on the sequences of receptors, adding only minor information about the ligand binding properties of the receptors. In this work, we compare a sequence-based classification of receptors to a ligand-based classification of the same group of receptors, and evaluate the potential to use sequence relatedness as a predictor for ligand interactions thus aiding the quest for ligands of orphan receptors. Results We present a classification of GPCRs that is purely based on their ligands, complementing sequence-based phylogenetic classifications of these receptors. Targets were hierarchically classified into phylogenetic trees, for both sequence space and ligand (substructure space. The overall organization of the sequence-based tree and substructure-based tree was similar; in particular, the adenosine receptors cluster together as well as most peptide receptor subtypes (e.g. opioid, somatostatin and adrenoceptor subtypes. In ligand space, the prostanoid and cannabinoid receptors are more distant from the other targets, whereas the tachykinin receptors, the oxytocin receptor, and serotonin receptors are closer to the other targets, which is indicative for ligand promiscuity. In 93% of the receptors studied, de-orphanization of a simulated orphan receptor using the ligands of related receptors performed better than random (AUC > 0.5 and for 35% of receptors de-orphanization performance was good (AUC > 0.7. Conclusions We constructed a phylogenetic classification of GPCRs that is solely based on the ligands of these receptors. The similarities and differences with traditional sequence-based classifications were investigated: our ligand

  18. Metabotropic glutamate receptor ligands as potential therapeutics for addiction

    OpenAIRE

    Olive, M.F.

    2009-01-01

    There is now compelling evidence that the excitatory amino acid neurotransmitter glutamate plays a pivotal role in drug addiction and alcoholism. As a result, there has been increasing interest in developing glutamate-based therapies for the treatment of addictive disorders. Receptors for glutamate are primarily divided into two classes: ionotropic glutamate receptors (iGluRs) that mediate fast excitatory glutamate transmission, and metabotropic glutamate receptors (mGluRs), which are G-prote...

  19. Perspectives on cognitive domains, H3 receptor ligands and neurological disease.

    Science.gov (United States)

    Hancock, Arthur A; Fox, Gerard B

    2004-10-01

    Histamine H(3) receptor agonists and antagonists have been evaluated in numerous in vitro and in vivo animal models to better understand how H(3) receptors modulate neurotransmitter function in the central nervous system. Likewise, behavioural models have explored the hypothesis that changes in neurotransmitter release could enhance cognitive function in human diseases. This review examines the reported effects of H(3) receptor ligands and how they influence cognitive behaviour. These data are interpreted on the basis of different cognitive domains that are relevant to neuropsychiatric diseases. Because of the diversity of H(3) receptors, their function and their influence on neurotransmitter systems, considerable promise exists for H(3) ligands to treat diseases in which aspects of learning and memory are impaired. However, because of the complexities of the histaminergic system and H(3) receptors and the lack of clinical data so far, proof of principle for use in human disease remains to be established.

  20. Modeling of cell adhesion and deformation mediated by receptor-ligand interactions.

    Science.gov (United States)

    Golestaneh, Amirreza F; Nadler, Ben

    2016-04-01

    The current work is devoted to studying adhesion and deformation of biological cells mediated by receptors and ligands in order to enhance the existing models. Due to the sufficient in-plane continuity and fluidity of the phospholipid molecules, an isotropic continuum fluid membrane is proposed for modeling the cell membrane. The developed constitutive model accounts for the influence of the presence of receptors on the deformation and adhesion of the cell membrane through the introduction of spontaneous area dilation. Motivated by physics, a nonlinear receptor-ligand binding force is introduced based on charge-induced dipole interaction. Diffusion of the receptors on the membrane is governed by the receptor-ligand interaction via Fick's Law and receptor-ligand interaction. The developed model is then applied to study the deformation and adhesion of a biological cell. The proposed model is used to study the role of the material, binding, spontaneous area dilation and environmental properties on the deformation and adhesion of the cell. PMID:26093646

  1. N,N'-dicyclohexylsulfamide and N,N'-diphenethylsulfamide are anticonvulsant sulfamides with affinity for the benzodiazepine binding site of the GABA(A) receptor and anxiolytic activity in mice.

    Science.gov (United States)

    Wasowski, Cristina; Gavernet, Luciana; Barrios, Ivana A; Villalba, Maria L; Pastore, Valentina; Samaja, Gisela; Enrique, Andrea; Bruno-Blanch, Luis E; Marder, Mariel

    2012-01-15

    A set of sulfamides designed, synthesized and evaluated against maximal electroshock seizure (MES) and pentilenetetrazol (PTZ) tests with promising results, were tested for their affinity for the benzodiazepine binding site of the GABA(A) receptor. The most active compounds, N,N'-dicyclohexylsulfamide (7) and N,N'-diphenethylsulfamide (10), competitively inhibited the binding of [(3)H]-flunitrazepam to the benzodiazepine binding site with K(i)±SEM values of 27.7±4.5μM (n=3) and 6.0±1.2μM (n=3), respectively. The behavioral actions of these sulfamides, i.p. administered in mice, were examined in the plus-maze, hole-board and locomotor activity assays. Compound 7 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the hole-board test (at 1 and 3mg/kg) and the plus-maze assay (at 1 and 3mg/kg). Compound 10 evidenced anxiolytic activity in the plus-maze and the hole-board tests at 1mg/kg. Locomotor activity of mice was not modified by compound 7 or 10 at the doses tested. Flumazenil, a non selective benzodiazepine binding site antagonist, was able to completely reverse the anxiolytic-like effects of these sulfamides, proving that the GABA(A) receptor is implicated in this action. Anxiety represents a major problem for people with epilepsy. The use of anxiolytic and anticonvulsant sulfamides would be beneficial to individuals who suffer from both disorders. PMID:22056620

  2. Ligands of estrogen receptors α and β, method of their preparation, and pharmaceuticals comprising them

    OpenAIRE

    Novák, P.; Sedlák, D. (David); Bartůněk, P. (Petr); Kotora, M. (Martin)

    2012-01-01

    The invention relates to novel ligands of the estrogen receptors α and β of general formula II, which are useful as an active substance of pharmaceuticals, for example pharmaceutical compositions useful for hormone replacement therapy, as well as for the treatment of tumors and inflammatory diseases. The invention also relates to a novel preparation method of these ligands comprising cyclotrimerization of ethynylestradiol with the appropriate diyne in an organic solvent. Further, th...

  3. The glucocorticoid receptor hormone binding domain mediates transcriptional activation in vitro in the absence of ligand.

    OpenAIRE

    Schmitt, J.; Stunnenberg, H G

    1993-01-01

    We show that recombinant rat glucocorticoid receptor (vvGR) expressed using vaccinia virus is indistinguishable from authentic GR with respect to DNA and hormone binding. In the absence of hormone, vvGR is mainly found in the cytoplasm in a complex with heat shock protein 90. Upon incubation with ligand, vvGR is released from this complex and translocated to the nucleus. Thus, the ligand binding domain displays the known biochemical properties. However, in vitro, transcription from a syntheti...

  4. Regulation of dendritic cell differentiation and function by estrogen receptor ligands

    OpenAIRE

    Kovats, Susan; Carreras, Esther

    2008-01-01

    Estrogen receptor (ER) ligands can modulate innate and adaptive immunity and hematopoiesis, which may explain the clear sex differences in immune responses during autoimmunity, infection or trauma. Dendritic cells (DC) are antigen-presenting cells important for initiation of innate and adaptive immunity, as well as immune tolerance. DC progenitors and terminally differentiated DC express ER, indicating the ER ligands may regulate DC at multiple developmental and functional stages. Although th...

  5. Biophysical characterization of G-protein coupled receptor-peptide ligand binding

    OpenAIRE

    Langelaan, David N.; Ngweniform, Pascaline; Rainey, Jan K.

    2011-01-01

    G-protein coupled receptors (GPCRs) are ubiquitous membrane proteins allowing intracellular response to extracellular factors that range from photons of light to small molecules to proteins. Despite extensive exploitation of GRCRs as therapeutic targets, biophysical characterization of GPCR-ligand interactions remains challenging. In this minireview, we focus on techniques which have been successfully employed for structural and biophysical characterization of peptide ligands binding to their...

  6. Functional characterization of the 1,5-benzodiazepine clobazam and its major active metabolite N-desmethylclobazam at human GABAA receptors expressed in Xenopus laevis oocytes

    DEFF Research Database (Denmark)

    Hammer, Harriet; Ebert, Bjarke; Jensen, Henrik S.;

    2015-01-01

    different in vivo adverse effects and addiction liability profile than the classic 1,4-benzodiazepines. In this study, it was investigated whether the in vitro pharmacological properties of clobazam and its major active metabolite N-desmethylclobazam could explain some of these clinical differences...

  7. Signal processing in the TGF-beta superfamily ligand-receptor network.

    Directory of Open Access Journals (Sweden)

    Jose M G Vilar

    2006-01-01

    Full Text Available The TGF-beta pathway plays a central role in tissue homeostasis and morphogenesis. It transduces a variety of extracellular signals into intracellular transcriptional responses that control a plethora of cellular processes, including cell growth, apoptosis, and differentiation. We use computational modeling to show that coupling of signaling with receptor trafficking results in a highly versatile signal-processing unit, able to sense by itself absolute levels of ligand, temporal changes in ligand concentration, and ratios of multiple ligands. This coupling controls whether the response of the receptor module is transient or permanent and whether or not different signaling channels behave independently of each other. Our computational approach unifies seemingly disparate experimental observations and suggests specific changes in receptor trafficking patterns that can lead to phenotypes that favor tumor progression.

  8. 3-Substituted phenylalanines as selective AMPA- and kainate receptor ligands

    DEFF Research Database (Denmark)

    Szymanska, Ewa; Pickering, Darryl S; Nielsen, Birgitte;

    2009-01-01

    On the basis of X-ray structures of ionotropic glutamate receptor constructs in complex with amino acid-based AMPA and kainate receptor antagonists, a series of rigid as well as flexible biaromatic alanine derivatives carrying selected hydrogen bond acceptors and donors have been synthesized in o...

  9. Photoaffinity ligand for dopamine D2 receptors: azidoclebopride

    Energy Technology Data Exchange (ETDEWEB)

    Niznik, H.B.; Guan, J.H.; Neumeyer, J.L.; Seeman, P.

    1985-02-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind (/sup 3/H)spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol.

  10. Genomic organization, annotation, and ligand-receptor inferences of chicken chemokines and chemokine receptor genes based on comparative genomics

    Directory of Open Access Journals (Sweden)

    Sze Sing-Hoi

    2005-03-01

    Full Text Available Abstract Background Chemokines and their receptors play important roles in host defense, organogenesis, hematopoiesis, and neuronal communication. Forty-two chemokines and 19 cognate receptors have been found in the human genome. Prior to this report, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to systematically identify chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach. Results Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. All of the chicken chemokines contained a conserved CC, CXC, CX3C, or XC motif, whereas all the chemokine receptors had seven conserved transmembrane helices, four extracellular domains with a conserved cysteine, and a conserved DRYLAIV sequence in the second intracellular domain. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and sequence homology. Conclusion The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. All identified chicken chemokines and their cognate receptors were identified in the chicken genome except CCR9, whose ligand was not identified in this study. The organization of these genes suggests that there were a substantial number of these genes present before divergence between aves and mammals and more gene duplications of CC, CXC, CCR, and CXCR subfamilies in mammals than in aves after the divergence.

  11. Determinants governing ligand specificity of the Vibrio harveyi LuxN quorum-sensing receptor.

    Science.gov (United States)

    Ke, Xiaobo; Miller, Laura C; Bassler, Bonnie L

    2015-01-01

    Quorum sensing is a process of bacterial cell-cell communication that relies on the production, release and receptor-driven detection of extracellular signal molecules called autoinducers. The quorum-sensing bacterium Vibrio harveyi exclusively detects the autoinducer N-((R)-3-hydroxybutanoyl)-L-homoserine lactone (3OH-C4 HSL) via the two-component receptor LuxN. To discover the principles underlying the exquisite selectivity LuxN has for its ligand, we identified LuxN mutants with altered specificity. LuxN uses three mechanisms to verify that the bound molecule is the correct ligand: in the context of the overall ligand-binding site, His210 validates the C3 modification, Leu166 surveys the chain-length and a strong steady-state kinase bias imposes an energetic hurdle for inappropriate ligands to elicit signal transduction. Affinities for the LuxN kinase on and kinase off states underpin whether a ligand will act as an antagonist or an agonist. Mutations that bias LuxN to the agonized, kinase off, state are clustered in a region adjacent to the ligand-binding site, suggesting that this region acts as the switch that triggers signal transduction. Together, our analyses illuminate how a histidine sensor kinase differentiates between ligands and exploits those differences to regulate its signaling activity.

  12. Lipoteichoic acid induces unique inflammatory responses when compared to other toll-like receptor 2 ligands.

    Directory of Open Access Journals (Sweden)

    Elizabeth M Long

    Full Text Available Toll-like receptors (TLRs recognize evolutionarily-conserved molecular patterns originating from invading microbes. In this study, we were interested in determining if microbial ligands, which use distinct TLR2-containing receptor complexes, represent unique signals to the cell and can thereby stimulate unique cellular responses. Using the TLR2 ligands, R-FSL1, S-FSL1, Pam2CSK4, Pam3CSK4, and lipoteichoic acid (LTA, we demonstrate that these ligands activate NF-kappaB and MAP Kinase pathways with ligand-specific differential kinetics in murine macrophages. Most strikingly, LTA stimulation of these pathways was substantially delayed when compared with the other TLR2 ligands. These kinetics differences were associated with a delay in the LTA-induced expression of a subset of genes as compared with another TLR2 ligand, R-FSL1. However, this did not translate to overall differences in gene expression patterns four hours following stimulation with different TLR2 ligands. We extended this study to evaluate the in vivo responses to distinct TLR2 ligands using a murine model of acute inflammation, which employs intravital microscopy to monitor leukocyte recruitment into the cremaster muscle. We found that, although R-FSL1, S-FSL1, Pam2CSK4, and Pam3CSK4 were all able to stimulate robust leukocyte recruitment in vivo, LTA remained functionally inert in this in vivo model. Therefore distinct TLR2 ligands elicit unique cellular responses, as evidenced by differences in the kinetic profiles of signaling and gene expression responses in vitro, as well as the physiologically relevant differences in the in vivo responses to these ligands.

  13. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    Science.gov (United States)

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  14. New halogenated tris-(phenylalkyl)amines as h5-HT2B receptor ligands.

    Science.gov (United States)

    Kapadia, Nirav; Ahmed, Shahrear; Harding, Wayne W

    2016-07-15

    A series of compounds in which various halogen substituents were incorporated into a phenyl ring of a tris-(phenylalkyl)amine scaffold, was synthesized and evaluated for affinity to h5-HT2 receptors. In general, all compounds were found to have good affinity for the 5-HT2B receptor and were selective over 5-HT2A and 5-HT2C receptors. Compound 9i was the most selective compound in this study and is the highest affinity 5-HT2B receptor ligand bearing a tris-(phenylalkyl)amine scaffold to date. PMID:27261181

  15. Therapeutic Potential of 5-HT2C Receptor Ligands

    Directory of Open Access Journals (Sweden)

    Nanna H. Jensen

    2010-01-01

    Full Text Available Serotonin 2C receptors are G protein-coupled receptors expressed by GABAergic, glutamatergic, and dopaminergic neurons. Anatomically, they are present in various brain regions, including cortical areas, hippocampus, ventral midbrain, striatum, nucleus accumbens, hypothalamus, and amygdala. A large body of evidence supports a critical role of serotonin 2C receptors in mediating the interaction between serotonergic and dopaminergic systems, which is at the basis of their proposed involvement in the regulation of mood, affective behavior, and memory. In addition, their expression in specific neuronal populations in the hypothalamus would be critical for their role in the regulation of feeding behavior. Modulation of these receptors has therefore been proposed to be of interest in the search for novel pharmacological strategies for the treatment of various pathological conditions, including schizophrenia and mood disorders, as well as obesity. More precisely, blockade of serotonin 2C receptors has been suggested to provide antidepressant and anxiolytic benefit, while stimulation of these receptors may offer therapeutic benefit for the treatment of psychotic symptoms in schizophrenia and obesity. In addition, modulation of serotonin 2C receptors may offer cognitive-enhancing potential, albeit still a matter of debate. In the present review, the most compelling evidence from the literature is presented and tentative hypotheses with respect to existing controversies are outlined.

  16. The Role of the Enterohepatic Circulation of Bile Salts and Nuclear Hormone Receptors in the Regulation of Cholesterol Homeostasis: Bile Salts as Ligands for Nuclear Hormone Receptors

    OpenAIRE

    Redinger, Richard N.

    2003-01-01

    The coordinated effect of lipid activated nuclear hormone receptors; liver X receptor (LXR), bound by oxysterol ligands and farnesoid X receptor (FXR), bound by bile acid ligands, act as genetic transcription factors to cause feed-forward cholesterol catabolism to bile acids and feedback repression of bile acid synthesis, respectively. It is the coordinated action of LXR and FXR, each dimerized to retinoid X receptor, that signal nuclear DNA response elements to encode proteins that prevent e...

  17. Heart Failure Therapeutics on the Basis of a Biased Ligand of the Angiotensin-2 Type 1 Receptor Rationale and Design of the BLAST-AHF Study (Biased Ligand of the Angiotensin Receptor Study in Acute Heart Failure)

    NARCIS (Netherlands)

    Felker, G. Michael; Butler, Javed; Collins, Sean P.; Cotter, Gad; Davison, Beth A.; Ezekowitz, Justin A.; Filippatos, Gerasimos; Levy, Phillip D.; Metra, Marco; Ponikowski, Piotr; Soergel, David G.; Teerlink, John R.; Violin, Jonathan D.; Voors, Adriaan A.; Pang, Peter S.

    2015-01-01

    The BLAST-AHF (Biased Ligand of the Angiotensin Receptor Study in Acute Heart Failure) study is designed to test the efficacy and safety of TRV027, a novel biased ligand of the angiotensin-2 type 1 receptor, in patients with acute heart failure (AHF). AHF remains a major public health problem, and n

  18. Dissecting the chemistry of nicotinic receptor-ligand interactions with infrared difference spectroscopy.

    Science.gov (United States)

    Ryan, Stephen E; Hill, Danny G; Baenziger, John E

    2002-03-22

    The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm(-1) indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm(-1) are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm(-1) indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm(-1) to 1690 and 1620 cm(-1), respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions. PMID:11782459

  19. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    Science.gov (United States)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  20. Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM

    Science.gov (United States)

    Pfreundschuh, Moritz; Alsteens, David; Wieneke, Ralph; Zhang, Cheng; Coughlin, Shaun R.; Tampé, Robert; Kobilka, Brian K.; Müller, Daniel J.

    2015-11-01

    A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution.

  1. Development of novel mixed ligand technetium complexes for imaging 5-HT1A neural system receptors

    International Nuclear Information System (INIS)

    The development of 99mTc complexes for imaging 5-HT1A neural system receptors using the 3 + 1 mixed ligand approach is described. Six novel complexes (I-VI) were designed using two different strategies. In complexes I-IV the pharmacophore 1-(2-methoxyphenyl)piperazine was attached to a monodentate thiol used as co-ligand and combined with tridentate dianionic aminothiols (SNS and NNS). On the other hand, complexes V and VI were obtained using thiophenol and 4-methoxy-thiophenol as co-ligand and a tridentate ligand (SNS) with the pharmacophore bound to the nitrogen through an alkyl chain. All complexes were prepared at tracer level using 99mTc-glucoheptonate as precursor. Ligand and co-ligand concentration, reaction time and temperature were optimized to achieve high substitution yield and radiochemical purity. Structure was studied at carrier level through the corresponding rhenium complexes. Complexes I and II presented the expected ReOLK structure and a distorted trigonal bipyramidal geometry. The structure of the other four complexes has not been completely elucidated yet. Biodistribution studies of all the complexes demonstrated selective brain uptake and retention. Uptake of complex I in receptor-rich hippocampus was significantly higher than that of the cerebellum (P = 0.05) 1 h post-injection. Oxorhenium complexes I and II showed affinity for the 5-HT1A receptor binding sites, with IC50 values in the nanomolar range. The results demonstrate the potential of the mixed ligand approach for the design of 99mTc complexes with the ability to bind neuroreceptors. However, the goal of imaging 5-HT1A receptors with technetium requires further development of complexes with improved biological profiles. (author)

  2. Preliminary Molecular Dynamic Simulations of the Estrogen Receptor Alpha Ligand Binding Domain from Antagonist to Apo

    Directory of Open Access Journals (Sweden)

    Adrian E. Roitberg

    2008-06-01

    Full Text Available Estrogen receptors (ER are known as nuclear receptors. They exist in the cytoplasm of human cells and serves as a DNA binding transcription factor that regulates gene expression. However the estrogen receptor also has additional functions independent of DNA binding. The human estrogen receptor comes in two forms, alpha and beta. This work focuses on the alpha form of the estrogen receptor. The ERα is found in breast cancer cells, ovarian stroma cells, endometrium, and the hypothalamus. It has been suggested that exposure to DDE, a metabolite of DDT, and other pesticides causes conformational changes in the estrogen receptor. Before examining these factors, this work examines the protein unfolding from the antagonist form found in the 3ERT PDB crystal structure. The 3ERT PDB crystal structure has the estrogen receptor bound to the cancer drug 4-hydroxytamoxifen. The 4-hydroxytamoxifen ligand was extracted before the simulation, resulting in new conformational freedom due to absence of van der Waals contacts between the ligand and the receptor. The conformational changes that result expose the binding clef of the co peptide beside Helix 12 of the receptor forming an apo conformation. Two key conformations in the loops at either end of the H12 are produced resulting in the antagonist to apo conformation transformation. The results were produced over a 42ns Molecular Dynamics simulation using the AMBER FF99SB force field.

  3. Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

    Energy Technology Data Exchange (ETDEWEB)

    Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai; Tao, Yong-guang; Tolbert, W. David; Simons, Jr., S. Stoney; Xu, H. Eric (NIH)

    2010-03-08

    A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

  4. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    OpenAIRE

    Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

    2016-01-01

    The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internali...

  5. Receptor activator of nuclear factor kappa B ligand and osteoprotegerin levels in gingival crevicular fluid

    OpenAIRE

    Fatemeh Sarlati; Mandana Sattari; Shilan Razzaghi; Malihe Nasiri

    2012-01-01

    Background: Osteoclastogenesis is coordinated by the interaction of three members of the tumor necrosis factor (TNF) superfamily: Osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL)/receptor activator of nuclear factor kappa B (RANK). The aim of this study was to investigate RANKL and OPG levels, and their relative ratio in gingival crevicular fluid (GCF) of patients with chronic and aggressive periodontitis, as well as healthy controls. Materials and Methods:...

  6. DMPD: Nucleic acid-sensing Toll-like receptors: beyond ligand search. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18321608 Nucleic acid-sensing Toll-like receptors: beyond ligand search. Miyake K. ...Adv Drug Deliv Rev. 2008 Apr 29;60(7):782-5. Epub 2008 Feb 15. (.png) (.svg) (.html) (.csml) Show Nucleic ac...id-sensing Toll-like receptors: beyond ligand search. PubmedID 18321608 Title Nucleic acid-sensing Toll-like

  7. NFkappaB Selectivity of Estrogen Receptor Ligands Revealed By Comparative Crystallographic Analyses

    Energy Technology Data Exchange (ETDEWEB)

    Nettles, K.W.; Bruning, J.B.; Gil, G.; Nowak, J.; Sharma, S.K.; Hahm, J.B.; Kulp, K.; Hochberg, R.B.; Zhou, H.; Katzenellenbogen, J.A.; Katzenllenbogen, B.S.; Kim, Y.; Joachmiak, A.; Greene, G.L.

    2009-05-22

    Our understanding of how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems that are common to steroid hormone receptors are circumvented by mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the structure of an apo steroid receptor that reveals a ligand-accessible channel allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NF{kappa}B-selective signaling through the estrogen receptor, thus revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define structural features of signaling specificity that would not be apparent in a single structure.

  8. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    Science.gov (United States)

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. PMID:25149566

  9. Peroxisome proliferator-activated receptor γ ligands regulate neural stem cell proliferation and differentiation in vitro and in vivo

    OpenAIRE

    Morales-García, José A.; Luna Medina, Rosario de; Alfaro-Cervello, Clara; Cortés-Canteli, Marta; Santos, Ángel; García-Verdugo, J. M.; Pérez Castillo, Ana

    2011-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated nuclear receptors and its ligands are known to control many physiological and pathological situations. Its role in the central nervous system has been under intense analysis during the last years. Here we show a novel function for PPARγ in controlling stem cell expansion in the adult mammalian brain. Adult rats treated with pioglitazone, a specific ligand of PPARγ, had elevated numbers of prolifer...

  10. Imaging GABAc Receptors with Ligand-Conjugated Quantum Dots

    Directory of Open Access Journals (Sweden)

    Ian D. Tomlinson

    2007-01-01

    Full Text Available We report a methodology for labeling the GABAc receptor on the surface membrane of intact cells. This work builds upon our earlier work with serotonin-conjugated quantum dots and our studies with PEGylated quantum dots to reduce nonspecific binding. In the current approach, a PEGylated derivative of muscimol was synthesized and attached via an amide linkage to quantum dots coated in an amphiphilic polymer derivative of a modified polyacrylamide. These conjugates were used to image GABAC receptors heterologously expressed in Xenopus laevis oocytes.

  11. Stochastic description of the ligand-receptor interaction of biologically active substances at extremely low doses.

    Science.gov (United States)

    Gurevich, Konstantin G; Agutter, Paul S; Wheatley, Denys N

    2003-04-01

    Signalling molecules can be effective at extraordinarily low concentrations (down to attomolar levels). To handle such cases, probabilistic methods have been used to describe the formal kinetics of action of biologically active substances in these low doses, although it has been necessary to review what is meant by such a term. The mean numbers of transformed/degraded molecules and their dispersions were calculated for the possible range of ligand-receptor binding schemes. We used both analytical equations and numerical simulations to calculate the coefficients of variation (ratio of standard deviation to mean) and demonstrated that the distribution of the coefficient is highly dependent on the reaction scheme. It may, therefore, be used as an additional factor for discriminating between cooperative and noncooperative models of ligand-receptor interaction over extreme ranges of ligand dilution. The relevance to signalling behaviour is discussed.

  12. Major advances in the development of histamine H4 receptor ligands.

    Science.gov (United States)

    Smits, Rogier A; Leurs, Rob; de Esch, Iwan J P

    2009-08-01

    The search for new and potent histamine H4 receptor ligands is leading to a steadily increasing number of scientific publications and patent applications. Several interesting and structurally diverse compounds have been found, but fierce IP competition for a preferred 2-aminopyrimidine scaffold is becoming apparent. Recent investigations into the role of the histamine H(4)R in (patho)physiology and the use of H4R ligands in in vivo disease models reveal enormous potential in the field of inflammation and allergy, among others. The development of ligands that display activity at two or more histamine receptor (HR) subtypes is another clinical opportunity that is currently being explored. Taken together, the histamine H4R field is gearing up for clinical studies and has the potential to deliver another generation of blockbuster drugs. PMID:19477292

  13. Ligand binding to G protein-coupled receptors in tethered cell membranes

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud;

    2003-01-01

    G protein-coupled receptors (GPCRs) constitute a large class of seven transmembrane proteins, which bind selectively agonists or antagonists with important consequences for cellular signaling and function. Comprehension of the molecular details of ligand binding is important for the understanding...

  14. Oestrogen receptor β ligand: a novel treatment to enhance endogenous functional remyelination

    OpenAIRE

    Crawford, Daniel K.; Mangiardi, Mario; Song, Bingbing; Patel, Rhusheet; Du, Sienmi; Michael V Sofroniew; Voskuhl, Rhonda R; Tiwari-Woodruff, Seema K.

    2010-01-01

    Demyelinating diseases, such as multiple sclerosis, are characterized by inflammatory demyelination and neurodegeneration of the central nervous system. Therapeutic strategies that induce effective neuroprotection and enhance intrinsic repair mechanisms are central goals for future therapy of multiple sclerosis. Oestrogens and oestrogen receptor ligands are promising treatments to prevent multiple sclerosis-induced neurodegeneration. In the present study we investigated the capacity of oestro...

  15. REACTIVITY PROFILE OF LIGANDS OF MAMMALIAN RETINOIC ACID RECEPTORS: A PRELIMINARY COREPA ANALYSIS

    Science.gov (United States)

    Retinoic acid and associated derivatives comprise a class of endogenous hormones that bind to and activate different families of retinoic acid receptors (RARs, RXRs), and control many aspects of vertebrate development. Identification of potential RAR and RXR ligands is of interes...

  16. Tension-compression asymmetry in the binding affinity of membrane-anchored receptors and ligands

    Science.gov (United States)

    Xu, Guang-Kui; Liu, Zishun; Feng, Xi-Qiao; Gao, Huajian

    2016-03-01

    Cell adhesion plays a crucial role in many biological processes of cells, e.g., immune responses, tissue morphogenesis, and stem cell differentiation. An essential problem in the molecular mechanism of cell adhesion is to characterize the binding affinity of membrane-anchored receptors and ligands under different physiological conditions. In this paper, a theoretical model is presented to study the binding affinity between a large number of anchored receptors and ligands under both tensile and compressive stresses, and corroborated by demonstrating excellent agreement with Monte Carlo simulations. It is shown that the binding affinity becomes lower as the magnitude of the applied stress increases, and drops to zero at a critical tensile or compressive stress. Interestingly, the critical compressive stress is found to be substantially smaller than the critical tensile stress for relatively long and flexible receptor-ligand complexes. This counterintuitive finding is explained by using the Euler instability theory of slender columns under compression. The tension-compression asymmetry in the binding affinity of anchored receptors and ligands depends subtly on the competition between the breaking and instability of their complexes. This study helps in understanding the role of mechanical forces in cell adhesion mediated by specific binding molecules.

  17. The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120)

    DEFF Research Database (Denmark)

    Hudson, Brian D; Shimpukade, Bharat; Milligan, Graeme;

    2014-01-01

    The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands ...

  18. Unnatural amino acids as probes of ligand-receptor interactions and their conformational consequences

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Ahern, Christopher A

    2013-01-01

    -edge synthetic and chemical biological approaches. Here we summarize recent advances in the use of site-directed incorporation of unnatural amino acids and chemical probes to study ligand-receptor interactions, determine the location of binding sites, and examine the downstream conformational consequences...

  19. Elimination of a ligand gating site generates a supersensitive olfactory receptor

    Science.gov (United States)

    Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I.

    2016-01-01

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors. PMID:27323929

  20. In Silico Docking of HNF-1a Receptor Ligands

    Directory of Open Access Journals (Sweden)

    Gumpeny Ramachandra Sridhar

    2012-01-01

    Full Text Available Background. HNF-1a is a transcription factor that regulates glucose metabolism by expression in various tissues. Aim. To dock potential ligands of HNF-1a using docking software in silico. Methods. We performed in silico studies using HNF-1a protein 2GYP·pdb and the following softwares: ISIS/Draw 2.5SP4, ARGUSLAB 4.0.1, and HEX5.1. Observations. The docking distances (in angstrom units: 1 angstrom unit (Å = 0.1 nanometer or  metres with ligands in decreasing order are as follows: resveratrol (3.8 Å, aspirin (4.5 Å, stearic acid (4.9 Å, retinol (6.0 Å, nitrazepam (6.8 Å, ibuprofen (7.9 Å, azulfidine (9.0 Å, simvastatin (9.0 Å, elaidic acid (10.1 Å, and oleic acid (11.6 Å. Conclusion. HNF-1a domain interacted most closely with resveratrol and aspirin

  1. Ligand-based receptor tyrosine kinase partial agonists: New paradigm for cancer drug discovery?

    Science.gov (United States)

    Riese, David J.

    2010-01-01

    Introduction Receptor tyrosine kinases (RTKs) are validated targets for oncology drug discovery and several RTK antagonists have been approved for the treatment of human malignancies. Nonetheless, the discovery and development of RTK antagonists has lagged behind the discovery and development of agents that target G-protein coupled receptors. In part, this is because it has been difficult to discover analogs of naturally-occurring RTK agonists that function as antagonists. Areas covered Here we describe ligands of ErbB receptors that function as partial agonists for these receptors, thereby enabling these ligands to antagonize the activity of full agonists for these receptors. We provide insights into the mechanisms by which these ligands function as antagonists. We discuss how information concerning these mechanisms can be translated into screens for novel small molecule- and antibody-based antagonists of ErbB receptors and how such antagonists hold great potential as targeted cancer chemotherapeutics. Expert opinion While there have been a number of important key findings into this field, the identification of the structural basis of ligand functional specificity is still of the greatest importance. While it is true that, with some notable exceptions, peptide hormones and growth factors have not proven to be good platforms for oncology drug discovery; addressing the fundamental issues of antagonistic partial agonists for receptor tyrosine kinases has the potential to steer oncology drug discovery in new directions. Mechanism based approaches are now emerging to enable the discovery of RTK partial agonists that may antagonize both agonist-dependent and –independent RTK signaling and may hold tremendous promise as targeted cancer chemotherapeutics. PMID:21532939

  2. Design and synthesis of carborane-containing estrogen receptor-beta (ERβ)-selective ligands.

    Science.gov (United States)

    Ohta, Kiminori; Ogawa, Takumi; Oda, Akifumi; Kaise, Asako; Endo, Yasuyuki

    2015-10-01

    Candidates for highly selective estrogen receptor-beta (ERβ) ligands (6a-c, 7a-c, 8a and 8b) were designed and synthesized based on carborane-containing ER ligands 1 and 2 as lead compounds. Among them, p-carboranylcyclohexanol derivatives 8a and 8b exhibited high ERβ selectivity in competitive binding assay: for example, 8a showed 56-fold selectivity for ERβ over ERα. Docking studies of 8a and 8b with the ERα and ERβ ligand-binding domains (LBDs) suggested that the p-carborane cage of the ligands is located close to key amino acid residues that influence ER-subtype selectivity, that is, Leu384 in the ERα LBD and Met336 in the ERβ LBD. The p-carborane cage in 8a and 8b appears to play a crucial role in the increased ERβ selectivity. PMID:26298498

  3. A python-based docking program utilizing a receptor bound ligand shape: PythDock.

    Science.gov (United States)

    Chung, Jae Yoon; Cho, Seung Joo; Hah, Jung-Mi

    2011-09-01

    PythDock is a heuristic docking program that uses Python programming language with a simple scoring function and a population based search engine. The scoring function considers electrostatic and dispersion/repulsion terms. The search engine utilizes a particle swarm optimization algorithm. A grid potential map is generated using the shape information of a bound ligand within the active site. Therefore, the searching area is more relevant to the ligand binding. To evaluate the docking performance of PythDock, two well-known docking programs (AutoDock and DOCK) were also used with the same data. The accuracy of docked results were measured by the difference of the ligand structure between x-ray structure, and docked pose, i.e., average root mean squared deviation values of the bound ligand were compared for fourteen protein-ligand complexes. Since the number of ligands' rotational flexibility is an important factor affecting the accuracy of a docking, the data set was chosen to have various degrees of flexibility. Although PythDock has a scoring function simpler than those of other programs (AutoDock and DOCK), our results showed that PythDock predicted more accurate poses than both AutoDock4.2 and DOCK6.2. This indicates that PythDock could be a useful tool to study ligand-receptor interactions and could also be beneficial in structure based drug design.

  4. Tools for investigating functional interactions between ligands and G-protein-coupled receptors.

    Science.gov (United States)

    Lerner, M R

    1994-04-01

    A general assay for evaluating functional interactions between ligands and G-protein-coupled receptors within minutes has been developed. The system uses the principles employed by animals such as reptiles, amphibians and fish to control their colors. In nature, activation of G-protein-coupled receptors expressed by skin cells called chromatophores effects pigment redistribution within the cells to change an animal's coloration. The in vitro 'chameleon in a dish' equivalent can use essentially any cloned G-protein-coupled receptor. PMID:7517590

  5. Labelling of central neural system receptor ligands with the fac-[Tc(CO)3]+ moiety

    International Nuclear Information System (INIS)

    During the period of the IAEA Co-ordinated Research Project on Development of Agents for the Imaging of CNS Receptors based on 99mTc, many efforts were made to find an improved system or alternative methods for the labelling of various central nervous system (CNS) receptor binding agents based on the fac-[Tc(CO)3]+ fragment. Within the same period the chemistry of the fac-[Tc(CO)3]+ fragment has been developed as a useful label more and more not only for the labelling of CNS receptor ligands but also for peptides, antibodies and other biologically active molecules such as B12. Especially the latter molecule is known to be taken up as well through the blood-brain barrier but is obviously not an CNS receptor ligand. One of the most important achievements over the whole period of the project has been the final formulation of a kit useful for the preparation of [99mTc(OH2)3(CO)3]+ without the requirement for using free CO. Much time was invested in that particular topic, since it will allow this relevant moiety to be applied not only on a routine basis but also for research into CNS ligands. A major achievement has thus been the commercial availability of these kits by the beginning of 2002. During the period of the project, a number of new systems were introduced, some of which were specially designed not only for CNS receptor ligands but also for other biomolecules. Among these is that for the syntheses of highly lipophilic ligands, the complex formation of which is based on classical co-ordination chemistry. In addition, the feasibility of the mixed ligand concept from a chemical point of view has been proved in principle. A number of complexes have been prepared where the CNS receptor ligand is attached to the monodentate ligand system. In principle it can also be attached to the bidentate moiety, allowing a screening of the biological behaviour as a function of the co-ligand. A major breakthrough could be achieved with the aqueous synthesis of cymantren

  6. Derivatives of serotonergic receptors ligands labeled with SPECT radionuclide for neutronal imaging

    International Nuclear Information System (INIS)

    Full text: Introduction: Serotonergic receptors are associated with a variety of pathophysiology of neuropsychiatric disorders. Serotonergic ligands have remained a very active area in the development of CNS drugs. In search of the ligands that recognize serotonergic receptor we have synthesized derivatives of methoxyphenylpiperazine. Long chain alkylation of methoxyphenylpiperazine was successfully carried out and a series of MPP based precursors were obtained which comprised of hydrocarbon chain of varied length. These derivatives were then conjugated to acyclic chelating system and efficiently labeled with SPECT radionuclide. Materials and Methods: Labeling was performed with high yield (>95%) and radiochemical purity (>98%) using very low ligand concentration. In vivo studies were done on Hela cell lines which overexpress serotonergic receptors. Further studies done includes in vivo distribution and gamma scintigraphy performed in rat and rabbit. Results: All the intermediates and final compounds were characterized by 1H, 13C NMR and Mass Spectroscopy. In vitro binding assays in rat hippocampal cultures demonstrated the high affinity of complexes for serotonergic receptors. Conclusion: We have optimized the synthesis of 2-methoxyphenylpiperazine based chelating agents. This series of imaging agents holds a promising future in imaging 5-HT receptors for the effective treatment of neuropathological disorders

  7. Structure of complement receptor 2 in complex with its C3d ligand.

    Science.gov (United States)

    Szakonyi, G; Guthridge, J M; Li, D; Young, K; Holers, V M; Chen, X S

    2001-06-01

    Complement receptor 2 (CR2/CD21) is an important receptor that amplifies B lymphocyte activation by bridging the innate and adaptive immune systems. CR2 ligands include complement C3d and Epstein-Barr virus glycoprotein 350/220. We describe the x-ray structure of this CR2 domain in complex with C3d at 2.0 angstroms. The structure reveals extensive main chain interactions between C3d and only one short consensus repeat (SCR) of CR2 and substantial SCR side-side packing. These results provide a detailed understanding of receptor-ligand interactions in this protein family and reveal potential target sites for molecular drug design. PMID:11387479

  8. Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands.

    Science.gov (United States)

    Naydenova, Emilia D; Todorov, Petar T; Mateeva, Polina I; Zamfirova, Rositza N; Pavlov, Nikola D; Todorov, Simeon B

    2010-11-01

    The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH2, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis--Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5-8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.

  9. GABA Acts as a Ligand Chaperone in the Early Secretory Pathway to Promote Cell Surface Expression of GABAA Receptors

    OpenAIRE

    Eshaq, Randa S.; Stahl, Letha D.; Stone, Randolph; Smith, Sheryl S.; Robinson, Lucy C.; Leidenheimer, Nancy J.

    2010-01-01

    GABA (γ-aminobutyric acid) is the primary inhibitory neurotransmitter in brain. The fast inhibitory effect of GABA is mediated through the GABAA receptor, a postsynaptic ligand-gated chloride channel. We propose that GABA can act as a ligand chaperone in the early secretory pathway to facilitate GABAA receptor cell surface expression. Forty-two hrs of GABA treatment increased the surface expression of recombinant receptors expressed in HEK 293 cells, an effect accompanied by an increase in GA...

  10. A strategy using NMR peptide structures of thromboxane A2 receptor as templates to construct ligand-recognition pocket of prostacyclin receptor

    Directory of Open Access Journals (Sweden)

    Ruan Ke-He

    2005-11-01

    Full Text Available Abstract Background: Prostacyclin receptor (IP and thromboxane A2 receptor (TP belong to rhodopsin-type G protein-coupling receptors and respectively bind to prostacyclin and thromboxane A2 derived from arachidonic acid. Recently, we have determined the extracellular loop (eLP structures of the human TP receptor by 2-D 1H NMR spectroscopy using constrained peptides mimicking the individual eLP segments. The studies have identified the segment along with several residues in the eLP domains important to ligand recognition, as well as proposed a ligand recognition pocket for the TP receptor. Results: The IP receptor shares a similar primary structure in the eLPs with those of the TP receptor. Forty percent residues in the second eLPs of the receptors are identical, which is the major region involved in forming the ligand recognition pocket in the TP receptor. Based on the high homology score, the eLP domains of the IP receptor were constructed by the homology modeling approach using the NMR structures of the TP eLPs as templates, and then configured to the seven transmembrane (TM domains model constructed using the crystal structure of the bovine rhodopsin as a template. A NMR structure of iloprost was docked into the modeled IP ligand recognition pocket. After dynamic studies, the segments and residues involved in the IP ligand recognition were proposed. A key residue, Arg173 involved in the ligand recognition for the IP receptor, as predicted from the modeling, was confirmed by site-directed mutagenesis. Conclusion: A 3-D model of the human IP receptor was constructed by homology modeling using the crystal structure of bovine rhodopsin TM domains and the NMR structures of the synthetic constrained peptides of the eLP domains of the TP receptor as templates. This strategy can be applied to molecular modeling and the prediction of ligand recognition pockets for other prostanoid receptors.

  11. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    International Nuclear Information System (INIS)

    Research highlights: → Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. → PXR undergoes dynamic deacetylation upon ligand-mediated activation. → SIRT1 partially mediates PXR deacetylation. → PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  12. Ligand-stimulated downregulation of the alpha interferon receptor: role of protein kinase D2.

    Science.gov (United States)

    Zheng, Hui; Qian, Juan; Varghese, Bentley; Baker, Darren P; Fuchs, Serge

    2011-02-01

    Alpha interferon (IFN-α) controls homeostasis of hematopoietic stem cells, regulates antiviral resistance, inhibits angiogenesis, and suppresses tumor growth. This cytokine is often used to treat cancers and chronic viral infections. The extent of cellular responses to IFN-α is limited by the IFN-induced ubiquitination and degradation of the IFN-α/β receptor chain 1 (IFNAR1) chain of the cognate receptor. IFNAR1 ubiquitination is facilitated by the βTrcp E3 ubiquitin ligase that is recruited to IFNAR1 upon its degron phosphorylation, which is induced by the ligand. Here we report identification of protein kinase D2 (PKD2) as a kinase that mediates the ligand-inducible phosphorylation of IFNAR1 degron and enables binding of βTrcp to the receptor. Treatment of cells with IFN-α induces catalytic activity of PKD2 and stimulates its interaction with IFNAR1. Expression and kinase activity of PKD2 are required for the ligand-inducible stimulation of IFNAR1 ubiquitination and endocytosis and for accelerated proteolytic turnover of IFNAR1. Furthermore, inhibition or knockdown of PKD2 robustly augments intracellular signaling induced by IFN-α and increases the efficacy of its antiviral effects. The mechanisms of the ligand-inducible elimination of IFNAR1 are discussed, along with the potential medical significance of this regulation. PMID:21173164

  13. Active regions' setting of the extracellular ligand-binding domain of human interleukin-6 receptor

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The reliable three dimensional (3-D) structure of the extracellular ligand-binding domain (V106-P322) of human interleukin-6 receptor (hIL-6R) has been constructed by means of computer-guided homology modeling techniques using the crystal structure of the extracellular ligand-binding region (K52-L251) of human growth hormone receptor (hGHR) as templet. The space location of some key residues which influence the combination ability between the receptor and the ligand has been observed and the effects of point mutagenesis of the four conservative cysteine residues on the space conformation are analyzed. The results show that the space conformation of the side-chain carboxyl of E305 plays a key role in the ligand-binding ability. Furthermore, the space conformation of the side-chain carboxyl of E305 is very important for the electrostatic potential complementarity between hIL-6R and hIL-6 according to the docking method.

  14. Bile acid derivatives as ligands of the farnesoid x receptor: molecular determinants for bile acid binding and receptor modulation.

    Science.gov (United States)

    Gioiello, Antimo; Cerra, Bruno; Mostarda, Serena; Guercini, Chiara; Pellicciari, Roberto; Macchiarulo, Antonio

    2014-01-01

    Bile acids are a peculiar class of steroidal compounds that never cease to amaze. From being simple detergents with a primary role in aiding the absorption of fats and fat-soluble vitamins, bile acids are now widely considered as crucial hormones endowed with genomic and non-genomic functions that are mediated by their interaction with several proteins including the nuclear receptor Farnesoid X Receptor (FXR). Taking advantages of the peculiar properties of bile acids in interacting with the FXR receptor, several biliary derivatives have been synthesized and tested as FXR ligands. The availability of these compounds has contributed to characterize the receptor from a structural, patho-physiological and therapeutic standpoint. Among these, obeticholic acid is a first-in-class FXR agonist that is demonstrating hepatoprotective effects upon FXR activation in patients with liver diseases such as primary biliary cirrhosis and nonalcoholic steatohepatitis. This review provides an historical overview of the rationale behind the discovery of obeticholic acid and chemical tools generated to depict the molecular features and bio-pharmacological relevance of the FXR receptor, as well as to summarize structure-activity relationships of bile acid-based FXR ligands so far reported. PMID:25388535

  15. Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering.

    Science.gov (United States)

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Studer, Romain A; Alvarez, Susana; de Lera, Angel R; Kuraku, Shigehiro; Bourguet, William; Schubert, Michael; Laudet, Vincent

    2016-03-01

    Whole genome duplications (WGDs) have been classically associated with the origin of evolutionary novelties and the so-called duplication-degeneration-complementation model describes the possible fates of genes after duplication. However, how sequence divergence effectively allows functional changes between gene duplicates is still unclear. In the vertebrate lineage, two rounds of WGDs took place, giving rise to paralogous gene copies observed for many gene families. For the retinoic acid receptors (RARs), for example, which are members of the nuclear hormone receptor (NR) superfamily, a unique ancestral gene has been duplicated resulting in three vertebrate paralogues: RARα, RARβ and RARγ. It has previously been shown that this single ancestral RAR was neofunctionalized to give rise to a larger substrate specificity range in the RARs of extant jawed vertebrates (also called gnathostomes). To understand RAR diversification, the members of the cyclostomes (lamprey and hagfish), jawless vertebrates representing the extant sister group of gnathostomes, provide an intermediate situation and thus allow the characterization of the evolutionary steps that shaped RAR ligand-binding properties following the WGDs. In this study, we assessed the ligand-binding specificity of cyclostome RARs and found that their ligand-binding pockets resemble those of gnathostome RARα and RARβ. In contrast, none of the cyclostome receptors studied showed any RARγ-like specificity. Together, our results suggest that cyclostome RARs cover only a portion of the specificity repertoire of the ancestral gnathostome RARs and indicate that the establishment of ligand-binding specificity was a stepwise event. This iterative process thus provides a rare example for the diversification of receptor-ligand interactions of NRs following WGDs. PMID:27069642

  16. Fluorinated azabicycloesters as muscarinic receptor ligands for application with PET

    International Nuclear Information System (INIS)

    Human muscarinic acetylcholine receptors (MAR) play an important role in a number of physiological and behavioral responses. A correlation has been established between changes in the MAR density and human memory as well as to other specific neurodegenerative disorders such as Huntington's chorea or Alzheimer's dementia. MAR density has been observed, also, to decrease under the effect of several chemical agents such as organophosphorus compounds, barbiturates, ethanol or antidepressants. Most of the studies on human MAR were done on post-mortem samples obtained at autopsy and stored for variable times which may not reflect the actual in vivo status of such receptors. To carry out preliminary in vivo studies, the choice will be directed primarily to experimental animals. However, animal models for many of the neurodegenerative disorders may be inadequate. Several studies showed a dramatically increasing number of dementia cases which is leading to decreased survival among this group. Such a dramatic increase in Alzheimer's dementia cases and the inability to determine the density and distribution of MAR in vivo have stimulated the interest of many researchers to investigate MAR mapping

  17. Structure-Based Evolution of Subtype-Selective Neurotensin Receptor Ligands

    OpenAIRE

    Schaab, Carolin; Kling, Ralf Christian; Einsiedel, Jürgen; Hübner, Harald; Clark, Tim; Seebach, Dieter; Gmeiner, Peter

    2014-01-01

    Subtype-selective agonists of the neurotensin receptor NTS2 represent a promising option for the treatment of neuropathic pain, as NTS2 is involved in the mediation of μ-opioid-independent anti-nociceptive effects. Based on the crystal structure of the subtype NTS1 and previous structure–activity relationships (SARs) indicating a potential role for the sub-pocket around Tyr11 of NT(8–13) in subtype-specific ligand recognition, we have developed new NTS2-selective ligands. Starting from NT(8–1...

  18. Differential ligand-dependent protein–protein interactions between nuclear receptors and a neuronal-specific cofactor

    OpenAIRE

    Greiner, Erich F.; Kirfel, Jutta; Greschik, Holger; Huang, DongYa; Becker, Peter; Kapfhammer, Josef P.; Schüle, Roland

    2000-01-01

    Nuclear receptors are transcription factors that require multiple protein–protein interactions to regulate target gene expression. We have cloned a 27-kDa protein, termed NIX1 (neuronal interacting factor X 1), that directly binds nuclear receptors in vitro and in vivo. Protein–protein interaction between NIX1 and ligand-activated or constitutive active nuclear receptors, including retinoid-related orphan receptor β (RORβ) (NR1F2), strictly depends on the conserved receptor C-terminal activat...

  19. Ligands for SPECT and PET imaging of muscarinic-cholinergic receptors of the heart and brain

    Energy Technology Data Exchange (ETDEWEB)

    Knapp, F.F. Jr.; McPherson, D.W.; Luo, H. [and others

    1995-06-01

    Interest in the potential use of cerebral SPECT and PET imaging for determination of the density and activity of muscarinic-cholinergic receptors (mAChR) has been stimulated by the changes in these receptors which occur in many neurological diseases. In addition, the important involvement of mAChR in modulating negative inotropic cardiac activity suggests that such receptor ligands may have important applications in evaluation of changes which may occur in cardiac disease. In this paper, the properties of several key muscarinic receptor ligands being developed or which have been used for clinical SPECT and PET are discussed. In addition, the ORNL development of the new iodinated IQNP ligand based on QNB and the results of in vivo biodistribution studies in rats, in vitro competitive binding studies and ex vivo autoradiographic experiments are described. The use of radioiodinated IQNP may offer several advantages in comparison to IQNB because of its easy and high yield preparation and high brain uptake and the potential usefulness of the {open_quotes}partial{close_quotes} subtype selective IONP isomers. We also describe the development of new IQNP-type analogues which offer the opportunity for radiolabeling with positron-emitting radioisotopes (carbon-11, fluorine-18 and bromine-76) for potential use with PET.

  20. Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Yongneng; Harrison, Chris B.; Freddolino, Peter L.; Schulten, Klaus; Mayer, Mark L. (UIUC); (NIH)

    2008-10-27

    NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.

  1. Domain architecture of a calcium-permeable AMPA receptor in a ligand-free conformation

    Directory of Open Access Journals (Sweden)

    Charles R. Midgett

    2012-01-01

    Full Text Available Ligand-gated ion channels couple the free energy of agonist binding to the gating of selective transmembrane ion pores, permitting cells to regulate ion flux in response to external chemical stimuli. However, the stereochemical mechanisms responsible for this coupling remain obscure. In the case of the ionotropic glutamate receptors (iGluRs, the modular nature of receptor subunits has facilitated structural analysis of the N-terminal domain (NTD, and of multiple conformations of the ligand-binding domain (LBD. Recently, the crystallographic structure of an antagonist-bound form of the receptor was determined. However, disulfide trapping of this conformation blocks channel opening, suggesting that channel activation involves additional quaternary packing arrangements. To explore the conformational space available to iGluR channels, we report here a second, clearly distinct domain architecture of homotetrameric, calcium-permeable AMPARs, determined by single-particle electron microscopy of untagged and fluorescently tagged constructs in a ligand-free state. It reveals a novel packing of NTD dimers, and a separation of LBD dimers across a central vestibule. In this arrangement, which reconciles diverse functional observations, agonist-induced cleft closure across LBD dimers can be converted into a twisting motion that provides a basis for receptor activation.

  2. International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

    Science.gov (United States)

    Christopoulos, Arthur; Changeux, Jean-Pierre; Catterall, William A; Fabbro, Doriano; Burris, Thomas P; Cidlowski, John A; Olsen, Richard W; Peters, John A; Neubig, Richard R; Pin, Jean-Philippe; Sexton, Patrick M; Kenakin, Terry P; Ehlert, Frederick J; Spedding, Michael; Langmead, Christopher J

    2014-10-01

    Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

  3. ONRLDB--manually curated database of experimentally validated ligands for orphan nuclear receptors: insights into new drug discovery.

    Science.gov (United States)

    Nanduri, Ravikanth; Bhutani, Isha; Somavarapu, Arun Kumar; Mahajan, Sahil; Parkesh, Raman; Gupta, Pawan

    2015-01-01

    Orphan nuclear receptors are potential therapeutic targets. The Orphan Nuclear Receptor Ligand Binding Database (ONRLDB) is an interactive, comprehensive and manually curated database of small molecule ligands targeting orphan nuclear receptors. Currently, ONRLDB consists of ∼11,000 ligands, of which ∼6500 are unique. All entries include information for the ligand, such as EC50 and IC50, number of aromatic rings and rotatable bonds, XlogP, hydrogen donor and acceptor count, molecular weight (MW) and structure. ONRLDB is a cross-platform database, where either the cognate small molecule modulators of a receptor or the cognate receptors to a ligand can be searched. The database can be searched using three methods: text search, advanced search or similarity search. Substructure search, cataloguing tools, and clustering tools can be used to perform advanced analysis of the ligand based on chemical similarity fingerprints, hierarchical clustering, binning partition and multidimensional scaling. These tools, together with the Tree function provided, deliver an interactive platform and a comprehensive resource for identification of common and unique scaffolds. As demonstrated, ONRLDB is designed to allow selection of ligands based on various properties and for designing novel ligands or to improve the existing ones. Database URL: http://www.onrldb.org/.

  4. Ligand-receptor binding kinetics in surface plasmon resonance cells: A Monte Carlo analysis

    CERN Document Server

    Carroll, Jacob; Forsten-Williams, Kimberly; Täuber, Uwe C

    2016-01-01

    Surface plasmon resonance (SPR) chips are widely used to measure association and dissociation rates for the binding kinetics between two species of chemicals, e.g., cell receptors and ligands. It is commonly assumed that ligands are spatially well mixed in the SPR region, and hence a mean-field rate equation description is appropriate. This approximation however ignores the spatial fluctuations as well as temporal correlations induced by multiple local rebinding events, which become prominent for slow diffusion rates and high binding affinities. We report detailed Monte Carlo simulations of ligand binding kinetics in an SPR cell subject to laminar flow. We extract the binding and dissociation rates by means of the techniques frequently employed in experimental analysis that are motivated by the mean-field approximation. We find major discrepancies in a wide parameter regime between the thus extracted rates and the known input simulation values. These results underscore the crucial quantitative importance of s...

  5. The phosphatase domains of CD45 are required for ligand induced T-cell receptor downregulation

    DEFF Research Database (Denmark)

    Kastrup, J; Lauritsen, Jens Peter Holst; Menné, C;

    2000-01-01

    Down-regulation of the T-cell receptor (TCR) plays an important role in modulating T-cell responses, both during T-cell development and in mature T cells. At least two distinct pathways exist for TCR down-regulation: down-regulation following TCR ligation; and down-regulation following activation...... of protein kinase C (PKC). Ligand-induced TCR down-regulation is dependent on protein tyrosine kinase (PTK) activity and seems to be closely related to T-cell activation. In addition, previous studies have indicated that ligand-induced TCR down-regulation is dependent on the expression of CD45, a...... transmembrane protein tyrosine phosphatase. The role of the different domains of CD45 in TCR down-regulation was investigated in this study. We found that the phosphatase domains of CD45 are required for efficient ligand-induced TCR down-regulation. In contrast, the extracellular domain of CD45 is dispensable...

  6. Labeling of receptor ligands with bromine radionuclides. Progress report, March 1, 1981-February 28, 1982

    International Nuclear Information System (INIS)

    In recent years there has been an interest in the use of various radioisotopes of bromine as labels for radiopharmaceuticals. Although radioisotopes of iodine have been used extensively as radiopharmaceutical labels, there are several advantages associated with the use of radiobromine as a label, due primarily to increased stability of bonds to the radiohalide and smaller steric perturbation resulting from substitution of the radiohalide. Methods of attaching radiobromine to receptor ligands with the potential of mapping estrogen receptors in mammary tumors and uteri were studied. Two ligands were studied extensively in vitro and in animal models; preliminary studies were also carried out in humans. To date, the only radioisotope of bromine used was bromine-77. In addition, a series of model compounds were labeled with bromine-77 using a recently described method for rapid bromination; the scope and limitations of this new rapid radiobromination technique were evaluated

  7. Structural Basis for Hydroxycholesterols as Natural Ligands of Orphan Nuclear Receptor ROR[gamma

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Lihua; Martynowski, Dariusz; Zheng, Songyang; Wada, Taira; Xie, Wen; Li, Yong (Pitt); (Xiamen)

    2010-09-03

    The retinoic acid-related orphan receptor {gamma} (ROR{gamma}) has important roles in development and metabolic homeostasis. Although the biological functions of ROR{gamma} have been studied extensively, no ligands for ROR{gamma} have been identified, and no structure of ROR{gamma} has been reported. In this study, we showed that hydroxycholesterols promote the recruitment of coactivators by ROR{gamma} using biochemical assays. We also report the crystal structures of the ROR{gamma} ligand-binding domain bound with hydroxycholesterols. The structures reveal the binding modes of various hydroxycholesterols in the ROR{gamma} pocket, with the receptors all adopting the canonical active conformation. Mutations that disrupt the binding of hydroxycholesterols abolish the constitutive activity of ROR{gamma}. Our observations suggest an important role for the endogenous hydroxycholesterols in modulating ROR{gamma}-dependent biological processes.

  8. Communication: Free energy of ligand-receptor systems forming multimeric complexes

    Science.gov (United States)

    Di Michele, Lorenzo; Bachmann, Stephan J.; Parolini, Lucia; Mognetti, Bortolo M.

    2016-04-01

    Ligand-receptor interactions are ubiquitous in biology and have become popular in materials in view of their applications to programmable self-assembly. Although complex functionalities often emerge from the simultaneous interaction of more than just two linker molecules, state of the art theoretical frameworks enable the calculation of the free energy only in systems featuring one-to-one ligand/receptor binding. In this Communication, we derive a general formula to calculate the free energy of systems featuring simultaneous direct interaction between an arbitrary number of linkers. To exemplify the potential and generality of our approach, we apply it to the systems recently introduced by Parolini et al. [ACS Nano 10, 2392 (2016)] and Halverson and Tkachenko [J. Chem. Phys. 144, 094903 (2016)], both featuring functionalized Brownian particles interacting via three-linker complexes.

  9. The Oligomeric States of the Purified Sigma-1 Receptor Are Stabilized by Ligands*

    Science.gov (United States)

    Gromek, Katarzyna A.; Suchy, Fabian P.; Meddaugh, Hannah R.; Wrobel, Russell L.; LaPointe, Loren M.; Chu, Uyen B.; Primm, John G.; Ruoho, Arnold E.; Senes, Alessandro; Fox, Brian G.

    2014-01-01

    Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[3H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed. PMID:24847081

  10. The oligomeric states of the purified sigma-1 receptor are stabilized by ligands.

    Science.gov (United States)

    Gromek, Katarzyna A; Suchy, Fabian P; Meddaugh, Hannah R; Wrobel, Russell L; LaPointe, Loren M; Chu, Uyen B; Primm, John G; Ruoho, Arnold E; Senes, Alessandro; Fox, Brian G

    2014-07-18

    Sigma-1 receptor (S1R) is a mammalian member of the ERG2 and sigma-1 receptor-like protein family (pfam04622). It has been implicated in drug addiction and many human neurological disorders, including Alzheimer and Parkinson diseases and amyotrophic lateral sclerosis. A broad range of synthetic small molecules, including cocaine, (+)-pentazocine, haloperidol, and small endogenous molecules such as N,N-dimethyltryptamine, sphingosine, and steroids, have been identified as regulators of S1R. However, the mechanism of activation of S1R remains obscure. Here, we provide evidence in vitro that S1R has ligand binding activity only in an oligomeric state. The oligomeric state is prone to decay into an apparent monomeric form when exposed to elevated temperature, with loss of ligand binding activity. This decay is suppressed in the presence of the known S1R ligands such as haloperidol, BD-1047, and sphingosine. S1R has a GXXXG motif in its second transmembrane region, and these motifs are often involved in oligomerization of membrane proteins. Disrupting mutations within the GXXXG motif shifted the fraction of the higher oligomeric states toward smaller states and resulted in a significant decrease in specific (+)-[(3)H]pentazocine binding. Results presented here support the proposal that S1R function may be regulated by its oligomeric state. Possible mechanisms of molecular regulation of interacting protein partners by S1R in the presence of small molecule ligands are discussed. PMID:24847081

  11. Peroxisome proliferator-activated receptor ligands as antiatherogenic agents: panacea or another Pandora's box?

    Science.gov (United States)

    Molavi, Behzad; Rasouli, Neda; Mehta, Jawahar L

    2002-01-01

    Peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor super family that modulate gene expression upon ligand activation. They are 3 major subtypes of PPARs: alpha, delta (also called beta), and gamma. PPAR-gamma is widely expressed in the cardiovascular system and is involved in the regulation of tissue inflammation and smooth muscle cell growth pathways as well as in lipoprotein metabolism and coagulation cascades. PPAR-gamma ligands of (e.g., rosigitazone and pioglitazone) have been shown to exert antiatherogenic effects both in vitro and in vivo. PPAR-alpha ligands (e.g., clofibrate and benzofibrate) modulate lipoprotein metabolism, and affect inflammation and coagulation cascade. These effects may be helpful in resolving the dilemma arising from studies that showed significant mortality and morbidity benefits of fibrates in the face of minimal changes in HDL-cholesterol levels. The role of PPAR-delta in atherogenesis remains largely unknown, although it appears that PPAR-delta activation affects lipoprotein metabolism. PPAR ligands appear to be promising agents in limiting atherosclerosis; however, large-scale clinical trials are required to assess their safety and efficacy before they can be added to the clinicians' arsenal of antiatherosclerotic agents. PMID:12000972

  12. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation.

    Science.gov (United States)

    Faria, Jerusa A Q A; de Andrade, Carolina; Goes, Alfredo M; Rodrigues, Michele A; Gomes, Dawidson A

    2016-09-01

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. PMID:27462018

  13. Effects of coumestrol on lipid and glucose metabolism as a farnesoid X receptor ligand

    International Nuclear Information System (INIS)

    In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4α. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism

  14. Differential effects of EGFR ligands on endocytic sorting of the receptor

    DEFF Research Database (Denmark)

    Roepstorff, Kirstine; Grandal, Michael Vibo; Henriksen, Lasse;

    2009-01-01

    Endocytic downregulation is a pivotal mechanism turning off signalling from the EGF receptor (EGFR). It is well established that whereas EGF binding leads to lysosomal degradation of EGFR, transforming growth factor (TGF)-alpha causes receptor recycling. TGF-alpha therefore leads to continuous...... recycling. EGF leads to lysosomal degradation of the majority but not all EGFRs. Amphiregulin does not target EGFR for lysosomal degradation but causes fast as well as slow EGFR recycling. The Cbl ubiquitin ligases, especially c-Cbl, are responsible for EGFR ubiquitination after stimulation with all ligands...

  15. Ligand-induced Coupling versus Receptor Pre-association: Cellular automaton simulations of FGF-2 binding

    OpenAIRE

    Gopalakrishnan, Manoj; Forsten-Williams, Kimberly; Tauber, Uwe C.

    2003-01-01

    The binding of basic fibroblast growth factor (FGF-2) to its cell surface receptor (CSR) and subsequent signal transduction is known to be enhanced by Heparan Sulfate Proteoglycans (HSPGs). HSPGs bind FGF-2 with low affinity and likely impact CSR-mediated signaling via stabilization of FGF-2-CSR complexes via association with both the ligand and the receptor. What is unknown is whether HSPG associates with CSR in the absence of FGF-2. In this paper, we determine conditions by which pre-associ...

  16. Structures of pattern recognition receptors reveal molecular mechanisms of autoinhibition, ligand recognition and oligomerization.

    Science.gov (United States)

    Chuenchor, Watchalee; Jin, Tengchuan; Ravilious, Geoffrey; Xiao, T Sam

    2014-02-01

    Pattern recognition receptors (PRRs) are essential sentinels for pathogens or tissue damage and integral components of the innate immune system. Recent structural studies have provided unprecedented insights into the molecular mechanisms of ligand recognition and signal transduction by several PRR families at distinct subcellular compartments. Here we highlight some of the recent discoveries and summarize the common themes that are emerging from these exciting studies. Better mechanistic understanding of the structure and function of the PRRs will improve future prospects of therapeutic targeting of these important innate immune receptors.

  17. High-throughput screening assay for new ligands at human melatonin receptors

    Institute of Scientific and Technical Information of China (English)

    Jian-hua YAN; Hao-ran SU; Jean A BOUTIN; M Pierre RENARD; Ming-wei WANG

    2008-01-01

    Aim: Melatonin (MT) is a neurohormone produced and secreted primarily by the pineal gland in a circadian manner, and mainly acta through 2 receptor subtypes: MT1 and MT2 in humans. The diversity in their tissue distribution is in favor of different functions for each receptor subtype. Selective modulators are therefore required to determine the physiological roles of these melatonin receptor sub-types and their implications in pathological processes. Methods: A homogenous MT1/MT2 receptor binding assay was established for high-throughput screening of new ligands at the hMT1 and/or hMT2 receptors. The functional properties (agonists or antagonists) were assessed by a conventional guanosine-5'[γ-35S] triphosphate (GTP-γS) assay. Results: Three hMT, receptor-selective small mol-ecule antagonists and 1 hMT2 receptor-selective small molecule antagonist with novel structural features were identified following a high-throughput screening campaign of 48 240 synthetic and natural compounds. Conclusion: The findings may assist in the expansion of chemical probes to these 2 receptor subtypes.

  18. The Prelude on Novel Receptor and Ligand Targets Involved in the Treatment of Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Venu Gopal Jonnalagadda

    2014-05-01

    Full Text Available Metabolic disorders are a group of disorders, due to the disruption of the normal metabolic process at a cellular level. Diabetes Mellitus and Tyrosinaemia are the majorly reported metabolic disorders. Among them, Diabetes Mellitus is a one of the leading metabolic syndrome, affecting 5 to 7 % of the population worldwide and mainly characterised by elevated levels of glucose and is associated with two types of physiological event disturbances such as impaired insulin secretion and insulin resistance. Up to now, various treatment strategies are like insulin, alphaglucosidase inhibitors, biguanides, incretins were being followed. Concurrently, various novel therapeutic strategies are required to advance the therapy of Diabetes mellitus. For the last few decades, there has been an extensive research in understanding the metabolic pathways involved in Diabetes Mellitus at the cellular level and having the profound knowledge on cell-growth, cell-cycle, and apoptosis at a molecular level provides new targets for the treatment of Diabetes Mellitus. Receptor signalling has been involved in these mechanisms, to translate the information coming from outside. To understand the various receptors involved in these pathways, we must have a sound knowledge on receptors and ligands involved in it. This review mainly summarises the receptors and ligands which are involved the Diabetes Mellitus. Finally, researchers have to develop the alternative chemical moieties that retain their affinity to receptors and efficacy. Diabetes Mellitus being a metabolic disorder due to the glucose surfeit, demands the need for regular exercise along with dietary changes.

  19. HER2-mediated anticancer drug delivery: strategies to prepare targeting ligands highly specific for the receptor.

    Science.gov (United States)

    Calce, Enrica; Monfregola, Luca; Saviano, Michele; De Luca, Stefania

    2015-01-01

    HER2 receptor, for its involvement in tumorigenesis, has been largely studied as topic in cancer research. In particular, the employment of trastuzumab (Herceptin), a humanized anti-HER2 antibody, showed several clinical benefits in the therapy against the breast cancer. Moreover, for its accessible extracellular domain, this receptor is considered an ideal target to deliver anticancer drugs for the receptormediated anticancer therapy. By now, monoclonal antibody and its fragments, affibody, and some peptides have been employed as targeting agents in order to deliver various drugs to HER2 positive tumor cells. In particular, the ability to perform a fast and reliable screening of a large number of peptide molecules would make possible the selection of highly specific compounds to the receptor target. In this regard, the availability of preparing a simplified synthetic model which is a good mimetic of the receptor target and can be used in a reliable screening method of ligands would be of a strategic importance for the development of selective HER2-targeting peptide molecules. Herein, we illustrate the importance of HER2-targeted anticancer therapies. We also report on a synthetic and effective mimetic of the receptor, which revealed to be a useful tool for the selection of specific HER2 ligands. PMID:25994863

  20. Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J;

    1995-01-01

    We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA, and cyto......We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA......, and cytosolic progesterone receptors (PR) measured by LBA were also studied. While ERc concentrations determined by LBA and EIA were highly correlated (r: 0.94), ERc values detected by LBA were approximately twice those found by EIA (median values of ERc: 155 vs. 64 fmol/mg cytosol protein, DCC vs. EIA......). The percentages of ERc positive tumors were 89% by LBA and 77% by EIA. The median fraction of total ER present as ERn was 63%. PR levels correlated positively with ERn concentrations (r: 0.73). We explore possible reasons why greater concentrations of ERc are determined by estradiol binding than by the ER-EIA kit...

  1. Synthesis and radiofluorination of putative NMDA receptor ligands

    International Nuclear Information System (INIS)

    In the course of this work on the synthesis of radioligands for the NMDA receptor the authentic standards and labeling precursors of four compounds with an amidine structure was performed. Synthesis of the precursors followed reaction conditions given in the literature and was successful. The imidoesters used for the synthesis were obtained from their nitriles in a Pinner synthesis, while 2-hydroxybenzylamine was synthesized in a reduction of 2-hydroxybenzonitrile using borane as a reducing agent. After a coupling reaction of the amine and the imidoester in DMF using triethylamine as base the precursors were obtained in good yields and purified by crystallization from methanol. The cyclic standard compound was synthesized directly from 2-(bromomethyl)- benzonitrile and 2-hydroxybenzylamine in a ring closing reaction. Similar to the other precursors, crystallization from methanol produced a pure compound. The authentic standards were synthesized starting from salicylaldehyde. In a four step synthesis the desired ortho-fluoroethoxybenzylamine was obtained in good yield. Coupling of the amine with the respective imidoester or in the case of the cyclic compound 2-(bromomethyl)-benzonitrile gave the desired product which was then purified by column chromatography or by crystallization from ethanol and water. For the labeling procedure 1-bromo-2-[18F]fluoroethane was synthesized following a previously published pathway starting from 1,2-dibromoethane. An alternative route of radiosynthesis for this prosthetic group was tested using ethyleneglycole- 1,2-ditosylate. The labeling reaction was performed on one of the precursors testing both DMF and DMSO as solvents and using NaOH as base. Yields of N-(2-fluoroethoxybenzyl)- cinnamamidine were about 78 % at 80 C after 30 minutes in DMSO. The desired product can now be synthesized in sufficient yields for in vitro and in vivo evaluation studies. Labeling on the cyclic precursor was attempted utilizing DMSO as solvent, but no

  2. Synthesis and radiofluorination of putative NMDA receptor ligands

    Energy Technology Data Exchange (ETDEWEB)

    Kronenberg, U.

    2011-01-15

    In the course of this work on the synthesis of radioligands for the NMDA receptor the authentic standards and labeling precursors of four compounds with an amidine structure was performed. Synthesis of the precursors followed reaction conditions given in the literature and was successful. The imidoesters used for the synthesis were obtained from their nitriles in a Pinner synthesis, while 2-hydroxybenzylamine was synthesized in a reduction of 2-hydroxybenzonitrile using borane as a reducing agent. After a coupling reaction of the amine and the imidoester in DMF using triethylamine as base the precursors were obtained in good yields and purified by crystallization from methanol. The cyclic standard compound was synthesized directly from 2-(bromomethyl)- benzonitrile and 2-hydroxybenzylamine in a ring closing reaction. Similar to the other precursors, crystallization from methanol produced a pure compound. The authentic standards were synthesized starting from salicylaldehyde. In a four step synthesis the desired ortho-fluoroethoxybenzylamine was obtained in good yield. Coupling of the amine with the respective imidoester or in the case of the cyclic compound 2-(bromomethyl)-benzonitrile gave the desired product which was then purified by column chromatography or by crystallization from ethanol and water. For the labeling procedure 1-bromo-2-[{sub 18}F]fluoroethane was synthesized following a previously published pathway starting from 1,2-dibromoethane. An alternative route of radiosynthesis for this prosthetic group was tested using ethyleneglycole- 1,2-ditosylate. The labeling reaction was performed on one of the precursors testing both DMF and DMSO as solvents and using NaOH as base. Yields of N-(2-fluoroethoxybenzyl)- cinnamamidine were about 78 % at 80 C after 30 minutes in DMSO. The desired product can now be synthesized in sufficient yields for in vitro and in vivo evaluation studies. Labeling on the cyclic precursor was attempted utilizing DMSO as solvent

  3. Natural ligands of nuclear receptors. Isolation, design, synthesis, biochemical decodification and potential therapeutic applications.

    OpenAIRE

    Ummarino, Raffaella

    2013-01-01

    Natural products have historically been a rich source of lead compounds in drug discovery. The biochemical investigation of marine organisms, through the deep collaboration between chemists and pharmacologists, focused on searching of new biologically active compounds, is a central issue of this kind of studies. My research work, described in this PhD thesis, has been developed in this research area and was addressed to the identification of new ligands of nuclear receptors, discovering ...

  4. Arrest functions of the MIF ligand/receptor axes in atherogenesis

    Directory of Open Access Journals (Sweden)

    Sabine eTillmann

    2013-05-01

    Full Text Available Macrophage migration inhibitory factor (MIF has been defined as an important chemokine-like function (CLF chemokine with an essential role in monocyte recruitment and arrest. Adhesion of monocytes to the vessel wall and their transendothelial migration are critical in atherogenesis and many other inflammatory diseases. Chemokines carefully control all steps of the monocyte recruitment process. Those chemokines specialized in controlling arrest are typically immobilized on the endothelial surface, mediating the arrest of rolling monocytes by chemokine receptor-triggered pathways. The chemokine receptor CXCR2 functions as an important arrest receptor on monocytes. An arrest function has been revealed for the bona fide CXCR2 ligands CXCL1 and CXCL8, but genetic studies also suggested that additional arrest chemokines are likely to be involved in atherogenic leukocyte recruitment. While CXCR2 is known to interact with numerous CXC chemokine ligands, the CLF-chemokine MIF, which structurally does not belong to the CXC chemokine sub-family, was surprisingly identified as a non-cognate ligand of CXCR2, responsible for critical arrest functions during the atherogenic process. MIF was originally identified as macrophage migration inhibitory factor, but is now known as a potent inflammatory cytokine with chemokine-like functions including chemotaxis and leukocyte arrest. This review will cover the mechanisms underlying these functions, including MIF’s effects on LFA1 integrin activity and signal transduction, and will discuss the structural similarities between MIF and the bona fide CXCR2 ligand CXCL8 while emphasizing the structural differences. As MIF also interacts with CXCR4, a chemokine receptor implicated in CXCL12-elicited lymphocyte arrest, the arrest potential of the MIF/CXCR4 axis will also be scrutinized as well as the recently identified role of pericyte MIF in attracting leukocytes exiting through venules as part of the pericyte 'motility

  5. Dopamine D3 receptor ligands for drug addiction treatment: update on recent findings.

    Science.gov (United States)

    Le Foll, Bernard; Collo, Ginetta; Rabiner, Eugenii A; Boileau, Isabelle; Merlo Pich, Emilio; Sokoloff, Pierre

    2014-01-01

    The dopamine D3 receptor is located in the limbic area and apparently mediates selective effects on motivation to take drugs and drug-seeking behaviors, so that there has been considerable interest on the possible use of D3 receptor ligands to treat drug addiction. However, only recently selective tools allowing studying this receptor have been developed. This chapter presents an overview of findings that were presented at a symposium on the conference Dopamine 2013 in Sardinia in May 2013. Novel neurobiological findings indicate that drugs of abuse can lead to significant structural plasticity in rodent brain and that this is dependent on the availability of functional dopamine D3 autoreceptor, whose activation increased phosphorylation in the ERK pathway and in the Akt/mTORC1 pathway indicating the parallel engagement of a series of intracellular signaling pathways all involved in cell growth and survival. Preclinical findings using animal models of drug-seeking behaviors confirm that D3 antagonists have a promising profile to treat drug addiction across drugs of abuse type. Imaging the D3 is now feasible in human subjects. Notably, the development of (+)-4-propyl-9-hydroxynaphthoxazine ligand used in positron emission tomography (PET) studies in humans allows to measure D3 and D2 receptors based on the area of the brain under study. This PET ligand has been used to confirm up-regulation of D3 sites in psychostimulant users and to reveal that tobacco smoking produces elevation of dopamine at the level of D3 sites. There are now novel antagonists being developed, but also old drugs such as buspirone, that are available to test the D3 hypothesis in humans. The first results of clinical investigations are now being provided. Overall, those recent findings support further exploration of D3 ligands to treat drug addiction. PMID:24968784

  6. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands

    Directory of Open Access Journals (Sweden)

    Zdzisław Chilmonczyk

    2015-08-01

    Full Text Available Serotonin (5-HT is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems, which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies.

  7. Expression of tumor necrosis factor related apoptosis inducing ligand receptor in glioblastoma

    Institute of Scientific and Technical Information of China (English)

    Dongling Gao; Zhongwei Zhao; Hongxin Zhang; Lan Zhang; Kuisheng Chen; Yunhan Zhang

    2008-01-01

    BACKGROUND: Receptors for tumor necrosis factor related apoptosis inducing ligand (TRAIL) include death receptor 4, death receptor 5, decoy receptor 1, and decoy receptor 2. Activation of death receptor 4 and 5 selectively kills tumor cells.OBJECTIVE: To detect TRAIL receptor expression in glioblastoma by immunohistochemistry and RT-PCR and to compare this expression to that in normal brain tissue.DESIGN: Observational analysis.SETTING: Department of Pathology, the First Affiliated Hospital of Zhengzhou University; Henan Tumor Pathology Key Laboratory.PARTICIPANTS: Twenty-five patients (17 males and 8 females) who received glioblastoma resection were selected from the Fifth Affiliated Hospital of Zhengzhou University, between September 2003 to June 2004. All glioblastoma samples were diagnosed pathologically. Twenty patients (12 males and 8 females) with craniocerebral injury who received normal brain tissue resection were selected in the same time period. There were no significant differences in sex and age between glioblastoma patients or between craniocerebral injury patients (P>0.05). All patients and appropriate relatives provided informed consent, and this study was approved by the local research ethics committee.METHODS: Polyclonal antibody against TRAIL receptors and an immunohistochemical kit (batch number: 200502) were purchased from Boster Company, Wuhan. Immunohistochemistry: Expression of death receptor 4, death receptor 5, decoy receptor 1, and decoy receptor 2 were observed in both glioblastoma and normal brain tissue. The experiment was performed according to the kit instructions, and positive staining was brown-yellow. Assessment: There were no positive signals (-); weakly positive signals, positive cells75% (++++). Evaluation: Expression levels of TRAIL receptors were estimated in both normal brain tissue and glioblastoma. Expression of decoy receptor 1 and decoy receptor 2 mRNA in glioblastoma were detected by reverse transcription polymerase

  8. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    Science.gov (United States)

    Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

    2016-01-01

    The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis. PMID:27463710

  9. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  10. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    Directory of Open Access Journals (Sweden)

    George Kourouniotis

    2016-07-01

    Full Text Available The binding of epidermal growth factor (EGF to EGF receptor (EGFR stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ and tagged a green fluorescent protein (GFP at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc, extracellular signal-regulated kinase (ERK and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  11. Trafficking of epidermal growth factor receptor ligands in polarized epithelial cells.

    Science.gov (United States)

    Singh, Bhuminder; Coffey, Robert J

    2014-01-01

    A largely unilamellar epithelial layer lines body cavities and organ ducts such as the digestive tract and kidney tubules. This polarized epithelium is composed of biochemically and functionally separate apical and basolateral surfaces. The epidermal growth factor receptor (EGFR) signaling pathway is a critical regulator of epithelial homeostasis and is perturbed in a number of epithelial disorders. It is underappreciated that in vivo EGFR signaling is most often initiated by cell-surface delivery and processing of one of seven transmembrane ligands, resulting in release of the soluble form that binds EGFR. In polarized epithelial cells, EGFR is restricted largely to the basolateral surface, and apical or basolateral ligand delivery therefore has important biological consequences. In vitro approaches have been used to study the biosynthesis, cell-surface delivery, proteolytic processing, and release of soluble EGFR ligands in polarized epithelial cells. We review these results, discuss their relevance to normal physiology, and demonstrate the pathophysiological consequences of aberrant trafficking. These studies have uncovered a rich diversity of apico-basolateral trafficking mechanisms among the EGFR ligands, provided insights into the pathogenesis of an inherited magnesium-wasting disorder of the kidney (isolated renal hypomagnesemia), and identified a new mode of EGFR ligand signaling via exosomes. PMID:24215440

  12. The evolution of the ligand/receptor couple: a long road from comparative endocrinology to comparative genomics

    OpenAIRE

    Markov, Gabriel V.; Paris, Mathilde; Bertrand, Stephanie; Laudet, Vincent

    2008-01-01

    The evolution of the ligand/receptor couple: a long road from comparative endocrinology to comparative genomics FRANCE (Markov, Gabriel V.) FRANCE Received: 2008-02-11 Revised: 2008-05-14 Accepted: 2008-06-11

  13. Non-canonical kinase signaling by the death ligand TRAIL in cancer cells : discord in the death receptor family

    NARCIS (Netherlands)

    Azijli, K.; Weyhenmeyer, B.; Peters, G. J.; de Jong, S.; Kruyt, F. A. E.

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based therapy is currently evaluated in clinical studies as a tumor cell selective pro-apoptotic approach. However, besides activating canonical caspase-dependent apoptosis by binding to TRAIL-specific death receptors, the TRAIL ligand

  14. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

    DEFF Research Database (Denmark)

    Hudson, Brian D; Christiansen, Elisabeth; Tikhonova, Irina G;

    2012-01-01

    When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL...... on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.-Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering...

  15. Potential clinical relevance of Eph receptors and ephrin ligands expressed in prostate carcinoma cell lines.

    Science.gov (United States)

    Fox, Brian P; Tabone, Christopher J; Kandpal, Raj P

    2006-04-21

    The family of Eph and ephrin receptors is involved in a variety of functions in normal cells, and the alterations in their expression profiles have been observed in several cancers. We have compared the transcripts for Eph receptors and ephrin ligands in cell lines established from normal prostate epithelium and several carcinoma cell lines isolated from prostate tumors of varying degree of metastasis. These cell lines included NPTX, CTPX, LNCaP, DU145, PC-3, and PC-3ML. The cell lines displayed characteristic pattern of expression for specific Eph receptors and ephrin ligands, thus allowing identification of Eph receptor signatures for a particular cell line. The sensitivity of these transcripts to genome methylation is also investigated by treating the cells with 5-aza-2'-deoxycytidine. The comparison of expression profiles revealed that normal prostate and primary prostate tumor cell lines differ in the expression of EphA3, EphB3, and ephrin A3 that are over-expressed in normal prostate. Furthermore, the transcript levels for EphA1 decrease progressively from normal prostate to primary prostate tumor cell line and metastatic tumor cells. A converse relationship was observed for ephrin B2. The treatment of cells with 5-aza-2'-deoxycytidine revealed the sensitivity of EphA3, EphA10, EphB3, and EphB6 to methylation status of genomic DNA. The utility of methylation specific PCR to identify prostate tumor cells and the importance of specific Eph receptors and ephrin ligands in initiation and progression of prostate tumor are discussed. PMID:16516143

  16. Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors

    Directory of Open Access Journals (Sweden)

    Akiyoshi Takahashi

    2016-06-01

    Full Text Available This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs related to research published in “Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish” (Takahashi et al., 2016 [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH, and β-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR.

  17. Novel aza-analogous ergoline derived scaffolds as potent serotonin 5-HT6 and dopamine D2 receptor ligands

    DEFF Research Database (Denmark)

    Krogsgaard-Larsen, Niels; Jensen, Anders A.; Schrøder, T.J.;

    2014-01-01

    By introducing distal substituents on a tetracyclic scaffold resembling the ergoline structure, two series of analogues were achieved exhibiting subnanomolar receptor binding affinities for the dopamine D2 and serotonin 5-HT6 receptor subtype, respectively. While the 5-HT6 ligands were antagonist......, the D2 ligands displayed intrinsic activities ranging from full agonism to partial agonism with low intrinsic activity. These structures could potentially be interesting for treatment of neurological diseases such as schizophrenia, Parkinson’s disease, and cognitive deficits....

  18. Evaluation of 3-Ethyl-3-(phenylpiperazinylbutyl)oxindoles as PET Ligands for the Serotonin 5-HT7 Receptor

    DEFF Research Database (Denmark)

    Herth, Matthias M; Andersen, Valdemar L; Hansen, Hanne D;

    2015-01-01

    We have investigated several oxindole derivatives in the pursuit of a 5-HT7 receptor PET ligand. Herein the synthesis, chiral separation, and pharmacological profiling of two possible PET candidates toward a wide selection of CNS-targets are detailed. Subsequent (11)C-labeling and in vivo evaluat...... evaluation in Danish landrace pigs showed that both ligands displayed high brain uptake. However, neither of the radioligands could be displaced by the 5-HT7 receptor selective inverse agonist SB-269970....

  19. Activation of epidermal growth factor receptor by metal-ligand complexes decreases levels of extracellular amyloid beta peptide.

    Science.gov (United States)

    Price, Katherine A; Filiz, Gulay; Caragounis, Aphrodite; Du, Tai; Laughton, Katrina M; Masters, Colin L; Sharples, Robyn A; Hill, Andrew F; Li, Qiao-Xin; Donnelly, Paul S; Barnham, Kevin J; Crouch, Peter J; White, Anthony R

    2008-01-01

    The epidermal growth factor receptor is a receptor tyrosine kinase expressed in a range of tissues and cell-types. Activation of the epidermal growth factor receptor by a number of ligands induces downstream signalling that modulates critical cell functions including growth, survival and differentiation. Abnormal epidermal growth factor receptor expression and activation is also involved in a number of cancers. In addition to its cognate ligands, the epidermal growth factor receptor can be activated by metals such as zinc (Zn) and copper (Cu). Due to the important role of these metals in a number of diseases including neurodegenerative disorders, therapeutic approaches are being developed based on the use of lipid permeable metal-complexing molecules. While these agents are showing promising results in animal models and clinical trials, little is known about the effects of metal-ligand complexes on cell signalling pathways. In this study, we investigated the effects of clioquinol (CQ)-metal complexes on activation of epidermal growth factor receptor. We show here that CQ-Cu complexes induced potent epidermal growth factor receptor phosphorylation resulting in downstream activation of extracellular signal-regulated kinase. Similar levels of epidermal growth factor receptor activation were observed with alternative lipid permeable metal-ligands including neocuproine and pyrrolidine dithiocarbamate. We found that CQ-Cu complexes induced a significant reduction in the level of extracellular Abeta1-40 in cell culture. Inhibition of epidermal growth factor receptor activation by PD153035 blocked extracellular signal-regulated kinase phosphorylation and restored Abeta1-40 levels. Activation of the epidermal growth factor receptor by CQ-Cu was mediated through up-regulation of src kinase activity by a cognate ligand-independent process involving membrane integrins. These findings provide the first evidence that metal-ligand complexes can activate the epidermal growth

  20. The CXCL12/CXCR4 axis promotes ligand-independent activation of the androgen receptor.

    Science.gov (United States)

    Kasina, Sathish; Macoska, Jill A

    2012-04-01

    The molecular mechanisms responsible for the transition of some prostate cancers from androgen ligand-dependent to androgen ligand-independent are incompletely established. Molecules that are ligands for G protein coupled receptors (GPCRs) have been implicated in ligand-independent androgen receptor (AR) activation. The purpose of this study was to examine whether CXCL12, the ligand for the GPCR, CXCR4, might mediate prostate cancer cell proliferation through AR-dependent mechanisms involving functional transactivation of the AR in the absence of androgen. The results of these studies showed that activation of the CXCL12/CXCR4 axis promoted: The nuclear accumulation of both wild-type and mutant AR in several prostate epithelial cell lines; AR-dependent proliferative responses; nuclear accumulation of the AR co-regulator SRC-1 protein; SRC-1:AR protein:protein association; co-localization of AR and SRC-1 on the promoters of AR-regulated genes; AR- and SRC-1 dependent transcription of AR-regulated genes; AR-dependent secretion of the AR-regulated PSA protein; P13K-dependent phosphorylation of AR; MAPK-dependent phosphorylation of SRC-1, and both MAPK- and P13K-dependent secretion of the PSA protein, in the absence of androgen. Taken together, these studies identify CXCL12 as a novel, non-steroidal growth factor that promotes the growth of prostate epithelial cells through AR-dependent mechanisms in the absence of steroid hormones. These findings support the development of novel therapeutics targeting the CXCL12/CXCR4 axis as an ancillary to those targeting the androgen/AR axis to effectively treat castration resistant/recurrent prostate tumors.

  1. Structure-activity relationships of constrained phenylethylamine ligands for the serotonin 5-HT2 receptors.

    Directory of Open Access Journals (Sweden)

    Vignir Isberg

    Full Text Available Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine class. Conformationally constrained phenethylamine analogs have demonstrated that for optimal activity the free lone pair electrons of the 2-oxygen must be oriented syn and the 5-oxygen lone pairs anti relative to the ethylamine moiety. Also the ethyl linker has been constrained providing information about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9-11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure showed that the 1,2-heterocyclized compounds can be accommodated in the binding site. Conformational analysis showed that 11 can only bind in a higher-energy conformation, which would explain its absent or low affinity. The amine and 2-oxygen interactions with D3.32 and S3.36, respectively, can form but shift the placement of the core scaffold. The constraints in 9-11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9-11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands.

  2. Peripheral benzodiazepine binding sites on striated muscles of the rat: Properties and effect of denervation

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, W.E.; Ickstadt, A. (Mainz Univ. (Germany, F.R.). Pharmakologisches Inst.); Hopf, H.Ch. (Mainz Univ. (Germany, F.R.))

    1985-01-01

    In order to test the hypothesis that peripheral benzodiazepine binding sites mediate some direct effects of benzodiazepines on striated muscles, the properties of specific /sup 3/H-Ro 5-4864 binding to rat biceps and rat diaphragm homogenates were investigated. In both tissues a single population of sites was found with a Ksub(D) value of 3 nmol/l. The density of these sites in both muscles was higher than the density in rat brain, but was considerably lower than in rat kidney. Competition experiments indicate a substrate specificity of specific /sup 3/H-Ro 5-4864 binding similar to the properties already demonstrated for the specific binding of this ligand to peripheral benzodiazepine binding sites in many other tissues. The properties of these sites in the rat diaphragm are not changed after motoric denervation by phrenicectomy. It is concluded that peripheral benzodiazepine binding sites are not involved in direct effects of benzodiazepines on striated muscles.

  3. Monitoring Solution Structures of Peroxisome Proliferator-Activated Receptor β/δ upon Ligand Binding.

    Science.gov (United States)

    Schwarz, Rico; Tänzler, Dirk; Ihling, Christian H; Sinz, Andrea

    2016-01-01

    Peroxisome proliferator-activated receptors (PPARs) have been intensively studied as drug targets to treat type 2 diabetes, lipid disorders, and metabolic syndrome. This study is part of our ongoing efforts to map conformational changes in PPARs in solution by a combination of chemical cross-linking and mass spectrometry (MS). To our best knowledge, we performed the first studies addressing solution structures of full-length PPAR-β/δ. We monitored the conformations of the ligand-binding domain (LBD) as well as full-length PPAR-β/δ upon binding of two agonists. (Photo-) cross-linking relied on (i) a variety of externally introduced amine- and carboxyl-reactive linkers and (ii) the incorporation of the photo-reactive amino acid p-benzoylphenylalanine (Bpa) into PPAR-β/δ by genetic engineering. The distances derived from cross-linking experiments allowed us to monitor conformational changes in PPAR-β/δ upon ligand binding. The cross-linking/MS approach proved highly advantageous to study nuclear receptors, such as PPARs, and revealed the interplay between DBD (DNA-binding domain) and LDB in PPAR-β/δ. Our results indicate the stabilization of a specific conformation through ligand binding in PPAR-β/δ LBD as well as full-length PPAR-β/δ. Moreover, our results suggest a close distance between the N- and C-terminal regions of full-length PPAR-β/δ in the presence of GW1516. Chemical cross-linking/MS allowed us gaining detailed insights into conformational changes that are induced in PPARs when activating ligands are present. Thus, cross-linking/MS should be added to the arsenal of structural methods available for studying nuclear receptors. PMID:26992147

  4. Epidermal growth factor receptor ligands as new extracellular targets for the metastasis-promoting S100A4 protein

    DEFF Research Database (Denmark)

    Klingelhöfer, Jörg; Møller, Henrik D.; Sumer, Eren U;

    2009-01-01

    that the most abundant peptide mimicked the F/YCC motif present in the epidermal growth factor domain of ErbB receptor ligands. S100A4 selectively interacted with a number of epidermal growth factor receptor (EGFR) ligands, demonstrating highest affinity for amphiregulin. Importantly, we found that S100A4...... stimulated EGFR/ErbB2 receptor signaling and enhanced the amphiregulin-mediated proliferation of mouse embryonic fibroblasts. S100A4-neutralizing antibodies, as well as EGFR- and ErbB2 receptor-specific tyrosine kinase inhibitors, blocked these effects. The present results suggest that extracellular S100A4...... regulates tumor progression by interacting with EGFR ligands, thereby enhancing EGFR/ErbB2 receptor signaling and cell proliferation. Structured digital abstract: * MINT-7256556: EGF (uniprotkb:P01133) binds (MI:0407) to S100A4 (uniprotkb:P26447) by far western blotting (MI:0047) * MINT-7256512: BC...

  5. SPOT-Ligand: Fast and effective structure-based virtual screening by binding homology search according to ligand and receptor similarity.

    Science.gov (United States)

    Yang, Yuedong; Zhan, Jian; Zhou, Yaoqi

    2016-07-01

    Structure-based virtual screening usually involves docking of a library of chemical compounds onto the functional pocket of the target receptor so as to discover novel classes of ligands. However, the overall success rate remains low and screening a large library is computationally intensive. An alternative to this "ab initio" approach is virtual screening by binding homology search. In this approach, potential ligands are predicted based on similar interaction pairs (similarity in receptors and ligands). SPOT-Ligand is an approach that integrates ligand similarity by Tanimoto coefficient and receptor similarity by protein structure alignment program SPalign. The method was found to yield a consistent performance in DUD and DUD-E docking benchmarks even if model structures were employed. It improves over docking methods (DOCK6 and AUTODOCK Vina) and has a performance comparable to or better than other binding-homology methods (FINDsite and PoLi) with higher computational efficiency. The server is available at http://sparks-lab.org. © 2016 Wiley Periodicals, Inc. PMID:27074979

  6. Development of novel mixed ligand technetium complexes (3 + 1 combination) for imaging central neural system receptors

    International Nuclear Information System (INIS)

    A series of mixed ligand oxotechnetium-99m complexes carrying the 1-(2-methoxyphenyl) piperazine moiety has been synthesized. For structural characterization, and for in vitro binding assays, the analogous oxorhenium or oxotechnetium-99 complexes were prepared. As demonstrated by appropriate competition binding tests in rat hippocampal preparations, all oxorhenium analogues showed affinity for the 5-HT1A receptor binding sites with 50% inhibitory concentration values in the nanomolar range (IC50=6-106nM). All 99mTcO[SN(R)S]/[S] complexes showed a significant brain uptake in rats at 2 min post-injection (0.24-1.31 dose/organ). The regional distribution is inhomogeneous but the ratio between areas rich and poor in 5-HT1A receptor was not high. Structural modifications to this system may further improve the biological profile of these compounds and eventually provide efficient 99mTc receptor imaging agents. (author)

  7. ReFlexIn: a flexible receptor protein-ligand docking scheme evaluated on HIV-1 protease.

    Directory of Open Access Journals (Sweden)

    Simon Leis

    Full Text Available For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation, that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1 protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.

  8. The different ligand-binding modes of relaxin family peptide receptors RXFP1 and RXFP2.

    Science.gov (United States)

    Scott, Daniel J; Rosengren, K Johan; Bathgate, Ross A D

    2012-11-01

    Relaxin and insulin-like peptide 3 (INSL3) are peptide hormones with a number of important physiological roles in reproduction, regulation of extracellular matrix turnover, and cardiovascular function. Relaxin and INSL3 mediate their actions through the closely related G-protein coupled receptors, relaxin family peptide receptors 1 and 2 (RXFP1 and RXFP2), respectively. These receptors have large extracellular domains (ECD) that contain high-affinity ligand-binding sites within their 10 leucine-rich repeat (LRR)-containing modules. Although relaxin can bind and activate both RXFP1 and RXFP2, INSL3 can only bind and activate RXFP2. To investigate whether this difference is related to the nature of the high-affinity ECD binding site or to differences in secondary binding sites involving the receptor transmembrane (TM) domain, we created a suite of constructs with RXFP1/2 chimeric ECD attached to single TM helices. We show that by changing as little as one LRR, representing four amino acid substitutions, we were able to engineer a high-affinity INSL3-binding site into the ECD of RXFP1. Molecular modeling of the INSL3-RXFP2 interaction based on extensive experimental data highlights the differences in the binding mechanisms of relaxin and INSL3 to the ECD of their cognate receptors. Interestingly, when the engineered RXFP1/2 ECD were introduced into full-length RXFP1 constructs, INSL3 exhibited only low affinity and efficacy on these receptors. These results highlight critical differences both in the ECD binding and in the coordination of the ECD-binding site with the TM domain, and provide new mechanistic insights into the binding and activation events of RXFP1 and RXFP2 by their native hormone ligands. PMID:22973049

  9. Peroxisome Proliferator-Activated Receptor γ (PPARγ) and Ligand Choreography: Newcomers Take the Stage.

    Science.gov (United States)

    Garcia-Vallvé, Santiago; Guasch, Laura; Tomas-Hernández, Sarah; del Bas, Josep Maria; Ollendorff, Vincent; Arola, Lluís; Pujadas, Gerard; Mulero, Miquel

    2015-07-23

    Thiazolidinediones (TZDs), such as rosiglitazone and pioglitazone, are peroxisome proliferator-activated receptor γ (PPARγ) full agonists that have been widely used in the treatment of type 2 diabetes mellitus. Despite the demonstrated beneficial effect of reducing glucose levels in the plasma, TZDs also induce several adverse effects. Consequently, the search for new compounds with potent antidiabetic effects but fewer undesired effects is an active field of research. Interestingly, the novel proposed mechanisms for the antidiabetic activity of PPARγ agonists, consisting of PPARγ Ser273 phosphorylation inhibition, ligand and receptor mutual dynamics, and the presence of an alternate binding site, have recently changed the view regarding the optimal characteristics for the screening of novel PPARγ ligands. Furthermore, transcriptional genomics could bring essential information about the genome-wide effects of PPARγ ligands. Consequently, facing the new mechanistic scenario proposed for these compounds is essential for resolving the paradoxes among their agonistic function, antidiabetic activities, and side effects and should allow the rational development of better and safer PPARγ-mediated antidiabetic drugs. PMID:25734377

  10. Emerging importance of chemokine receptor CXCR3 and its ligands in cardiovascular diseases.

    Science.gov (United States)

    Altara, Raffaele; Manca, Marco; Brandão, Rita D; Zeidan, Asad; Booz, George W; Zouein, Fouad A

    2016-04-01

    The CXC chemokines, CXCL4, -9, -10, -11, CXCL4L1, and the CC chemokine CCL21, activate CXC chemokine receptor 3 (CXCR3), a cell-surface G protein-coupled receptor expressed mainly by Th1 cells, cytotoxic T (Tc) cells and NK cells that have a key role in immunity and inflammation. However, CXCR3 is also expressed by vascular smooth muscle and endothelial cells, and appears to be important in controlling physiological vascular function. In the last decade, evidence from pre-clinical and clinical studies has revealed the participation of CXCR3 and its ligands in multiple cardiovascular diseases (CVDs) of different aetiologies including atherosclerosis, hypertension, cardiac hypertrophy and heart failure, as well as in heart transplant rejection and transplant coronary artery disease (CAD). CXCR3 ligands have also proven to be valid biomarkers for the development of heart failure and left ventricular dysfunction, suggesting an underlining pathophysiological relation between levels of these chemokines and the development of adverse cardiac remodelling. The observation that several of the above-mentioned chemokines exert biological actions independent of CXCR3 provides both opportunities and challenges for developing effective drug strategies. In this review, we provide evidence to support our contention that CXCR3 and its ligands actively participate in the development and progression of CVDs, and may additionally have utility as diagnostic and prognostic biomarkers. PMID:26888559

  11. Peroxisome Proliferator-Activated Receptor γ (PPARγ) and Ligand Choreography: Newcomers Take the Stage.

    Science.gov (United States)

    Garcia-Vallvé, Santiago; Guasch, Laura; Tomas-Hernández, Sarah; del Bas, Josep Maria; Ollendorff, Vincent; Arola, Lluís; Pujadas, Gerard; Mulero, Miquel

    2015-07-23

    Thiazolidinediones (TZDs), such as rosiglitazone and pioglitazone, are peroxisome proliferator-activated receptor γ (PPARγ) full agonists that have been widely used in the treatment of type 2 diabetes mellitus. Despite the demonstrated beneficial effect of reducing glucose levels in the plasma, TZDs also induce several adverse effects. Consequently, the search for new compounds with potent antidiabetic effects but fewer undesired effects is an active field of research. Interestingly, the novel proposed mechanisms for the antidiabetic activity of PPARγ agonists, consisting of PPARγ Ser273 phosphorylation inhibition, ligand and receptor mutual dynamics, and the presence of an alternate binding site, have recently changed the view regarding the optimal characteristics for the screening of novel PPARγ ligands. Furthermore, transcriptional genomics could bring essential information about the genome-wide effects of PPARγ ligands. Consequently, facing the new mechanistic scenario proposed for these compounds is essential for resolving the paradoxes among their agonistic function, antidiabetic activities, and side effects and should allow the rational development of better and safer PPARγ-mediated antidiabetic drugs.

  12. Crystallization and preliminary X-ray analysis of the human androgen receptor ligand-binding domain with a coactivator-like peptide and selective androgen receptor modulators

    International Nuclear Information System (INIS)

    The human androgen receptor ligand-binding domain has been crystallized as a ternary complex with a coactivator-like undecapeptide and two different synthetic ligands. The ligand-binding domain of the human androgen receptor has been cloned, overproduced and crystallized in the presence of a coactivator-like 11-mer peptide and two different nonsteroidal ligands. The crystals of the two ternary complexes were isomorphous and belonged to space group P212121, with one molecule in the asymmetric unit. They diffracted to 1.7 and 1.95 Å resolution, respectively. Structure determination of these two complexes will help in understanding the mode of binding of selective nonsteroidal androgens versus endogenous steroidal ligands and possibly the origin of their tissue selectivity

  13. GluVII:06--a highly conserved and selective anchor point for non-peptide ligands in chemokine receptors

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Schwartz, Thue W

    2006-01-01

    to be crucially important for the binding and action of a number of non-peptide ligands in for example the CCR1, CCR2 and CCR5 receptors. It is proposed that in chemokine receptors in general GluVII:06 serves as a selective anchor point for the centrally located, positively charged nitrogen of the small molecule...

  14. The relaxin family peptide receptors and their ligands : new developments and paradigms in the evolution from jawless fish to mammals

    NARCIS (Netherlands)

    Yegorov, Sergey; Bogerd, Jan; Good, Sara V

    2014-01-01

    Relaxin family peptide receptors (Rxfps) and their ligands, relaxin (Rln) and insulin-like (Insl) peptides, are broadly implicated in the regulation of reproductive and neuroendocrine processes in mammals. Most placental mammals harbour genes for four receptors, namely rxfp1, rxfp2, rxfp3 and rxfp4.

  15. An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells.

    Science.gov (United States)

    Wang, Kai; Li, Yan; Jiang, Yi-Zhou; Dai, Cai-Feng; Patankar, Manish S; Song, Jia-Sheng; Zheng, Jing

    2013-10-28

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor mediates many biological processes. Herein, we investigated if 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, an endogenous AhR ligand) regulated proliferation and migration of human ovarian cancer cells via AhR. We found that AhR was widely present in many histotypes of ovarian cancer tissues. ITE suppressed OVCAR-3 cell proliferation and SKOV-3 cell migration in vitro, which were blocked by AhR knockdown. ITE also suppressed OVCAR-3 cell growth in mice. These data suggest that the ITE might potentially be used for therapeutic intervention for at least a subset of human ovarian cancer.

  16. Emerging Roles for CSF-1 Receptor and its Ligands in the Nervous System.

    Science.gov (United States)

    Chitu, Violeta; Gokhan, Şölen; Nandi, Sayan; Mehler, Mark F; Stanley, E Richard

    2016-06-01

    The colony-stimulating factor-1 receptor (CSF-1R) kinase regulates tissue macrophage homeostasis, osteoclastogenesis, and Paneth cell development. However, recent studies in mice have revealed that CSF-1R signaling directly controls the development and maintenance of microglia, and cell autonomously regulates neuronal differentiation and survival. While the CSF-1R-cognate ligands, CSF-1 and interleukin-34 (IL-34) compete for binding to the CSF-1R, they are expressed in a largely non-overlapping manner by mature neurons. The recent identification of a dominantly inherited, adult-onset, progressive dementia associated with inactivating mutations in the CSF-1R highlights the importance of CSF-1R signaling in the brain. We review the roles of the CSF-1R and its ligands in microglial and neural development and function, and their relevance to our understanding of neurodegenerative disease.

  17. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    Science.gov (United States)

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M. PMID:23325100

  18. Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity.

    Science.gov (United States)

    Nguyen, Duy P; Miyaoka, Yuichiro; Gilbert, Luke A; Mayerl, Steven J; Lee, Brian H; Weissman, Jonathan S; Conklin, Bruce R; Wells, James A

    2016-01-01

    Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants. PMID:27363581

  19. Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity

    Science.gov (United States)

    Nguyen, Duy P.; Miyaoka, Yuichiro; Gilbert, Luke A.; Mayerl, Steven J.; Lee, Brian H.; Weissman, Jonathan S.; Conklin, Bruce R.; Wells, James A.

    2016-01-01

    Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants. PMID:27363581

  20. Ligand Binding Affinities of Arctigenin and Its Demethylated Metabolites to Estrogen Receptor Alpha

    Directory of Open Access Journals (Sweden)

    Masao Hattori

    2013-01-01

    Full Text Available Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (−-arctigenin, the aglycone of arctiin, was demethylated to (−-dihydroxyenterolactone (DHENL by Eubacterium (E. sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (−-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (−-arctigenin using a ligand binding screen assay method. The IC50 value of (2R,3R-2-(4-hydroxy-3-methoxybenzyl-3-(3,4-dihydroxybenzyl-butyrolactone was 7.9 × 10−4 M.

  1. Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region

    DEFF Research Database (Denmark)

    Madsen, Mette; Møller, Holger J; Nielsen, Marianne Jensby;

    2004-01-01

    CD163 is the macrophage receptor for endocytosis of haptoglobin.hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteine rich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163...

  2. Characterization of a ligand binding site in the human transient receptor potential ankyrin 1 pore.

    Science.gov (United States)

    Klement, Göran; Eisele, Lina; Malinowsky, David; Nolting, Andreas; Svensson, Mats; Terp, Gitte; Weigelt, Dirk; Dabrowski, Michael

    2013-02-19

    The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule.

  3. NOP Receptor Ligands as Potential Agents for Inflammatory and Autoimmune Diseases

    Directory of Open Access Journals (Sweden)

    Elaine C. Gavioli

    2011-01-01

    Full Text Available Nociceptin/orphanin FQ (N/OFQ is a seventeen-amino acid peptide that is the endogenous ligand of a G-protein-coupled receptor (NOP. Various immune cells express the precursor protein and secrete N/OFQ as well as display binding sites for this peptide. The functional capacity of NOP receptor was demonstrated in vitro and in vivo studies by the ability of N/OFQ to induce chemotaxis of immune cells, to regulate the expression of cytokines and other inflammatory mediators, and to control cellular and humoral immunity. In this context, N/OFQ could modulate the outcome of some inflammatory diseases, such as sepsis and autoimmune pathologies by mechanisms not clearly elucidated yet. In fact, human body fluid revealed increased levels of N/OFQ under sepsis, arthritis, and Parkinson's diagnose. Preclinical studies pointed to the blockade of NOP receptor signaling as successful in treating these experimental conditions. Further preclinical and clinical studies are required to investigate the potential of NOP ligands in treating inflammatory diseases.

  4. Dual ligand/receptor interactions activate urothelial defenses against uropathogenic E. coli.

    Science.gov (United States)

    Liu, Yan; Mémet, Sylvie; Saban, Ricardo; Kong, Xiangpeng; Aprikian, Pavel; Sokurenko, Evgeni; Sun, Tung-Tien; Wu, Xue-Ru

    2015-11-09

    During urinary tract infection (UTI), the second most common bacterial infection, dynamic interactions take place between uropathogenic E. coli (UPEC) and host urothelial cells. While significant strides have been made in the identification of the virulence factors of UPEC, our understanding of how the urothelial cells mobilize innate defenses against the invading UPEC remains rudimentary. Here we show that mouse urothelium responds to the adhesion of type 1-fimbriated UPEC by rapidly activating the canonical NF-κB selectively in terminally differentiated, superficial (umbrella) cells. This activation depends on a dual ligand/receptor system, one between FimH adhesin and uroplakin Ia and another between lipopolysaccharide and Toll-like receptor 4. When activated, all the nuclei (up to 11) of a multinucleated umbrella cell are affected, leading to significant amplification of proinflammatory signals. Intermediate and basal cells of the urothelium undergo NF-κB activation only if the umbrella cells are detached or if the UPEC persistently express type 1-fimbriae. Inhibition of NF-κB prevents the urothelium from clearing the intracellular bacterial communities, leading to prolonged bladder colonization by UPEC. Based on these data, we propose a model of dual ligand/receptor system in innate urothelial defenses against UPEC.

  5. Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses.

    Science.gov (United States)

    Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang

    2008-10-14

    High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins. PMID:18840687

  6. Synthetic Ligands of Cannabinoid Receptors Affect Dauer Formation in the Nematode Caenorhabditis elegans

    Science.gov (United States)

    Reis Rodrigues, Pedro; Kaul, Tiffany K.; Ho, Jo-Hao; Lucanic, Mark; Burkewitz, Kristopher; Mair, William B.; Held, Jason M.; Bohn, Laura M.; Gill, Matthew S.

    2016-01-01

    Under adverse environmental conditions the nematode Caenorhabditis elegans can enter an alternate developmental stage called the dauer larva. To identify lipophilic signaling molecules that influence this process, we screened a library of bioactive lipids and found that AM251, an antagonist of the human cannabinoid (CB) receptor, suppresses dauer entry in daf-2 insulin receptor mutants. AM251 acted synergistically with glucose supplementation indicating that the metabolic status of the animal influenced the activity of this compound. Similarly, loss of function mutations in the energy-sensing AMP-activated kinase subunit, aak-2, enhanced the dauer-suppressing effects of AM251, while constitutive activation of aak-2 in neurons was sufficient to inhibit AM251 activity. Chemical epistasis experiments indicated that AM251 acts via G-protein signaling and requires the TGF-β ligand DAF-7, the insulin peptides DAF-28 and INS-6, and a functional ASI neuron to promote reproductive growth. AM251 also required the presence of the SER-5 serotonin receptor, but in vitro experiments suggest that this may not be via a direct interaction. Interestingly, we found that other antagonists of mammalian CB receptors also suppress dauer entry, while the nonselective CB receptor agonist, O-2545, not only inhibited the activity of AM251, but also was able to promote dauer entry when administered alone. Since worms do not have obvious orthologs of CB receptors, the effects of synthetic CBs on neuroendocrine signaling in C. elegans are likely to be mediated via another, as yet unknown, receptor mechanism. However, we cannot exclude the existence of a noncanonical CB receptor in C. elegans. PMID:27172180

  7. Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses.

    Science.gov (United States)

    Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang

    2008-10-14

    High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.

  8. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  9. Anthranilic acid derivatives as nuclear receptor modulators--development of novel PPAR selective and dual PPAR/FXR ligands.

    Science.gov (United States)

    Merk, Daniel; Lamers, Christina; Weber, Julia; Flesch, Daniel; Gabler, Matthias; Proschak, Ewgenij; Schubert-Zsilavecz, Manfred

    2015-02-01

    Nuclear receptors, especially the peroxisome proliferator activated receptors (PPARs) and the farnesoid X receptor (FXR) fulfill crucial roles in metabolic balance. Their activation offers valuable therapeutic potential which has high clinical relevance with the fibrates and glitazones as PPAR agonistic drugs. With growing knowledge about the various functions of nuclear receptors in many disorders, new selective or dual ligands of these pharmaceutical targets are however still required. Here we report the class of anthranilic acid derivatives as novel selective PPAR or dual FXR/PPAR ligands. We identified distinct molecular determinants that govern selectivity for each PPAR subtype or FXR as well as the amplitude of activation of the respective receptors. We thereby discovered several lead compounds for further optimization and developed a highly potent dual PPARα/FXR partial agonist that might have a beneficial synergistic effect on lipid homeostasis by simultaneous activation of two nuclear receptors involved in lipid metabolism. PMID:25583100

  10. Expressions of chemokine receptor CXCR4 and its ligand CXCL12 in salivary adenoid cystic carcinoma

    Institute of Scientific and Technical Information of China (English)

    徐晓刚; 吕春堂; 周中华

    2004-01-01

    Objective: To examine expressions of chemokine receptor CXCR4 and its ligand CXCL12 in primary focus and lymphogenous metastasis of salivary adenoid cystic carcinoma (ACC) with lung metastasis. Methods: Using immunohistochemical hypersensitivity catalyzed signal amplification (CSA), expressions of chemokine receptor CXCR4 and ligand CXCL12 were detected in tissue specimens from 20 cases of primary cancer focus and lymphogenous metastasis of salivary adenoid cystic carcinoma, of which 7 cases were associated with lung metastasis and 3 with lympogenons metastasis. Twenty cases of tongue carcinoma (including 10 cases with lymphogenous metastasis) and 15 cases of mucoepidermoid carcinoma (including 5 cases with lymphogenous metastasis) were used as the malignant control group; and salivary mixed tumor ( n =10), tongue leukoceratosis ( n = 10) and cervical lymph node reactive hyperplasia ( n = 10) were used as the benign control group. Results: Expression of CXCR4 in the tissues and lymph metastases of oral and maxillofacial salivary ACC, mucoepidermoid carcinoma and tongue carcinoma was significantly higher than that of the benign control group ( P < 0.05); expression of CXCR4 in the primary focus of ACC was significantly higher than that of the malignant control group; and expression of CXCR4 in the ACC with lung metastasis was 87.1% (6/7), significantly higher than that without lung metastasis( P <0.01 ). There was evident positive expression of CXCL12 in endotheliocytes of microvessels within cancer and paracancer tissues and significantly high expression of CXCL12 in lymphogenous metastasis( P < 0.05). Conclusion: Chemokine receptor CXCR4 and its ligand CXCL12 may be associated with local invasion and lymphogenous metastasis of oral and maxillofacial cancer, especially with lung metastasis of salivary ACC.

  11. Characterization of apela, a novel endogenous ligand of apelin receptor, in the adult heart.

    Science.gov (United States)

    Perjés, Ábel; Kilpiö, Teemu; Ulvila, Johanna; Magga, Johanna; Alakoski, Tarja; Szabó, Zoltán; Vainio, Laura; Halmetoja, Eveliina; Vuolteenaho, Olli; Petäjä-Repo, Ulla; Szokodi, István; Kerkelä, Risto

    2016-01-01

    The G protein-coupled apelin receptor regulates important processes of the cardiovascular homeostasis, including cardiac development, cardiac contractility, and vascular tone. Most recently, a novel endogenous peptide ligand for the apelin receptor was identified in zebrafish, and it was named apela/elabela/toddler. The peptide was originally considered as an exclusively embryonic regulator, and so far its function in the adult organism remains elusive. We show here that apela is predominantly expressed in the non-cardiomyocyte fraction in the adult rodent heart. We also provide evidence that apela binds to apelin receptors in the heart. Using isolated adult rat hearts, we demonstrate, that just like the fellow receptor agonist apelin, apela increases cardiac contractility and induces coronary vasodilation already in the nanomolar level. The inotropic effect, as revealed by Western blot analysis, is accompanied by a significant increase in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Pharmacological inhibition of ERK1/2 activation markedly attenuates the apela-induced inotropy. Analysis of samples from infarcted mouse hearts showed that expression of both apela and apelin receptor is induced in failing mouse hearts and correlate with left ventricular ejection fraction. Hence, we conclude that apela is present in the adult heart, is upregulated in post-infarction cardiac remodeling, and increases cardiac contractility in an ERK1/2-dependent manner.

  12. Synthesis and Evaluation of Mefway Analogs as Ligands for Serotonin 5HT1A Receptors

    OpenAIRE

    Thio, Joanne P.; Liang, Christopher; Bajwa, Alisha K; Wooten, Dustin W; Christian, Bradley T; Mukherjee, Jogeshwar

    2014-01-01

    18F-Mefway (N-{2-[4-(2′-methoxyphenyl)piperazinyl]ethyl}-N-(2-pyridyl)-N-(4′-18F-fluoro-methylcyclohexane)carboxamide) was developed and evaluated for use as a PET ligand for imaging 5-HT1A receptors. Ongoing studies of 18F-Mefway have shown it to be an effective PET radiotracer. We have synthesized isomers of Mefway by changing the position of the methyl-group in attempts to evaluate stability for imaging purposes. 2-Methyl-, 3-methyl-, and 4-methyl-cyclohexane-1-carboxylic acids and 3-carbo...

  13. Development of small molecule non-peptide formyl peptide receptor (FPR) ligands and molecular modeling of their recognition

    OpenAIRE

    Schepetkin I.A.; Klebnikov A.I.; Giovannoni M.P.; Kirpotina L.N.; Cilibrizzi A.; Quinn M.T.

    2014-01-01

    Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) expressed on a variety of cell types. These receptors play an important role in the regulation of inflammatory reactions and sensing cellular damage. They have also been implicated in the pathogenesis of various diseases, including neurodegenerative diseases, cataract formation, and atherogenesis. Thus, FPR ligands, both agonists and antagonists, may represent novel therapeutics for modulating host defense and innate immu...

  14. Use and abuse of benzodiazepines*

    OpenAIRE

    1983-01-01

    Benzodiazepines are widely used for the treatment of anxiety, insomnia, and certain neuromuscular and convulsive disorders. However, their widespread availability has given rise to fears that they are over-prescribed. The problem is compounded by the fact that there is no universal agreement among medical practitioners as to the clinical indications warranting the use of these drugs. Although most industrialized countries exercise control over the sale and manufacture of benzodiazepines, many...

  15. Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution

    OpenAIRE

    Park, Yoonseong; KIM, YOUNG-JOON; Adams, Michael E.

    2002-01-01

    G-protein coupled receptors (GPCRs) are ancient, ubiquitous sensors vital to environmental and physiological signaling throughout organismal life. With the publication of the Drosophila genome, numerous “orphan” GPCRs have become available for functional analysis. Here we characterize two groups of GPCRs predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of −PRXamide (PRXa). Assuming ligand-receptor coevolution, two alternative hypotheses were construct...

  16. Effects of benzodiazepines and non-benzodiazepine compounds on the GABA-induced response in frog isolated sensory neurones.

    Science.gov (United States)

    Yakushiji, T; Fukuda, T; Oyama, Y; Akaike, N

    1989-11-01

    1. The effects of benzodiazepines and non-benzodiazepine compounds on the gamma-aminobutyric acid (GABA)-induced chloride current (ICl) were studied in frog isolated sensory neurones by use of a concentration-jump (termed 'concentration-clamp') technique, under single-electrode voltage-clamp conditions. The drugs used were classified into four categories as follows: full benzodiazepine receptor agonists (diazepam, clonazepam, nitrazepam, midazolam, clotiazepam and etizolam), partial agonists (CL 218,872, Ro 16-6028, Ro 17-1812 and Ro 23-0364), inverse agonists (Ro 15-3505, FG 7142 and beta-CCE) and a benzodiazepine receptor antagonist, Ro 15-1788 (flumazenil). 2. All full agonists at concentrations of 3 x 10(-6) M or less increased dose-dependently the peak amplitude of ICl elicited by 3 x 10(-6) M GABA to twice to three times larger than the control. However, no further augmentation of the GABA response was observed at concentrations of 1 x 10(-5) M or higher. Partial agonists also showed a dose-dependent augmentation of the GABA response at concentrations ranging from 3 x 10(-8) M to 3 x 10(-5) M, but their efficacies of augmentation of the GABA response were only about half or less of those of full agonists. Of the inverse agonists, beta-CCE had a unique dose-dependent effect on the GABA response. Beta-CCE reduced dose-dependently the GABA response at concentrations of less than 3 x 10(-6) M, but augmented it at concentrations of 3 x 10(-5) M and 6 x 10(-5) M. The inverse agonists reduced dose-dependently the GABA response. The benzodiazepine antagonist, flumazenil, slightly augmented the GABA response at concentrations between 3 x 10 7M and 3 x 10 5 M. 3. These results show clear differences in the effects on the GABA response between these four categories of compounds known to affect the benzodiazepine recognition site of the GABA/ benzodiazepine receptor-chloride channel complex. Our experimental system of frog isolated sensory neurones and a 'concentration

  17. Functional Selectivity of CB2 Cannabinoid Receptor Ligands at a Canonical and Noncanonical Pathway.

    Science.gov (United States)

    Dhopeshwarkar, Amey; Mackie, Ken

    2016-08-01

    The CB2 cannabinoid receptor (CB2) remains a tantalizing, but unrealized therapeutic target. CB2 receptor ligands belong to varied structural classes and display extreme functional selectivity. Here, we have screened diverse CB2 receptor ligands at canonical (inhibition of adenylyl cyclase) and noncanonical (arrestin recruitment) pathways. The nonclassic cannabinoid (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940) was the most potent agonist for both pathways, while the classic cannabinoid ligand (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran JWH133) was the most efficacious agonist among all the ligands profiled in cyclase assays. In the cyclase assay, other classic cannabinoids showed little [(-)-trans-Δ(9)-tetrahydrocannabinol and (-)-(6aR,7,10,10aR)-tetrahydro-6,6,9-trimethyl-3-(1-methyl-1-phenylethyl)-6H-dibenzo[b,d]pyran-1-ol] (KM233) to no efficacy [(6aR,10aR)-1-methoxy-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromene(L759633) and (6aR,10aR)-3-(1,1-dimethylheptyl)-6a,7,8,9,10,10a-hexahydro-1-methoxy-6,6-dimethyl-9-methylene-6H-dibenzo[b,d]pyran]L759656. Most aminoalkylindoles, including [(3R)-​2,​3-​dihydro-​5-​methyl-​3-​(4-​morpholinylmethyl)pyrrolo[1,​2,​3-​de]-​1,​4-​benzoxazin-​6-​yl]-​1-​naphthalenyl-​methanone,​ monomethanesulfonate (WIN55212-2), were moderate efficacy agonists. The cannabilactone 3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c)chromen-6-one (AM1710) was equiefficacious to CP55940 to inhibit adenylyl cyclase, albeit with lower potency. In the arrestin recruitment assays, all classic cannabinoid ligands failed to recruit arrestins, indicating a bias toward G-protein coupling for this class of compound. All aminoalkylindoles tested, except for WIN55212-2 and (1-​pentyl-​1H-​indol-​3-​yl)(2,​2,​3,​3-​tetramethylcyclopropyl)-​methanone (UR144), failed

  18. Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form

    Energy Technology Data Exchange (ETDEWEB)

    Hosoi, Takayuki; Sawyer, S.T.; Krantz, S.B. (Vanderbilt Univ. School of Medicine, Nashville, TN (USA))

    1991-01-01

    Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-((3-((4-((p-azido-m-({sup 125}I)iodophenyl)azo)benzoyl)amino)propanoyl)oxy)-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to {sup 125}I-EP. Recently, D'Andrea and co-workers cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands cross-linked with {sup 125}I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.

  19. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

    CERN Document Server

    Winckler, Pascale; Giannone, Gregory; De Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

    2013-01-01

    Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule F\\"orster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-...

  20. Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: receptor homology modelling, ligand docking, and molecular dynamics results validated by experimental studies

    Science.gov (United States)

    Córdova-Sintjago, Tania; Villa, Nancy; Fang, Lijuan; Booth, Raymond G.

    2014-02-01

    The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists - in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

  1. Drug-likeness approach of 2-aminopyrimidines as histamine H3 receptor ligands

    Directory of Open Access Journals (Sweden)

    Sadek B

    2014-09-01

    Full Text Available Bassem Sadek,1 Annemarie Schreeb,2 Johannes Stephan Schwed,2,3 Lilia Weizel,2 Holger Stark3 1Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates; 2Biocenter, Institute of Pharmaceutical Chemistry, Johann-Wolfgang Goethe University, Frankfurt, Germany; 3Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Duesseldorf, Germany Abstract: A small series of compounds containing derivatives of 2,4-diamino- and 2,4,6-triaminopyrimidine (compounds 2–7 was synthesized and tested for binding affinity to human histamine H3 receptors (hH3Rs stably expressed in HEK-293 cells and human H4Rs (hH4Rs co-expressed with Gαi2 and Gβ1γ2 subunits in Sf9 cells. Working in part from the lead compound 6-(4-methylpiperazin-1-yl-N4-(3-(piperidin-1-ylpropylpyrimidine-2,4-diamine (compound 1 with unsatisfactory affinity and selectivity to hH3Rs, our structure-activity relationship studies revealed that replacement of 4-methylpiperazino by N-benzylamine and substitution of an amine group at the 2-position of the 2-aminopyrimidine core structure with 3-piperidinopropoxyphenyl moiety as an hH3R pharmacophore resulted in N4-benzyl-N2-(4-(3-(piperidin-1-ylpropoxyphenylpyrimidine-2,4-diamine (compound 5 with high hH3R affinity (ki =4.49±1.25 nM and H3R receptor subtype selectivity of more than 6,500×. Moreover, initial metric analyses were conducted based on their target-oriented drug-likeness for predictively quantifying lipophilicity, ligand efficiency, lipophilicity-dependent ligand efficiency, molecular size-independent efficiency, and topological molecular polar surface. As to the development of potential H3R ligands, results showed that integration of the hH3R pharmacophore in hH4R-affine structural scaffolds resulted in compounds with high hH3R affinity (4.5–650 nM, moderate to low hH4R affinity (4,500–30,000 nM, receptor subtype selectivity

  2. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook;

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and...

  3. Ligand binding alters dimerization and sequestering of urokinase receptors in raft-mimicking lipid mixtures.

    Science.gov (United States)

    Ge, Yifan; Siegel, Amanda P; Jordan, Rainer; Naumann, Christoph A

    2014-11-01

    Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.

  4. Roles of cell and microvillus deformation and receptor-ligand binding kinetics in cell rolling.

    Science.gov (United States)

    Pawar, Parag; Jadhav, Sameer; Eggleton, Charles D; Konstantopoulos, Konstantinos

    2008-10-01

    Polymorphonuclear leukocyte (PMN) recruitment to sites of inflammation is initiated by selectin-mediated PMN tethering and rolling on activated endothelium under flow. Cell rolling is modulated by bulk cell deformation (mesoscale), microvillus deformability (microscale), and receptor-ligand binding kinetics (nanoscale). Selectin-ligand bonds exhibit a catch-slip bond behavior, and their dissociation is governed not only by the force but also by the force history. Whereas previous theoretical models have studied the significance of these three "length scales" in isolation, how their interplay affects cell rolling has yet to be resolved. We therefore developed a three-dimensional computational model that integrates the aforementioned length scales to delineate their relative contributions to PMN rolling. Our simulations predict that the catch-slip bond behavior and to a lesser extent bulk cell deformation are responsible for the shear threshold phenomenon. Cells bearing deformable rather than rigid microvilli roll slower only at high P-selectin site densities and elevated levels of shear (>or=400 s(-1)). The more compliant cells (membrane stiffness=1.2 dyn/cm) rolled slower than cells with a membrane stiffness of 3.0 dyn/cm at shear rates >50 s(-1). In summary, our model demonstrates that cell rolling over a ligand-coated surface is a highly coordinated process characterized by a complex interplay between forces acting on three distinct length scales.

  5. A D-peptide ligand of nicotine acetylcholine receptors for brain-targeted drug delivery.

    Science.gov (United States)

    Wei, Xiaoli; Zhan, Changyou; Shen, Qing; Fu, Wei; Xie, Cao; Gao, Jie; Peng, Chunmei; Zheng, Ping; Lu, Weiyue

    2015-03-01

    Lysosomes of brain capillary endothelial cells are implicated in nicotine acetylcholine receptor (nAChR)-mediated transcytosis and act as an enzymatic barrier for the transport of peptide ligands to the brain. A D-peptide ligand of nAChRs (termed (D)CDX), which binds to nAChRs with an IC50 value of 84.5 nM, was developed by retro-inverso isomerization. (D)CDX displayed exceptional stability in lysosomal homogenate and serum, and demonstrated significantly higher transcytosis efficiency in an in vitro blood-brain barrier monolayer compared with the parent L-peptide. When modified on liposomal surface, (D)CDX facilitated significant brain-targeted delivery of liposomes. As a result, brain-targeted delivery of (D)CDX modified liposomes enhanced therapeutic efficiency of encapsulated doxorubicin for glioblastoma. This study illustrates the importance of ligand stability in nAChRs-mediated transcytosis, and paves the way for developing stable brain-targeted entities.

  6. Structural insights into human peroxisome proliferator activated receptor delta (PPAR-delta selective ligand binding.

    Directory of Open Access Journals (Sweden)

    Fernanda A H Batista

    Full Text Available Peroxisome proliferator activated receptors (PPARs δ, α and γ are closely related transcription factors that exert distinct effects on fatty acid and glucose metabolism, cardiac disease, inflammatory response and other processes. Several groups developed PPAR subtype specific modulators to trigger desirable effects of particular PPARs without harmful side effects associated with activation of other subtypes. Presently, however, many compounds that bind to one of the PPARs cross-react with others and rational strategies to obtain highly selective PPAR modulators are far from clear. GW0742 is a synthetic ligand that binds PPARδ more than 300-fold more tightly than PPARα or PPARγ but the structural basis of PPARδ:GW0742 interactions and reasons for strong selectivity are not clear. Here we report the crystal structure of the PPARδ:GW0742 complex. Comparisons of the PPARδ:GW0742 complex with published structures of PPARs in complex with α and γ selective agonists and pan agonists suggests that two residues (Val312 and Ile328 in the buried hormone binding pocket play special roles in PPARδ selective binding and experimental and computational analysis of effects of mutations in these residues confirms this and suggests that bulky substituents that line the PPARα and γ ligand binding pockets as structural barriers for GW0742 binding. This analysis suggests general strategies for selective PPARδ ligand design.

  7. NRLiSt BDB, the manually curated nuclear receptors ligands and structures benchmarking database.

    Science.gov (United States)

    Lagarde, Nathalie; Ben Nasr, Nesrine; Jérémie, Aurore; Guillemain, Hélène; Laville, Vincent; Labib, Taoufik; Zagury, Jean-François; Montes, Matthieu

    2014-04-10

    Nuclear receptors (NRs) constitute an important class of drug targets. We created the most exhaustive NR-focused benchmarking database to date, the NRLiSt BDB (NRs ligands and structures benchmarking database). The 9905 compounds and 339 structures of the NRLiSt BDB are ready for structure-based and ligand-based virtual screening. In the present study, we detail the protocol used to generate the NRLiSt BDB and its features. We also give some examples of the errors that we found in ChEMBL that convinced us to manually review all original papers. Since extensive and manually curated experimental data about NR ligands and structures are provided in the NRLiSt BDB, it should become a powerful tool to assess the performance of virtual screening methods on NRs, to assist the understanding of NR's function and modulation, and to support the discovery of new drugs targeting NRs. NRLiSt BDB is freely available online at http://nrlist.drugdesign.fr .

  8. Multi-scale Simulation of Receptor-Ligand-Mediated Adhesion of Two (PMN) Leukocytes

    Science.gov (United States)

    Gupta, Vijay; Konstantopoulos, Kostas; Eggleton, Charles

    2008-11-01

    Leukocytes are recruited from the bloodstream to the site of inflammation through interactions between cell surface receptors and complementary ligands expressed on the surface of the endothelium. PMNs rolling on activated endothelium can mediate secondary capture of PMNs flowing in the free stream through homotypic interactions. This interaction is mediated by L-selectin binding to PSGL-1 between the free-stream and adherent PMNs. Both L-selectin and PSGL-1 molecules are concentrated on the tips of PMN microvilli. It has been demonstrated that steady application of a threshold level of shear rate is necessary to support PMN homotypic aggregation in bulk suspension. A reduction of shear rate below a threshold value diminishes the probability of cell adhesion. Cell aggregation is a complex phenomenon involving the interplay of bond kinetics and hydrodynamics. We simulate PSGL-1--L-selectin-mediated homotypic leukocyte adhesion-dissociation under an externally applied force field using the Immersed Boundary Method. We investigate the influence of membrane elasticity and kinetic parameters on contact area, bond dynamics, average number of bonds formed and their respective life time. A Hookean spring model is used to characterize receptor-ligand bonds and their stochastic nature is simulated using the Monte Carlo technique.

  9. Analysis of ligand-receptor cross-linked fragments by mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Son, C.D. [University of Tennessee, Knoxville (UTK); Sargsyan, H. [City University of New York (CUNY); Hurst, Gregory {Greg} B [ORNL; Naider, F. [City University of New York (CUNY); Becker, J.M. [University of Tennessee, Knoxville (UTK)

    2005-01-01

    G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The a-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable a-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to crosslinked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an a-factor analog with the photoaffinity group p-benzoyl-L-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of a-factor and a region of Ste2p covering residues 251 294.

  10. Crystallographic Identification and Functional Characterization of Phospholipids as Ligands for the Orphan Nuclear Receptor Steroidogenic Factor-1

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yong; Choi, Mihwa; Cavey, Greg; Daugherty, Jennifer; Suino, Kelly; Kovach, Amanda; Bingham, Nathan C.; Kliewer, Steven A.; Xu, H.Eric (Van Andel); (U. of Texas-SMED)

    2010-11-10

    The orphan nuclear receptor steroidogenic factor 1 (SF-1) regulates the differentiation and function of endocrine glands. Although SF-1 is constitutively active in cell-based assays, it is not known whether this transcriptional activity is modulated by ligands. Here, we describe the 1.5 {angstrom} crystal structure of the SF-1 ligand binding domain in complex with an LXXLL motif from a coregulator protein. The structure reveals the presence of a phospholipid ligand in a surprisingly large pocket ({approx}1600 {angstrom}{sup 3}), with the receptor adopting the canonical active conformation. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid abolish SF-1/coactivator interactions and significantly reduce SF-1 transcriptional activity. These findings provide evidence that SF-1 is regulated by endogenous ligands and suggest an unexpected relationship between phospholipids and endocrine development and function.

  11. Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hae; Jun, Hee-jin; Hoang, Minh-Hien; Jia, Yaoyao [Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Department of Biotechnology, Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Han, Xiang Hua [College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763 (Korea, Republic of); Lee, Dong-Ho [Department of Biotechnology, Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Lee, Hak-Ju [Division of Green Business Management, Department of Forest Resources Utilization, Korean Forest Research Institute, Seoul 130-712 (Korea, Republic of); Hwang, Bang Yeon, E-mail: byhwang@chungbuk.ac.kr [College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763 (Korea, Republic of); Lee, Sung-Joon, E-mail: junelee@korea.ac.kr [Division of Food Bioscience and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Department of Biotechnology, Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Catalposide is a novel ligand for PPAR{alpha}. Black-Right-Pointing-Pointer Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid {beta}-oxidation and synthesis. Black-Right-Pointing-Pointer Catalposdie reduces hepatic triacylglycerides. Black-Right-Pointing-Pointer Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPAR{alpha}) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPAR{alpha} agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPAR{alpha} agonist, identified from reporter gene assay-based activity screening with approximately 900 natural plant and seaweed extracts. Results of time-resolved fluorescence resonance energy transfer analyses suggested that the compound interacted directly with the ligand-binding domain of PPAR{alpha}. Cultured hepatocytes stimulated with catalposide exhibited significantly reduced cellular triglyceride concentrations, by 21%, while cellular uptake of fatty acids was increased, by 70% (P < 0.05). Quantitative PCR analysis revealed that the increase in cellular fatty acid uptake was due to upregulation of fatty acid transporter protein-4 (+19% vs. the control) in cells stimulated with catalposide. Additionally, expression of genes related to fatty acid oxidation and high-density lipoprotein metabolism were upregulated, while that of genes related to fatty acid synthesis were suppressed. In conclusion, catalposide is hypolipidemic by activation of PPAR{alpha} via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

  12. 60 YEARS OF POMC: Melanocortin receptors: evolution of ligand selectivity for melanocortin peptides.

    Science.gov (United States)

    Dores, Robert M; Liang, Liang; Davis, Perry; Thomas, Alexa L; Petko, Bogdana

    2016-05-01

    The evolution of the melanocortin receptors (MCRs) is linked to the evolution of adrenocorticotrophic hormone (ACTH), the melanocyte-stimulating hormones (MSHs), and their common precursor pro-opiomelanocortin (POMC). The origin of the MCRs and POMC appears to be grounded in the early radiation of the ancestral protochordates. During the genome duplications that have occurred during the evolution of the chordates, the organization plan for POMC was established, and features that have been retained include, the high conservation of the amino acid sequences of α-MSH and ACTH, and the presence of the HFRW MCR activation motif in all of the melanocortin peptides (i.e. ACTH, α-MSH, β-MSH, γ-MSH, and δ-MSH). For the MCRs, the chordate genome duplication events resulted in the proliferation of paralogous receptor genes, and a divergence in ligand selectivity. While most gnathostome MCRs can be activated by either ACTH or the MSHs, teleost and tetrapod MC2R orthologs can only be activated by ACTH. The appearance of the accessory protein, MRAP1, paralleled the emergence of teleost and tetrapods MC2R ligand selectivity, and the dependence of these orthologs on MRAP1 for trafficking to the plasma membrane. The accessory protein, MRAP2, does not affect MC2R ligand selectivity, but does influence the functionality of MC4R orthologs. In this regard, the roles that these accessory proteins may play in the physiology of the five MCRs (i.e. MC1R, MC2R, MC3R, MC4R, and MC5R) are discussed. PMID:26792827

  13. Successful virtual screening for a submicromolar antagonist of the neurokinin-1 receptor based on a ligand-supported homology model.

    Science.gov (United States)

    Evers, Andreas; Klebe, Gerhard

    2004-10-21

    The neurokinin-1 (NK1) receptor belongs to the family of G-protein-coupled receptors (GPCRs), which represents one of the most relevant target families in small-molecule drug design. In this paper, we describe a homology modeling of the NK1 receptor based on the high-resolution X-ray structure of rhodopsin and the successful virtual screening based on this protein model. The NK1 receptor model has been generated using our new MOBILE (modeling binding sites including ligand information explicitly) approach. Starting with preliminary homology models, it generates improved models of the protein binding pocket together with bound ligands. Ligand information is used as an integral part in the homology modeling process. For the construction of the NK1 receptor, antagonist CP-96345 was used to restrain the modeling. The quality of the obtained model was validated by probing its ability to accommodate additional known NK1 antagonists from structurally diverse classes. On the basis of the generated model and on the analysis of known NK1 antagonists, a pharmacophore model was deduced, which subsequently guided the 2D and 3D database search with UNITY. As a following step, the remaining hits were docked into the modeled binding pocket of the NK1 receptor. Finally, seven compounds were selected for biochemical testing, from which one showed affinity in the submicromolar range. Our results suggest that ligand-supported homology models of GPCRs may be used as effective platforms for structure-based drug design.

  14. Ligand-specific Deactivation Time Course of GluN1/GluN2D NMDA Receptors

    Energy Technology Data Exchange (ETDEWEB)

    K Vance; N Simorowski; S Traynelis; H Furukawa

    2011-12-31

    N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors that mediate a majority of excitatory synaptic transmission. One unique property of GluN1/GluN2D NMDA receptors is an unusually prolonged deactivation time course following the removal of L-glutamate. Here we show, using x-ray crystallography and electrophysiology, that the deactivation time course of GluN1/GluN2D receptors is influenced by the conformational variability of the ligand-binding domain (LBD) as well as the structure of the activating ligand. L-glutamate and L-CCG-IV induce significantly slower deactivation time courses compared with other agonists. Crystal structures of the isolated GluN2D LBD in complex with various ligands reveal that the binding of L-glutamate induces a unique conformation at the backside of the ligand-binding site in proximity to the region at which the transmembrane domain would be located in the intact receptors. These data suggest that the activity of the GluN1/GluN2D NMDA receptor is controlled distinctively by the endogenous neurotransmitter L-glutamate.

  15. The GABA-A benzodiazepine receptor complex: Role of pet and spect in neurology and psychiatry; Der GABA-A-benzodiazepinrezeptorkomplex: Rolle von PET und SPECT in Neurologie und Psychiatrie

    Energy Technology Data Exchange (ETDEWEB)

    Juengling, F.D. [Abt. fuer Nuklearmedizin, Radiologie III, Universitaetsklinik Ulm (Germany); Schaefer, M.; Heinz, A. [Klinik fuer Psychiatrie und Psychotherapie, Charite, Humboldt-Univ. zu Berlin (Germany)

    2002-09-01

    Nuclear medicine imaging techniques such as positron emission tomography (PET) and single photon emission tomography (SPECT) for selective depiction of GABA-A-benzodiazepine receptor (GBZR) binding are complementary investigations in the diagnostic process of neurological and psychiatric disorders. This review summarizes the current knowledge about options and limitations of PET and SPECT for in vivo diagnostics in neurology and psychiatry. The growing importance of GBZR-imaging for the understanding of pathophysiology and pharmacological treatment in different psychiatric syndromes is discussed. (orig.) [German] Mit der Entwicklung selektiver Liganden fuer den GABA-A-Benzodiazepinrezeptorkomplex (GBZR) hat die nuklearmedizinische Bildgebung mittels positronen-emissionstomographie (PET) und single-photon-emissionscomputertomographie (SPECT) einen festen Stellenwert fuer Klinik und Forschung in der Neurologie und Psychiatrie erlangt. Die vorliegende Ueberblicksarbeit fasst den aktuellen Wissensstand von Anwendungsmoeglichkeiten und -grenzen der nuklearmedizinischen Bildgebung der GBZR in vivo zusammen und beleuchtet ihren klinischen Nutzen. Die wachsende Bedeutung fuer das Verstaendnis der Pathophysiologie und pharmakotherapeutischer Konzepte unterschiedlicher psychiatrischer Erkrankungen wird herausgestellt. (orig.)

  16. Therapeutic and Adverse Effects of a Non-Steroidal Glucocorticoid Receptor Ligand in a Mouse Model of Multiple Sclerosis

    OpenAIRE

    Simone Wüst; Denise Tischner; Michael John; Tuckermann, Jan P; Christiane Menzfeld; Uwe-Karsten Hanisch; Jens van den Brandt; Fred Lühder; Reichardt, Holger M.

    2009-01-01

    BACKGROUND: Dissociating glucocorticoid receptor (GR) ligands hold great promise for treating inflammatory disorders since it is assumed that they exert beneficial activities mediated by transrepression but avoid adverse effects of GR action requiring transactivation. Here we challenged this paradigm by investigating 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (CpdA), a dissociating non-steroidal GR ligand, in the context of experimental autoimmune encephalomyelitis (EAE), an...

  17. Killer Cell Immunoglobulin-Like Receptor-Ligand Matching and Outcomes after Unrelated Cord Blood Transplantation in Acute Myeloid Leukemia.

    Science.gov (United States)

    Rocha, Vanderson; Ruggeri, Annalisa; Spellman, Stephen; Wang, Tao; Sobecks, Ronald; Locatelli, Franco; Askar, Medhat; Michel, Gerard; Arcese, William; Iori, Anna Paola; Purtill, Duncan; Danby, Robert; Sanz, Guillermo F; Gluckman, Eliane; Eapen, Mary

    2016-07-01

    The effect of killer cell immunoglobulin-like receptor (KIR)-ligand matching on outcomes after unrelated cord blood (CB) transplantation was studied in 461 patients with acute myeloid leukemia, categorizing KIR ligand for HLA-C groups C1 and C2 and Bw4. Donor-recipient HLA matching considered allele-level matching at HLA-A, -B, -C, and -DRB1. Separate analyses were conducted for 6-7/8 HLA-matched and 3-5/8 HLA-matched transplants because HLA matching confounded KIR-ligand matching (ie, KIR-ligand mismatching was less likely with better HLA matching). All patients received single CB unit and myeloablative conditioning. There were no significant differences in nonrelapse mortality (NRM), relapse, and overall mortality by KIR-ligand match status. However, among recipients of 3-5/8 HLA-matched transplants, NRM (HR, 2.26; P = .008) and overall mortality (HR, 1.78; P = .008) but not relapse were higher with KIR-ligand mismatched (host-versus-graft direction) compared with KIR-ligand matched transplants. These data do not support selecting CB units based on KIR-ligand match status for transplants mismatched at 1 or 2 HLA loci. Although transplants mismatched at 3 or more HLA loci are not recommended, avoiding KIR-ligand mismatching in this setting lowers mortality risks. PMID:27090957

  18. GTP synthases. Proton pumping and phosphorylation in ligand-receptor-G alpha-protein complexes.

    Science.gov (United States)

    Nederkoorn, P H; Timmerman, H; Donné-Op Den Kelder, G M; Timms, D; Wilkinson, A J; Kelly, D R; Broadley, K J; Davies, R H

    1996-01-01

    A structural model for a ligand-receptor-Gs alpha-protein complex to function as a GTP synthase is presented. The mechanism which is dependent on the movement and rotation of the G alpha-protein alpha 2-helix is seen to involve the delivery of, at least, one proton to the phosphorylation site in the rotation of this helix. The cycle is driven by a ligand-mediated proton pump through the alpha-helices of the receptor, attachment of the conserved Tyr-Arg-Tyr receptor proton shuttle being made to an aspartate group on the Gs alpha-protein terminal sidechain, which is itself linked to the Asn-Gln interaction known to control movement and rotation of the alpha 2-helix between .GDP and .GTP structures. The energetics of proton transfer through the shuttle mechanism and delivery of a proton to the aspartate group are shown to be sufficient to rupture this controlling interaction and its associated backbone bond. The complex leads to full spatial and energetic definition of the receptor proton shuttle mechanism, while there is a striking association of further Tyrosine and Arginine residues in the vicinity of the Gs alpha-protein Asn-Gln interaction. Calculations at the HF 6-31G** level confirm that a critical balance between ion pair and neutral forms of Tyr-Arg interactions under multiply hydrogen bonded conditions in a hydrophobic environment controls proton transfer and recovery mechanisms. The intrinsic preference of the neutral Tyr-Arg form over the ion-pair is 14.0 kcal/mol. Activation of the Tyrosine oxygen atom in the neutral form by single-NH or -OH groups reduces this difference by some 6.4-8.6 kcal/mol but the dominance of the neutral form is maintained. The expected slight overestimates are consistent with the maximum activation enthalpy of 11.0-12.0 kcal/ mol required to initiate proton transfer through the shuttle. The extended form of the shuttle with the Arginine acting competitively between the two Tyrosine residues allows interpretation of observed

  19. In vivo (/sup 3/H)flunitrazepam binding: imaging of receptor regulation

    Energy Technology Data Exchange (ETDEWEB)

    Ciliax, B.J.; Penney, J.B. Jr.; Young, A.B.

    1986-08-01

    The use of (/sup 3/H)flunitrazepam as a ligand to measure alterations in benzodiazepine receptors in vivo in rats was investigated. Animals were injected with (/sup 3/H)flunitrazepam i.v., arterial samples of (/sup 3/H)flunitrazepam were obtained and, later, the animals were sacrificed to assay brain binding. (/sup 3/H)flunitrazepam enters the brain rapidly and binds to benzodiazepine receptors. About two-thirds of this binding is blocked by predosing the animals with 5 mg/kg of clonazepam. The amount of remaining (nonspecific) binding correlates very well (r = 0.88) with the amount of radioactivity found in plasma at the time of death. A series of rats were lesioned unilaterally with kainic acid in the caudate-putamen several months before the infusion of (/sup 3/H)flunitrazepam. In vivo autoradiography in lesioned rats showed that benzodiazepine binding in globus pallidus and substantia nigra on the side of the lesion was increased significantly as compared to the intact side. The observed changes in benzodiazepine binding were similar to those observed previously in lesioned rats using in vitro techniques. Thus, benzodiazepine receptor regulation can be imaged quantitatively using in vivo binding techniques.

  20. In vivo [3H]flunitrazepam binding: imaging of receptor regulation

    International Nuclear Information System (INIS)

    The use of [3H]flunitrazepam as a ligand to measure alterations in benzodiazepine receptors in vivo in rats was investigated. Animals were injected with [3H]flunitrazepam i.v., arterial samples of [3H]flunitrazepam were obtained and, later, the animals were sacrificed to assay brain binding. [3H]flunitrazepam enters the brain rapidly and binds to benzodiazepine receptors. About two-thirds of this binding is blocked by predosing the animals with 5 mg/kg of clonazepam. The amount of remaining (nonspecific) binding correlates very well (r = 0.88) with the amount of radioactivity found in plasma at the time of death. A series of rats were lesioned unilaterally with kainic acid in the caudate-putamen several months before the infusion of [3H]flunitrazepam. In vivo autoradiography in lesioned rats showed that benzodiazepine binding in globus pallidus and substantia nigra on the side of the lesion was increased significantly as compared to the intact side. The observed changes in benzodiazepine binding were similar to those observed previously in lesioned rats using in vitro techniques. Thus, benzodiazepine receptor regulation can be imaged quantitatively using in vivo binding techniques

  1. Distribution and dynamics of rat basophilic leukemia immunoglobulin E receptors (FcepsilonRI) on planar ligand-presenting surfaces.

    Science.gov (United States)

    Spendier, Kathrin; Carroll-Portillo, Amanda; Lidke, Keith A; Wilson, Bridget S; Timlin, Jerilyn A; Thomas, James L

    2010-07-21

    There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. We have quantified the spatiotemporal dynamics of the redistribution of immunoglobulin E-loaded receptors (IgE-FcepsilonRI) on rat basophilic leukemia-2H3 mast cells in contact with fluid and gel-phase membranes displaying ligands for immunoglobulin E, using total internal reflection fluorescence microscopy. To clearly separate the kinetics of receptor redistribution from cell spreading, and to precisely define the initial contact time (+/-50 ms), micropipette cell manipulation was used to bring individual cells into contact with surfaces. On ligand-free surfaces, there are micron-scale heterogeneities in fluorescence that likely reflect regions of the cell that are more closely apposed to the substrate. When ligands are present, receptor clusters form with this same size scale. The initial rate of accumulation of receptors into the clusters is consistent with diffusion-limited trapping with D approximately 10(-1) microm2/s. These results support the hypothesis that clusters form by diffusion to cell-surface contact regions. Over longer timescales (>10 s), individual clusters moved with both diffusive and directed motion components. The dynamics of the cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes.

  2. Ligand binding reduces SUMOylation of the peroxisome proliferator-activated receptor γ (PPARγ activation function 1 (AF1 domain.

    Directory of Open Access Journals (Sweden)

    Rolf Diezko

    Full Text Available Peroxisome proliferator-activated receptor gamma (PPARγ is a ligand-activated nuclear receptor regulating adipogenesis, glucose homeostasis and inflammatory responses. The activity of PPARγ is controlled by post-translational modifications including SUMOylation and phosphorylation that affects its biological and molecular functions. Several important aspects of PPARγ SUMOylation including SUMO isoform-specificity and the impact of ligand binding on SUMOylation remain unresolved or contradictory. Here, we present a comprehensive study of PPARγ1 SUMOylation. We show that PPARγ1 can be modified by SUMO1 and SUMO2. Mutational analyses revealed that SUMOylation occurs exclusively within the N-terminal activation function 1 (AF1 domain predominantly at lysines 33 and 77. Ligand binding to the C-terminal ligand-binding domain (LBD of PPARγ1 reduces SUMOylation of lysine 33 but not of lysine 77. SUMOylation of lysine 33 and lysine 77 represses basal and ligand-induced activation by PPARγ1. We further show that lysine 365 within the LBD is not a target for SUMOylation as suggested in a previous report, but it is essential for full LBD activity. Our results suggest that PPARγ ligands negatively affect SUMOylation by interdomain communication between the C-terminal LBD and the N-terminal AF1 domain. The ability of the LBD to regulate the AF1 domain may have important implications for the evaluation and mechanism of action of therapeutic ligands that bind PPARγ.

  3. Synthetic Peptide Ligands of the Antigen Binding Receptor Induce Programmed Cell Death in a Human B-Cell Lymphoma

    Science.gov (United States)

    Renschler, Markus F.; Bhatt, Ramesh R.; Dower, William J.; Levy, Ronald

    1994-04-01

    Peptide ligands for the antigen binding site of the surface immunoglobulin receptor of a human B-cell lymphoma cell line were identified with the use of filamentous phage libraries displaying random 8- and 12-amino acid peptides. Corresponding synthetic peptides bound specifically to the antigen binding site of this immunoglobulin receptor and blocked the binding of an anti-idiotype antibody. The ligands, when conjugated to form dimers or tetramers, induced cell death by apoptosis in vitro with an IC50 between 40 and 200 nM. This effect was associated with specific stimulation of intracellular protein tyrosine phosphorylation.

  4. Feedbacks, Receptor Clustering, and Receptor Restriction to Single Cells yield large Turing Spaces for Ligand-receptor based Turing Models

    OpenAIRE

    Kurics, Tamás; Menshykau, Denis; Iber, Dagmar

    2014-01-01

    Turing mechanisms can yield a large variety of patterns from noisy, homogenous initial conditions and have been proposed as patterning mechanism for many developmental processes. However, the molecular components that give rise to Turing patterns have remained elusive, and the small size of the parameter space that permits Turing patterns to emerge makes it difficult to explain how Turing patterns could evolve. We have recently shown that Turing patterns can be obtained with a single ligand i...

  5. Rat neuronal nicotinic acetylcholine receptors containing a7 subunit: pharmacological properties of ligand binding and function

    Institute of Scientific and Technical Information of China (English)

    Yingxian XIAO; Galya R ABDRAKHMANOVA; Maryna BAYDYUK; Susan HERNANDEZ; Kenneth J KELLAR

    2009-01-01

    Aim: To compare pharmacological properties of heterologously expressed homomeric a7 nicotinic acetylcholine receptors (a.7 nAChRs) with those of native nAChRs containing a.7 subunit (a.7* nAChRs) in rat hippocampus and cerebral cortex. Methods: We established a stably transfected HEK-293 cell line that expresses homomeric rat a7 nAChRs. We studies ligand binding profiles and functional properties of nAChRs expressed in this cell line and native rat a.7* nAChRs in rat hippocampus and cerebral cortex. We used [125IJ-a-bungarotoxin to compare ligand binding profiles in these cells with those in rat hippocampus and cerebral cortex. The functional properties of the a.7 nAChRs expressed in this cell line were studied using whole-cell current recording.Results: The newly established cell line, KXa7Rl, expresses homomeric a7 nAChRs that bind [125I]-a-bungarotoxin with a Kd value of 0.38±0.06 nmol/L, similar to Kj values of native rat a.7* nAChRs from hippocampus (Kd=0.28±0.03 nmol/L) and cerebral cortex (Kd=0.33±0.05 nmol/L). Using whole-cell current recording, the homomeric a7 nAChRs expressed in the cells were activated by acetylcholine and (-)-nicotine with EC50 values of 280±19 nmol/L and 180±40 nmol/L, respectively. The acetylcholine activated currents were potently blocked by two selective antagonists of a.7 nAChRs, a-bungarotoxin (IC5o=19±2 nmol/L) and methyllycaconitine (IC50=100±10 pmol/L). A comparative study of ligand binding profiles, using 13 nicotinic ligands, showed many similarities between the homomeric a.7 nAChRs and native a.7* receptors in rat brain, but it also revealed several notable differences.Conclusion: This newly established stable cell line should be very useful for studying the properties of homomeric a7 nAChRs and comparing these properties to native a.7* nAChRs.

  6. Ligand-dependent inhibition of beta-catenin/TCF signaling by androgen receptor.

    Science.gov (United States)

    Chesire, Dennis R; Isaacs, William B

    2002-12-01

    Beta-catenin signaling may contribute to prostate cancer (CaP) progression. Although beta-catenin is known to upregulate T cell factor (TCF) target gene expression in CaP cells, recent evidence demonstrates its capacity to enhance ligand-dependent androgen receptor (AR) function. Thus, we wished to further understand the interaction between these two pathways. We find in both CaP cells (CWR22-Rv1, LAPC-4, DU145) and non-CaP cells (HEK-293, TSU, SW480, HCT-116) that beta-catenin/TCF-related transcription (CRT), as measured by activation of a synthetic promoter and that of cyclin D1, is inhibited by androgen treatment. This inhibition is AR-dependent, as it only occurs in cells expressing AR endogenously or transiently, and is abrogated by AR antagonists. Additional analyses convey that the ligand-dependent nature of CRT suppression depends on transactivation-competent AR in the nucleus, but not on indirect effects stemming from AR target gene expression. Given the recent work identifying an AR/beta-catenin interaction, and from our finding that liganded AR does not prompt gross changes in the constitutive nuclear localization of TCF4 or mutant beta-catenin, we hypothesized that transcription factor (i.e. AR and TCF) competition for beta-catenin recruitment may explain, in part, androgen-induced suppression of CRT. To address this idea, we expressed an AR mutant lacking its DNA-binding domain (DBD). This receptor could not orchestrate ligand-dependent CRT repression, thereby providing support for those recent data implicating the AR DBD/LBD as necessary for beta-catenin interaction. Further supporting this hypothesis, TCF/LEF over-expression counteracts androgen-induced suppression of CRT, and requires beta-catenin binding activity to do so. Interestingly, TCF4 over-expression potently antagonizes AR function; however, this inhibition may occur independently of beta-catenin/TCF4 interaction. These results from TCF4 over-expression analyses, taken together, provide

  7. (/sup 3/H)-(Thr4,Gly7)OT: a highly selective ligand for central and peripheral OT receptors

    Energy Technology Data Exchange (ETDEWEB)

    Elands, J.; Barberis, C.; Jard, S.

    1988-01-01

    Oxytocin receptors in rat hippocampal synaptic plasma membranes were compared with mammary gland and uterine oxytocin receptors. For this purpose, a highly specific oxytocic agonist (Thr4,Gly7)oxytocin was tritiated. We demonstrated that this ligand labels oxytocin receptors selectively. Scatchard analyses revealed a high affinity for all the oxytocin receptors investigated, with equilibrium dissociation constants between 1.0 and 2.0 nM. Binding appeared to take place at a single population of receptor sites. Competition experiments confirmed the high affinity of arginine vasopressin for hippocampal oxytocin receptors but also revealed that mammary gland and uterine oxytocin receptors do not discriminate more efficiently between oxytocin and arginine vasopressin. This lack in specificity is not affected by applying different concentrations of Mg ions.

  8. Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization.

    Directory of Open Access Journals (Sweden)

    Irina M Kuznetsova

    Full Text Available In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA and ANS - bovine serum albumin (BSA interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.

  9. Revealing the sequence and resulting cellular morphology of receptor-ligand interactions during Plasmodium falciparum invasion of erythrocytes.

    Directory of Open Access Journals (Sweden)

    Greta E Weiss

    2015-02-01

    Full Text Available During blood stage Plasmodium falciparum infection, merozoites invade uninfected erythrocytes via a complex, multistep process involving a series of distinct receptor-ligand binding events. Understanding each element in this process increases the potential to block the parasite's life cycle via drugs or vaccines. To investigate specific receptor-ligand interactions, they were systematically blocked using a combination of genetic deletion, enzymatic receptor cleavage and inhibition of binding via antibodies, peptides and small molecules, and the resulting temporal changes in invasion and morphological effects on erythrocytes were filmed using live cell imaging. Analysis of the videos have shown receptor-ligand interactions occur in the following sequence with the following cellular morphologies; 1 an early heparin-blockable interaction which weakly deforms the erythrocyte, 2 EBA and PfRh ligands which strongly deform the erythrocyte, a process dependant on the merozoite's actin-myosin motor, 3 a PfRh5-basigin binding step which results in a pore or opening between parasite and host through which it appears small molecules and possibly invasion components can flow and 4 an AMA1-RON2 interaction that mediates tight junction formation, which acts as an anchor point for internalization. In addition to enhancing general knowledge of apicomplexan biology, this work provides a rational basis to combine sequentially acting merozoite vaccine candidates in a single multi-receptor-blocking vaccine.

  10. /sup 125/I-spiperone: a novel ligand for D/sub 2/ dopamine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Gundlach, A.L.; Largent, B.L.; Synder, S.H.

    1984-11-05

    /sup 125/I-Spiperone binds with high affinity K/sub D/ 0.3 nM) to a single specific site (B/sub max/ 34 pmole/g wet weight) in homogenates of rat corpus striatum. Specific binding is about 40-60 percent of total binding and is displaced stereo-specifically by butaclamol and clopenthixol. Neuroleptic drugs of various classes are potent inhibitors of /sup 125/I-spiperone binding (/sub i/'s 1-10 nM). Selective dopamine antagonists such as sulpiride (K/sub i/ 50 nM) and dopamine agonists such as apomorphine (K/sub i/ 200 nM) are also potent inhibitors. The drugs specificity of /sup 125/I-spiperone binding correlates well with that of /sup 3/H-spiperone binding, providing good evidence that /sup 125/I-spiperone labels D/sub 2/ dopamine receptors in striatal membranes. /sup 125/I-Spiperone, with its high specific activity (2200 Ci/mmol) may prove to be a useful ligand in studies examining D/sub 2/ dopamine receptors in soluble preparations and by autoradiography. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a /sup 123/I-labeled form for imaging of dopamine receptors, in vivo, using single photon tomography. 18 references, 4 figures, 1 table.

  11. GABAB receptor ligands for the treatment of alcohol use disorder: preclinical and clinical evidence

    Directory of Open Access Journals (Sweden)

    Roberta eAgabio

    2014-06-01

    Full Text Available The present paper summarizes the preclinical and clinical studies conducted to define the anti-alcohol pharmacological profile of the prototypic GABAB receptor agonist, baclofen, and its therapeutic potential for treatment of alcohol use disorder (AUD. Numerous studies have reported baclofen-induced suppression of alcohol drinking (including relapse- and binge-like drinking and alcohol reinforcing, motivational, stimulating, and rewarding properties in rodents and monkeys. The majority of clinical surveys conducted to date – including case reports, retrospective chart reviews, and randomized placebo-controlled studies – suggest the ability of baclofen to suppress alcohol consumption, craving for alcohol, and alcohol withdrawal symptomatology in alcohol-dependent patients. The recent identification of a positive allosteric modulatory binding site, together with the synthesis of in vivo effective ligands, represents a novel, and likely more favorable, option for pharmacological manipulations of the GABAB receptor. Accordingly, data collected to date suggest that positive allosteric modulators of the GABAB receptor reproduce several anti-alcohol effects of baclofen and display a higher therapeutic index (with larger separation – in terms of doses – between anti-alcohol effects and sedation.

  12. Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells.

    LENUS (Irish Health Repository)

    O'Callaghan, G

    2012-02-03

    Fas ligand (FasL\\/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E(2) (PGE(2)), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE(2) increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E(2)-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE(2) positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE(2).

  13. Drug-likeness approach of 2-aminopyrimidines as histamine H3 receptor ligands.

    Science.gov (United States)

    Sadek, Bassem; Schreeb, Annemarie; Schwed, Johannes Stephan; Weizel, Lilia; Stark, Holger

    2014-01-01

    A small series of compounds containing derivatives of 2,4-diamino- and 2,4,6-triaminopyrimidine (compounds 2-7) was synthesized and tested for binding affinity to human histamine H3 receptors (hH3Rs) stably expressed in HEK-293 cells and human H4Rs (hH4Rs) co-expressed with Gαi2 and Gβ1γ2 subunits in Sf9 cells. Working in part from the lead compound 6-(4-methylpiperazin-1-yl)-N (4)-(3-(piperidin-1-yl)propyl)pyrimidine-2,4-diamine (compound 1) with unsatisfactory affinity and selectivity to hH3Rs, our structure-activity relationship studies revealed that replacement of 4-methylpiperazino by N-benzylamine and substitution of an amine group at the 2-position of the 2-aminopyrimidine core structure with 3-piperidinopropoxyphenyl moiety as an hH3R pharmacophore resulted in N (4)-benzyl-N (2)-(4-(3-(piperidin-1-yl)propoxy)phenyl)pyrimidine-2,4-diamine (compound 5) with high hH3R affinity (k(i) =4.49 ± 1.25 nM) and H3R receptor subtype selectivity of more than 6,500×. Moreover, initial metric analyses were conducted based on their target-oriented drug-likeness for predictively quantifying lipophilicity, ligand efficiency, lipophilicity-dependent ligand efficiency, molecular size-independent efficiency, and topological molecular polar surface. As to the development of potential H3R ligands, results showed that integration of the hH3R pharmacophore in hH4R-affine structural scaffolds resulted in compounds with high hH3R affinity (4.5-650 nM), moderate to low hH4R affinity (4,500-30,000 nM), receptor subtype selectivity (ratio hH4R/hH3R; 8-6,500), and promising calculated drug-likeness properties. PMID:25278747

  14. Probing the structure and function of the estrogen receptor ligand binding domain by analysis of mutants with altered transactivation characteristics.

    OpenAIRE

    Eng, F C; Lee, H.S.; Ferrara, J; Willson, T M; White, J H

    1997-01-01

    We have developed a genetic screen for the yeast Saccharomyces cerevisiae to isolate estrogen receptor (ER) mutants with altered transactivation characteristics. Use of a "reverse" ER, in which the mutagenized ligand binding domain was placed at the N terminus of the receptor, eliminated the isolation of truncated constitutively active mutants. A library was screened with a low-affinity estrogen, 2-methoxyestrone (2ME), at concentrations 50-fold lower than those required for activation of the...

  15. The Effects of Exogenous and Endogenous Ligands of the Aryl Hydrocarbon Receptor on the Activation of Autoimmune Diabetes

    OpenAIRE

    Abu-Rizq, Hana'A

    2012-01-01

    The aryl-hydrocarbon receptor (AhR) is an important receptor found in immune cells. Itfunctions as a detector of environmental toxins, naturally occurring dietary products, andendogenous tryptophan derivatives for induction of gene transcription responses. Previousreports have implicated stimulation of AhR by various ligands in promoting T cellactivation or regulatory function, with effects on autoimmune disease models. Also, effectsof Ah toxins or natural products on increasing or suppressin...

  16. Discovery of novel ligands for mouse olfactory receptor MOR42-3 using an in silico screening approach and in vitro validation.

    Directory of Open Access Journals (Sweden)

    Selvan Bavan

    Full Text Available The ligands for many olfactory receptors remain largely unknown despite successful heterologous expression of these receptors. Understanding the molecular receptive range of olfactory receptors and deciphering the olfactory recognition code are hampered by the huge number of odorants and large number of olfactory receptors, as well as the complexity of their combinatorial coding. Here, we present an in silico screening approach to find additional ligands for a mouse olfactory receptor that allows improved definition of its molecular receptive range. A virtual library of 574 odorants was screened against a mouse olfactory receptor MOR42-3. We selected the top 20 candidate ligands using two different scoring functions. These 40 odorant candidate ligands were then tested in vitro using the Xenopus oocyte heterologous expression system and two-electrode voltage clamp electrophysiology. We experimentally confirmed 22 of these ligands. The candidate ligands were screened for both agonist and antagonist activity. In summary, we validated 19 agonists and 3 antagonists. Two of the newly identified antagonists were of low potency. Several previously known ligands (mono- and dicarboxylic acids are also confirmed in this study. However, some of the newly identified ligands were structurally dissimilar compounds with various functional groups belonging to aldehydes, phenyls, alkenes, esters and ethers. The high positive predictive value of our in silico approach is promising. We believe that this approach can be used for initial deorphanization of olfactory receptors as well as for future comprehensive studies of molecular receptive range of olfactory receptors.

  17. An integrated methodology for data processing in dynamic force spectroscopy of ligand-receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Odorico, M.; Teulon, J.-M.; Berthoumieu, O. [CEA-Valrho, DSV-DIEP-SBTN, BP 17171, Bagnols sur Ceze 30207 (France); Chen, S.-W. [13 avenue de la Mayre, Bagnols sur Ceze 30200 (France); Parot, P.; Pellequer, J.-L. [CEA-Valrho, DSV-DIEP-SBTN, BP 17171, Bagnols sur Ceze 30207 (France)

    2007-10-15

    Dynamic force spectroscopy (DFS), using atomic force microscopy (AFM), is a powerful tool to study ligand-receptor binding. The interaction mode of two binding partners is investigated by exploring stochastic behaviors of bond rupture events. However, to define a rupture event from force-distance measurements is not conclusive or unique in literature. To reveal the influence of event identification methods, we have developed an efficient protocol to manage tremendous amount of data by implementing different choices of peak selection from the force-distance curve. This data processing software simplifies routinely experimental procedures such as cantilever spring constant and force-distance curve calibrations, statistical treatments of data, and analysis distributions of rupture events. In the present work, we took available experimental data from a complex between a chelate metal compound and a monoclonal antibody as a study system.

  18. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Bokoch, Michael P; Zou, Yaozhong; Rasmussen, Søren Gøgsig Faarup;

    2010-01-01

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation...... extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known...... conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive...

  19. Role of Killer Immunoglobulin-Like Receptor and Ligand Matching in Donor Selection

    Directory of Open Access Journals (Sweden)

    Meral Beksaç

    2012-01-01

    Full Text Available Despite all efforts to improve HLA typing and immunosuppression, it is still impossible to prevent severe graft versus host disease (GVHD which can be fatal. GVHD is not always associated with graft versus malignancy and can prevent stem cell transplantation from reaching its goals. Overall T-cell alloreactivity is not the sole mechanism modulating the immune defense. Innate immune system has its own antigens, ligands, and mediators. The bridge between HLA and natural killer (NK cell-mediated reactions is becoming better understood in the context of stem cell transplantation. Killer immunoglobulin-like receptors (KIRs constitute a wide range of alleles/antigens segregated independently from the HLA alleles and classified into two major haplotypes which imprints the person's ability to suppress or to amplify T-cell alloreactivity. This paper will summarize the impact of both activating and inhibitory KIRs and their ligands on stem cell transplantation outcome. The ultimate goal is to develop algorithms based on KIR profiles to select donors with maximum antileukemic and minimum antihost effects.

  20. Ligand fishing using new chitosan based functionalized Androgen Receptor magnetic particles.

    Science.gov (United States)

    Marszałł, Michał Piotr; Sroka, Wiktor Dariusz; Sikora, Adam; Chełminiak, Dorota; Ziegler-Borowska, Marta; Siódmiak, Tomasz; Moaddel, Ruin

    2016-08-01

    Superparamagnetic nanoparticles with chemically modified chitosan has been proposed as a potential support for the immobilization of the androgen receptor (AR). The study involved comparison of different AR carriers like commercially available magnetic beads coated with silica (BcMag) and chitosan coated nanoparticles with different amount of amino groups. The immobilization was carried out through covalent immobilization of the AR through the terminal amino group or through available carboxylic acids. The initial characterization of the AR coated magnetic beads was carried out with dihydrotestosterone, a known AR ligand. Subsequently, chitosan modified nanporticles with long-distanced primary amino groups (Fe3O4CS-(NH2)3) (upto 8.34mM/g) were used for further study to isolate known AR ligands (bicalutamide, flutamide, hydroxyflutamide and levonogestrel) from a mixture of tested compounds in ammonium acetate buffer [10mM, pH 7.4]. The results showed that the selected nanoparticles are a promising semi-quantitative tool for the identification of high affinity compounds to AR and might be of special importance in the identification of novel agonists or antiandrogens. PMID:27156644

  1. Effect of adrenergic receptor ligands on metaiodobenzylguanidine uptake and storage in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Babich, J.W. [Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts (United States)]|[Department of Radiology, Harvard Medical School, Boston, Massachusetts (United States); Graham, W. [Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts (United States); Fischman, A.J. [Division of Nuclear Medicine, Department of Radiology, Massachusetts General Hospital, Boston, Massachusetts (United States)]|[Department of Radiology, Harvard Medical School, Boston, Massachusetts (United States)

    1997-05-01

    The effects of adrenergic receptor ligands on uptake and storage of the radiopharmaceutical [{sup 125}I]metaiodobenzylguanidine (MIBG) were studied in the human neuroblastoma cell line SK-N-SH. For uptake studies, cells were with varying concentrations of {alpha}-agonist (clonidine, methoxamine, and xylazine), {alpha}-antagonist (phentolamine, tolazoline, phenoxybenzamine, yohimbine, and prazosin), {beta}-antagonist (propranolol, atenolol), {beta}-agonist (isoprenaline and salbutamol), mixed {alpha}/{beta} antagonist (labetalol), or the neuronal blocking agent guanethidine, prior to the addition of [{sup 125}I]MIBG (0.1 {mu}M). The incubation was continued for 2 h and specific cell-associated radioactivity was measured. For the storage studies, cells were incubated with [{sup 125}I]MIBG for 2 h, followed by replacement with fresh medium with or without drug (MIBG, clonidine, or yohimbine). Cell-associated radioactivity was measured at various times over the next 20 h. Propanolol reduced [{sup 125}I]MIBG uptake by approximately 30% (P<0.01) at all concentrations tested, most likely due to nonspecific membrane changes. In conclusion, the results of this study establish that selected adrenergic ligands can significantly influence the pattern of uptake and storage of MIBG in cultured neuroblastoma cells, most likely through inhibition of uptake or through noncompetitive inhibition. The potential inplications of these findings justify further study. (orig./VHE). With 4 figs., 1 tab.

  2. Discovery of allosteric modulators for GABAA receptors by ligand-directed chemistry.

    Science.gov (United States)

    Yamaura, Kei; Kiyonaka, Shigeki; Numata, Tomohiro; Inoue, Ryuji; Hamachi, Itaru

    2016-10-01

    The fast inhibitory actions of γ-aminobutyric acid (GABA) are mainly mediated by GABAA receptors (GABAARs) in the brain. The existence of multiple ligand-binding sites and a lack of structural information have hampered the efficient screening of drugs capable of acting on GABAARs. We have developed semisynthetic fluorescent biosensors for orthosteric and allosteric GABAAR ligands on live cells via coupling of affinity-based chemical labeling reagents to a bimolecular fluorescence quenching and recovery system. These biosensors were amenable to the high-throughput screening of a chemical library, leading to the discovery of new small molecules capable of interacting with GABAARs. Electrophysiological measurements revealed that one hit, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), was a novel negative allosteric modulator capable of strongly suppressing GABA-induced chloride currents. Thus, these semisynthetic biosensors represent versatile platforms for screening drugs to treat GABAAR-related neurological disorders, and this strategy can be extended to structurally complicated membrane proteins. PMID:27526031

  3. CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands.

    Science.gov (United States)

    Lamas, Bruno; Richard, Mathias L; Leducq, Valentin; Pham, Hang-Phuong; Michel, Marie-Laure; Da Costa, Gregory; Bridonneau, Chantal; Jegou, Sarah; Hoffmann, Thomas W; Natividad, Jane M; Brot, Loic; Taleb, Soraya; Couturier-Maillard, Aurélie; Nion-Larmurier, Isabelle; Merabtene, Fatiha; Seksik, Philippe; Bourrier, Anne; Cosnes, Jacques; Ryffel, Bernhard; Beaugerie, Laurent; Launay, Jean-Marie; Langella, Philippe; Xavier, Ramnik J; Sokol, Harry

    2016-06-01

    Complex interactions between the host and the gut microbiota govern intestinal homeostasis but remain poorly understood. Here we reveal a relationship between gut microbiota and caspase recruitment domain family member 9 (CARD9), a susceptibility gene for inflammatory bowel disease (IBD) that functions in the immune response against microorganisms. CARD9 promotes recovery from colitis by promoting interleukin (IL)-22 production, and Card9(-/-) mice are more susceptible to colitis. The microbiota is altered in Card9(-/-) mice, and transfer of the microbiota from Card9(-/-) to wild-type, germ-free recipients increases their susceptibility to colitis. The microbiota from Card9(-/-) mice fails to metabolize tryptophan into metabolites that act as aryl hydrocarbon receptor (AHR) ligands. Intestinal inflammation is attenuated after inoculation of mice with three Lactobacillus strains capable of metabolizing tryptophan or by treatment with an AHR agonist. Reduced production of AHR ligands is also observed in the microbiota from individuals with IBD, particularly in those with CARD9 risk alleles associated with IBD. Our findings reveal that host genes affect the composition and function of the gut microbiota, altering the production of microbial metabolites and intestinal inflammation. PMID:27158904

  4. Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

    Science.gov (United States)

    Wu, Qing-Ping; Zhang, Lei; Shao, Xiao-Xia; Wang, Jia-Hui; Gao, Yu; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-04-01

    Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1. PMID:26767372

  5. Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system.

    Science.gov (United States)

    Lemaire, Géraldine; Mnif, Wissem; Pascussi, Jean-Marc; Pillon, Arnaud; Rabenoelina, Fanja; Fenet, Hélène; Gomez, Elena; Casellas, Claude; Nicolas, Jean-Claude; Cavaillès, Vincent; Duchesne, Marie-Josèphe; Balaguer, Patrick

    2006-06-01

    Pregnane X receptor (PXR, NR1I2) is activated by various chemically unrelated compounds, including environmental pollutants and drugs. We proceeded here to in vitro screening of 28 pesticides with a new reporter system that detects human pregnane X receptor (hPXR) activators. The cell line was obtained by a two-step stable transfection of cervical cancer HeLa cells. The first transfected cell line, HG5LN, contained an integrated luciferase reporter gene under the control of a GAL4 yeast transcription factor-binding site. The second cell line HGPXR was derived from HG5LN and stably expressed hPXR ligand-binding domain fused to GAL4 DNA-binding domain (DBD). The HG5LN cells were used as a control to detect nonspecific activities. Pesticides from various chemical classes were demonstrated, for the first time, to be hPXR activators: (1) herbicides: pretilachlor, metolachlor, and alachlor chloracetanilides, oxadiazon oxiconazole, and isoproturon urea; (2) fungicides: bupirimate and fenarimol pyrimidines, propiconazole, fenbuconazole, prochloraz conazoles, and imazalil triazole; and (3) insecticides: toxaphene organochlorine, permethrin pyrethroid, fipronil pyrazole, and diflubenzuron urea. Pretilachlor, metolachlor, bupirimate, and oxadiazon had an affinity for hPXR equal to or greater than the positive control rifampicin. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 expression in a primary culture of human hepatocytes. HGPXR, with HG5LN as a reference, was grafted onto nude mice to assess compound bioavailability through in vivo quantification of hPXR activation. Altogether, our data indicate that HGPXR cells are an efficient tool for identifying hPXR ligands and establishing pesticides as hPXR activators.

  6. Synthesis and binding characteristics of [(3)H]neuromedin N, a NTS2 receptor ligand.

    Science.gov (United States)

    Tóth, Fanni; Mallareddy, Jayapal Reddy; Tourwé, Dirk; Lipkowski, Andrzej W; Bujalska-Zadrozny, Magdalena; Benyhe, Sándor; Ballet, Steven; Tóth, Géza; Kleczkowska, Patrycja

    2016-06-01

    Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [(3)H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454nM in rat brain and 425nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8±30.18nM, while the Bmax value corresponded to 3.8±0.2pmol/mg protein in rat brain membranes. The specific binding of [(3)H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na(+) ions was observed on the binding of [(3)H]NN, resulting in an IC50 of 150.6mM. In [(35)S]GTPγS binding experiments the Emax value was 112.3±1.4% in rat brain and 112.9±2.4% in rat spinal cord membranes and EC50 values of 0.7nM and 0.79nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [(3)H]NN labels NTS2 receptors in rat brain membranes. PMID:26707235

  7. Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226

    International Nuclear Information System (INIS)

    Crystal structure of the ligand-binding domain of androgen receptor in complex with LGD2226. The androgen receptor (AR) is a ligand-inducible steroid hormone receptor that mediates androgen action, determining male sexual phenotypes and promoting spermatogenesis. As the androgens play a dominant role in male sexual development and function, steroidal androgen agonists have been used clinically for some years. However, there is a risk of potential side effects and most steroidal androgens cannot be dosed orally, which limits the use of these substances. 1,2-Dihydro-6-N,N-bis(2,2,2-trifluoroethyl) amino-4-trifluoromethyl-2-quinolinone (LGD2226) is a synthetic nonsteroidal ligand and a novel selective AR modulator. The crystal structure of the complex of LGD2226 with the androgen receptor ligand-binding domain (AR LBD) at 2.1 Å was solved and compared with the structure of the AR LBD–R1881 complex. It is hoped that this will aid in further explaining the selectivity of LGD2226 observed in in vitro and in vivo assays and in developing more selective and effective therapeutic agents

  8. Serum Levels of Soluble Fas Ligand and Soluble Fas Receptor in Patients with Chronic Congestive Heart Failure

    Institute of Scientific and Technical Information of China (English)

    李刚; 令狐华; 魏良明

    2002-01-01

    @@ To understand the pathophysiologic significances of soluble Fas Ligand (sFasL) and soluble Fas receptor (sFas) in chronic congestive heart failure (CHF) and to determine the relationship of circulating levels of sFasL and sFas to the severity of CHF, the serum sFasL and sfas levels were evaluated in patients with CHF.

  9. Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain

    NARCIS (Netherlands)

    Stoorvogel, W; Kerstens, S; Fritzsche, I; den Hartigh, JC; Oud, R; van der Heyden, MAG; Henegouwen, PMPVE

    2004-01-01

    Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the sec

  10. Identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Jacob Lauwring, E-mail: jla@mb.au.dk [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark); Schrøder, Tenna Juul; Christensen, Søren [H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark); Strandbygård, Dorthe [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark); Pallesen, Lone Tjener [Aarhus University, Ole Worms Allé 3, 8000 Aarhus C (Denmark); García-Alai, Maria Marta [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark); Lindberg, Samsa; Langgård, Morten; Eskildsen, Jørgen Calí; David, Laurent; Tagmose, Lena; Simonsen, Klaus Baek; Maltas, Philip James; Rønn, Lars Christian Biilmann; Jong, Inge E. M. de; Malik, Ibrahim John; Egebjerg, Jan; Karlsson, Jens-Jacob [H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark); Uppalanchi, Srinivas; Sakumudi, Durga Rao; Eradi, Pradheep [GVK BioScience, Plot No. 28 A, IDA Nacharam, Hyderabad 500 076 (India); Watson, Steven P., E-mail: jla@mb.au.dk [H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark); Thirup, Søren, E-mail: jla@mb.au.dk [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark)

    2014-02-01

    The identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex are reported. Sortilin is a type I membrane glycoprotein belonging to the vacuolar protein sorting 10 protein (Vps10p) family of sorting receptors and is most abundantly expressed in the central nervous system. Sortilin has emerged as a key player in the regulation of neuronal viability and has been implicated as a possible therapeutic target in a range of disorders. Here, the identification of AF40431, the first reported small-molecule ligand of sortilin, is reported. Crystals of the sortilin–AF40431 complex were obtained by co-crystallization and the structure of the complex was solved to 2.7 Å resolution. AF40431 is bound in the neurotensin-binding site of sortilin, with the leucine moiety of AF40431 mimicking the binding mode of the C-terminal leucine of neurotensin and the 4-methylumbelliferone moiety of AF40431 forming π-stacking with a phenylalanine.

  11. Endocytosis via galactose receptors in vivo. Ligand size directs uptake by hepatocytes and/or liver macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Schlepper-Schaefer, J.; Huelsmann, D.; Djovkar, A.; Meyer, H.E.; Herbertz, L.; Kolb, H.; Kolb-Bachofen, V.

    1986-01-01

    The intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues is rat liver was followed. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au/sub 5/), 17 nm (Au/sub 17/), 50 nm (Au/sub 50/) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au/sub 5/ in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc.

  12. Molecular interaction studies of hemostasis: fibrinogen ligand-human platelet receptor interactions

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Imshik; Marchant, Roger E

    2003-10-15

    The interactions between fibrinogen ligands and platelet receptor {alpha}{sub IIb}{beta}{sub 3} were studied under physiological conditions by atomic force microscopy (AFM). Two linear peptide sequences in fibrinogen, RGD and HHLGGAKQAGDV, play central roles in the regulation of hemostasis and thrombosis by facilitating adhesion and aggregation of platelets. In order to measure the interactions (i.e., debonding force), oligopeptides, GSSSGaaa, where aaa is -RGDSPA or -HHLGGAKQAGDV, were synthesized and grafted on to the surface of AFM probe tips. The interaction forces between a peptide-modified AFM probe tip and platelet surface were determined from pN to nN levels using AFM force measurements. Our results show that the zero kinetic off-rate, K{sub off}(0), for RGDSPA is significantly smaller than that for HHLGGAKQAGDV, under the consideration of flexible receptor surfaces. From our analysis, the K{sub off}(0), the single molecular binding energy E{sub b}, and the transition state x{sub b}, were extracted from the data, and estimated to be 1.53 s{sup -1}, -2.64x10{sup -20} J and 1.03 A for the RGD-{alpha}{sub IIb}{beta}{sub 3} system, and 47.58 s{sup -1}, 2.67x10{sup -20}, 1.09 A for the HHLGGAKQAGDV-{alpha}{sub IIb}{beta}{sub 3} system, respectively.

  13. Brownian nanoimaging of interface dynamics and ligand-receptor binding at cell surfaces in 3-D.

    Science.gov (United States)

    Kuznetsov, Igor R; Evans, Evan A

    2013-04-01

    We describe a method for nanoimaging interfacial dynamics and ligand-receptor binding at surfaces of live cells in 3-D. The imaging probe is a 1-μm diameter glass bead confined by a soft laser trap to create a "cloud" of fluctuating states. Using a facile on-line method of video image analysis, the probe displacements are reported at ~10 ms intervals with bare precisions (±SD) of 4-6 nm along the optical axis (elevation) and 2 nm in the transverse directions. We demonstrate how the Brownian distributions are analyzed to characterize the free energy potential of each small probe in 3-D taking into account the blur effect of its motions during CCD image capture. Then, using the approach to image interactions of a labeled probe with lamellae of leukocytic cells spreading on cover-glass substrates, we show that deformations of the soft distribution in probe elevations provide both a sensitive long-range sensor for defining the steric topography of a cell lamella and a fast telemetry for reporting rare events of probe binding with its surface receptors. Invoking established principles of Brownian physics and statistical thermodynamics, we describe an off-line method of super resolution that improves precision of probe separations from a non-reactive steric boundary to ~1 nm.

  14. Increased expression of cytokines, soluble cytokine receptors, soluble apoptosis ligand and apoptosis in dengue.

    Science.gov (United States)

    Arias, Julia; Valero, Nereida; Mosquera, Jesús; Montiel, Milagros; Reyes, Eduardo; Larreal, Yraima; Alvarez-Mon, Melchor

    2014-03-01

    Several studies have been performed to determine biomarkers that define the risk factors to developing severe forms of dengue. In this study, the levels of TNF-α, IL-6, IL-1, IL-17, soluble interleukin-1 receptor like 1 protein (sST2), soluble TNF-related apoptosis-inducing ligand (sTRAIL), IL-12 and soluble receptors for TNF (sTNF-RI and sTNF-RII) were determined by ELISA in dengue patients and monocyte/macrophage cultures. Dengue was classified as dengue without warning symptoms (DNWS), with warning symptoms (DWWS) and severe dengue (SD). High values of IL-6, sTNFRI, sTNFRII and sST2 were observed in DWWS and/or SD and IL-12 and sTRAIL in DNWS. TNF-α and IL-17 were increased not associated to the disease severity. High production of TNF-α, IL-1β, IL-12, IL-17, sST2 and sTRAIL and apoptosis expression were observed in dengue monocyte/macrophage cultures. This study shows that beneficial or deleterious biomarkers can be present in dengue regardless the disease severity and that monocytes may be in part the source of studied molecules.

  15. EANM procedure guidelines for brain neurotransmission SPECT/PET using dopamine D2 receptor ligands, version 2

    DEFF Research Database (Denmark)

    Van Laere, Koen; Varrone, Andrea; Booij, Jan;

    2010-01-01

    The guidelines summarize the current views of the European Association of Nuclear Medicine Neuroimaging Committee (ENC). The aims of the guidelines are to assist nuclear medicine practitioners in making recommendations, performing, interpreting and reporting the results of clinical dopamine D2...... receptor SPECT or PET studies, and to achieve a high quality standard of dopamine D2 receptor imaging, which will increase the impact of this technique in neurological practice.The present document is an update of the first guidelines for SPECT using D2 receptor ligands labelled with (123)I [1] and was...

  16. Expression cloning of cDNA encoding a seven-helix receptor from human placenta with affinity for opioid ligands

    OpenAIRE

    1992-01-01

    Here we report the expression cloning of cDNA encoding a putative opioid receptor from a human placenta cDNA library. Placental opioid receptors are of the kappa type. As the dynorphin opioid peptides are kappa-selective, a dynorphin ligand was used in an affinity-enrichment (panning) procedure to select transiently transfected COS-7 cells expressing kappa receptor binding sites. The cloned cDNA encodes a 440-residue protein of the seven-helix guanine nucleotide-binding protein (G-protein)-co...

  17. Selective modulation of Wnt ligands and their receptors in adipose tissue by chronic hyperadiponectinemia.

    Directory of Open Access Journals (Sweden)

    Nobuhiko Wada

    Full Text Available BACKGROUND: Adiponectin-transgenic mice had many small adipocytes in both subcutaneous and visceral adipose tissues, and showed higher sensitivity to insulin, longer life span, and reduced chronic inflammation. We hypothesized that adiponectin regulates Wnt signaling in adipocytes and thereby modulates adipocyte proliferation and chronic inflammation in adipose tissue. MATERIALS AND METHODS: We examined the expression of all Wnt ligands and their receptors and the activity of Wnt signaling pathways in visceral adipose tissue from wild-type mice and two lines of adiponectin-transgenic mice. The effects of adiponectin were also investigated in cultured 3T3-L1 cells. RESULTS: The Wnt5b, Wnt6, Frizzled 6 (Fzd6, and Fzd9 genes were up-regulated in both lines of transgenic mice, whereas Wnt1, Wnt2, Wnt5a, Wnt9b, Wnt10b, Wnt11, Fzd1, Fzd2, Fzd4, Fzd7, and the Fzd coreceptor low-density-lipoprotein receptor-related protein 6 (Lrp6 were reduced. There was no difference in total β-catenin levels in whole-cell extracts, non-phospho-β-catenin levels in nuclear extracts, or mRNA levels of β-catenin target genes, indicating that hyperadiponectinemia did not affect canonical Wnt signaling. In contrast, phosphorylated calcium/calmodulin-dependent kinase II (p-CaMKII and phosphorylated Jun N-terminal kinase (p-JNK were markedly reduced in adipose tissue from the transgenic mice. The adipose tissue of the transgenic mice consisted of many small cells and had increased expression of adiponectin, whereas cyclooxygenase-2 expression was reduced. Wnt5b expression was elevated in preadipocytes of the transgenic mice and decreased in diet-induced obese mice, suggesting a role in adipocyte differentiation. Some Wnt genes, Fzd genes, and p-CaMKII protein were down-regulated in 3T3-L1 cells cultured with a high concentration of adiponectin. CONCLUSION: Chronic hyperadiponectinemia selectively modulated the expression of Wnt ligands, Fzd receptors and LRP coreceptors

  18. Orthogonal Optical Control of a G Protein-Coupled Receptor with a SNAP-Tethered Photochromic Ligand.

    Science.gov (United States)

    Broichhagen, Johannes; Damijonaitis, Arunas; Levitz, Joshua; Sokol, Kevin R; Leippe, Philipp; Konrad, David; Isacoff, Ehud Y; Trauner, Dirk

    2015-10-28

    The covalent attachment of synthetic photoswitches is a general approach to impart light sensitivity onto native receptors. It mimics the logic of natural photoreceptors and significantly expands the reach of optogenetics. Here we describe a novel photoswitch design-the photoswitchable orthogonal remotely tethered ligand (PORTL)-that combines the genetically encoded SNAP-tag with photochromic ligands connected to a benzylguanine via a long flexible linker. We use the method to convert the G protein-coupled receptor mGluR2, a metabotropic glutamate receptor, into a photoreceptor (SNAG-mGluR2) that provides efficient optical control over the neuronal functions of mGluR2: presynaptic inhibition and control of excitability. The PORTL approach enables multiplexed optical control of different native receptors using distinct bioconjugation methods. It should be broadly applicable since SNAP-tags have proven to be reliable, many SNAP-tagged receptors are already available, and photochromic ligands on a long leash are readily designed and synthesized. PMID:27162996

  19. Design and Characterization of Superpotent Bivalent Ligands Targeting Oxytocin Receptor Dimers via a Channel-Like Structure.

    Science.gov (United States)

    Busnelli, Marta; Kleinau, Gunnar; Muttenthaler, Markus; Stoev, Stoytcho; Manning, Maurice; Bibic, Lucka; Howell, Lesley A; McCormick, Peter J; Di Lascio, Simona; Braida, Daniela; Sala, Mariaelvina; Rovati, G Enrico; Bellini, Tommaso; Chini, Bice

    2016-08-11

    Dimeric/oligomeric states of G-protein coupled receptors have been difficult to target. We report here bivalent ligands consisting of two identical oxytocin-mimetics that induce a three order magnitude boost in G-protein signaling of oxytocin receptors (OTRs) in vitro and a 100- and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish. Through receptor mutagenesis and interference experiments with synthetic peptides mimicking transmembrane helices (TMH), we show that such superpotent behavior follows from the binding of the bivalent ligands to dimeric receptors based on a TMH1-TMH2 interface. Moreover, in this arrangement, only the analogues with a well-defined spacer length (∼25 Å) precisely fit inside a channel-like passage between the two protomers of the dimer. The newly discovered oxytocin bivalent ligands represent a powerful tool for targeting dimeric OTR in neurodevelopmental and psychiatric disorders and, in general, provide a framework to untangle specific arrangements of G-protein coupled receptor dimers. PMID:27420737

  20. Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

    Institute of Scientific and Technical Information of China (English)

    GENG De-chun; XU Yao-zeng; YANG Hui-lin; ZHU Guang-ming; WANG Xian-bin; ZHU Xue-song

    2011-01-01

    Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL)induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA)was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK),phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of >100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

  1. Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

    Science.gov (United States)

    Antoine, Thomas; Ott, David; Ebell, Katharina; Hansen, Kerrin; Henry, Luc; Becker, Frank; Hannus, Stefan

    2016-06-01

    G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. PMID:26954998

  2. Computational Analysis of the Ligand Binding Site of the Extracellular ATP Receptor, DORN1.

    Science.gov (United States)

    Nguyen, Cuong The; Tanaka, Kiwamu; Cao, Yangrong; Cho, Sung-Hwan; Xu, Dong; Stacey, Gary

    2016-01-01

    DORN1 (also known as P2K1) is a plant receptor for extracellular ATP, which belongs to a large gene family of legume-type (L-type) lectin receptor kinases. Extracellular ATP binds to DORN1 with strong affinity through its lectin domain, and the binding triggers a variety of intracellular activities in response to biotic and abiotic stresses. However, information on the tertiary structure of the ligand binding site of DORN1is lacking, which hampers efforts to fully elucidate the mechanism of receptor action. Available data of the crystal structures from more than 50 L-type lectins enable us to perform an in silico study of molecular interaction between DORN1 and ATP. In this study, we employed a computational approach to develop a tertiary structure model of the DORN1 lectin domain. A blind docking analysis demonstrated that ATP binds to a cavity made by four loops (defined as loops A B, C and D) of the DORN1 lectin domain with high affinity. In silico target docking of ATP to the DORN1 binding site predicted interaction with 12 residues, located on the four loops, via hydrogen bonds and hydrophobic interactions. The ATP binding pocket is structurally similar in location to the carbohydrate binding pocket of the canonical L-type lectins. However, four of the residues predicted to interact with ATP are not conserved between DORN1 and the other carbohydrate-binding lectins, suggesting that diversifying selection acting on these key residues may have led to the ATP binding activity of DORN1. The in silico model was validated by in vitro ATP binding assays using the purified extracellular lectin domain of wild-type DORN1, as well as mutated DORN1 lacking key ATP binding residues. PMID:27583834

  3. Structural and functional insights into the ligand-binding domain of a nonduplicated retinoid X nuclear receptor from the invertebrate chordate amphioxus

    OpenAIRE

    Tocchini-Valentini, Guiseppe D.; Rochel, Natacha; Escriva, Hector; Germain, Pierre; Peluso-Iltis, Carole; Paris, Mathilde; Sanglier-Cianferani, Sarah; Van Dorsselaer, Alain; Moras, Dino; Laudet, Vincent

    2009-01-01

    Retinoid X nuclear receptors (RXRs), as well as their insect orthologue, ultraspiracle protein (USP), play an important role in the transcription regulation mediated by the nuclear receptors as the common partner of many other nuclear receptors. Phylogenetic and structural studies have shown that the several evolutionary shifts have modified the ligand binding ability of RXRs. To understand the vertebrate-specific character of RXRs, we have studied the RXR ligand-binding domain of the cephalo...

  4. Identification and pharmacological characterization of a series of new 1H-4-substituted-imidazoyl histamine H3 receptor ligands.

    Science.gov (United States)

    Yates, S L; Phillips, J G; Gregory, R; Pawlowski, G P; Fadnis, L; Khan, M A; Ali, S M; Tedford, C E

    1999-05-01

    A new series of 1H-4-substituted imidazole compounds were synthesized and identified as potent and selective histamine (HA) H3 receptor ligands. These ligands establish that HA H3 antagonists exhibit stereoselective and conformational preferences in their binding to the HA H3 receptor. Structure-activity relationships were determined in vitro by HA H3 receptor-binding affinities using [3H]Nalpha-methylhistamine and rat cerebral cortical tissue homogenates. Several derivatives containing olefin, amide, and acetylene functional groups were identified as potent HA H3 receptor ligands. In the olefin series, GT-2227 (4-(6-cyclohexylhex-cis-3-enyl)imidazole) was identified as a potent HA H3 receptor ligand with a Ki of 4.2 +/- 0.6 nM, while the trans isomer (GT-2228) displayed a reduced potency (Ki = 15.2 +/- 2.4 nM). GT-2227 was also found to have excellent central nervous system penetration in an ex vivo binding paradigm (ED50 = 0.7 mg/kg i.p.). In the acetylene series, GT-2260 and GT-2286 both exhibited high affinity (Ki = 2.9 +/- 0.2 and 0.95 +/- 0.3 nM) and excellent central nervous system penetration profiles (ED50 = 0.43 and 0.48 mg/kg i.p., respectively). As a prototype for the series, GT-2227 showed high affinity for the human HA H3 receptor (3.2 nM) and minimal affinity for the human HA H1 (Ki = 13,407 +/- 540 nM) and H2 (Ki = 4,469 +/- 564 nM) receptor subtypes. GT-2227 also showed good selectivity for the HA H3 receptor over a broad spectrum of other neurotransmitter receptors (IC50 >/= 1 microM). Furthermore, GT-2227 improved acquisition in a cognitive paradigm without behavioral excitation or effect on spontaneous locomotor activity. In summary, the present studies demonstrate the development of novel HA H3-selective ligands, and lend support for the use of such agents in the treatment of disorders associated with cognitive or attentional deficits.

  5. Strategy for improved [{sup 11}C]DAA1106 radiosynthesis and in vivo peripheral benzodiazepine receptor imaging using microPET, evaluation of [{sup 11}C]DAA1106

    Energy Technology Data Exchange (ETDEWEB)

    Probst, Katrin C. [Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom)]|[BHF Carotid Imaging Group, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom)]. E-mail: kp296@wbic.cam.ac.uk; Izquierdo, David [Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom)]|[BHF Carotid Imaging Group, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom); Bird, Joseph L.E. [BHF Carotid Imaging Group, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom)]|[Department of Medicine, Cardiovascular Medicine Division, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom); Brichard, Laurent; Franck, Dominic; Fryer, Tim D.; Clark, John C. [Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom); Davies, John R. [Cardiovascular Medicine Division, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom); Richards, Hugh K. [Neurology Unit, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom); Davenport, Anthony P. [Clinical Pharmacology Unit, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom); Weissberg, Peter L. [Cardiovascular Medicine Division, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom); Warburton, Elizabeth A. [BHF Carotid Imaging Group, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom)]|[Department of Clinical Neurosciences, University of Cambridge, Addenbrooke' s Hospital, CB2 2QQ Cambridge (United Kingdom)

    2007-05-15

    Introduction: The peripheral benzodiazepine receptor (PBR) has shown considerable potential as a clinical marker of neuroinflammation and tumour progression. [{sup 11}C]DAA1106 ([{sup 11}C]N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)-acetamide) is a promising positron emission tomography (PET) radioligand for imaging PBRs. Methods: A four-step synthetic route was devised to prepare DAA1123, the precursor for [{sup 11}C]DAA1106. Two robust, high yielding methods for radiosynthesis based on [{sup 11}C]-O-methylation of DAA1123 were developed and implemented on a nuclear interface methylation module, producing [{sup 11}C]DAA1106 with up to 25% radiochemical yields at end-of-synthesis based on [{sup 11}C]CH{sub 3}I trapped. Evaluation of [{sup 11}C]DAA1106 for in vivo imaging was performed in a rabbit model with microPET, and the presence of PBR receptor in the target organ was further corroborated by immunohistochemistry. Results: The standard solution method produced 2.6-5.2 GBq (n=19) of [{sup 11}C]DAA1106, whilst the captive solvent method produced 1.6-6.3 GBq (n=10) of [{sup 11}C]DAA1106. Radiochemical purities obtained were 99% and specific radioactivity at end-of-synthesis was up to 200 GBq/{mu}mol for both methods. Based on radiochemical product, shorter preparation times and simplicity of synthesis, the captive solvent method was chosen for routine productions of [{sup 11}C]DAA1106. In vivo microPET [{sup 11}C]DAA1106 scans of rabbit kidney demonstrated high levels of binding in the cortex. The subsequent introduction of nonradioactive DAA1106 (0.2 {mu}mol) produced considerable displacement of the radioactive signal in this region. The presence of PBR in kidney cortex was further corroborated by immunohistochemistry. Conclusions: A robust, high yielding captive solvent method of [{sup 11}C]DAA1106 production was developed which enabled efficacious in vivo imaging of PBR expressing tissues in an animal model.

  6. Cloning, expression, and ligand-binding characterization of two neuropeptide Y receptor subtypes in orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Wang, Fei; Chen, Weimin; Lin, Haoran; Li, Wensheng

    2014-12-01

    As one of the most important multifunctional peptides, neuropeptide Y (NPY) performs its physiological functions through different subtype receptors. In this study, full-length cDNAs of two NPY receptors (YRs) in orange-spotted grouper (Epinephelus coioides) were cloned and named npy8br (y8b) and npy2r (y2). Phylogenetic analysis indicated that the Y8b receptor is an ortholog of the teleostean Y8b receptor, which belongs to the Y1 subfamily, and the Y2 receptor is an ortholog of the teleostean Y2 receptor, which belongs to the Y2 subfamily. Both of the YRs have G protein-coupled receptor family profiles. Multiple alignments demonstrate that the extracellular loop regions of YRs have distinctive residues of each species. Expression profile analysis revealed that the grouper Y8b receptor mRNA is primarily expressed in the brain, stomach and intestine, while the grouper Y2 receptor mRNA is primarily expressed in the brain, ovary, liver and heart. Double immunofluorescence analysis determined that the grouper YRs interact with the grouper NPY around the human embryonic kidney 293T cell surface. Furthermore, site-directed mutagenesis in a phage display system revealed that Asp(6.59) might be a common NPY-binding site, while Asp(2.68) of the Y8b receptor and Glu(5.24) of the Y2 receptor could be likely involved in subtype-specific binding. Combining the expression profile and ligand-binding feature, the grouper Y8b receptor could be involved in regulating food intake via the brain-gut axis and the grouper Y2 receptor might play a role in balancing the regulatory activity of the Y8b receptor and participate in metabolism in the liver and ovary.

  7. The selenium metabolite methylselenol regulates the expression of ligands that trigger immune activation through the lymphocyte receptor NKG2D

    DEFF Research Database (Denmark)

    Hagemann-Jensen, Michael Henrik; Uhlenbrock, Franziska Katharina; Kehlet, Stephanie;

    2014-01-01

    early during malignant transformation, and enables recognition and elimination of tumors by activating the lymphocyte receptor NKG2D. CH3SeH regulated NKG2D ligands both on the transcriptional and the posttranscriptional level: CH3SeH induced the transcription of MICA/B and ULBP2 mRNA, however......, the induction of cell-surface expression was restricted to the ligands MICA/B. Remarkably, our studies showed that CH3SeH inhibited ULBP2 surface transport through inhibition of the autophagic transport pathway. Finally, we identified extracellular calcium as being essential for CH3SeH -regulation of NKG2D...

  8. Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jørgensen, M U; Emr, S D; Winther, Jakob R.

    1999-01-01

    Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand....... The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p...

  9. New 3-, 8-disubstituted pyrazolo[5,1-c][1,2,4]benzotriazines useful for studying the interaction with the HBp-3 area (hydrogen bond point area) in the benzodiazepine site on the gamma-aminobutyric acid type A (GABAA) receptor

    OpenAIRE

    G. Guerrini; G. CICIANI; Bruni, F.; Selleri, S.; F. Melani; Daniele, S.; Martini, C; A. Costanzo

    2011-01-01

    The pharmacophoric model using ADLR procedure, based on pyrazolo[5,1-c][1,2,4]benzotriazine system, studied in our laboratory, allowed us to identify the essential interaction points (HBp-1, HBp-2, and Lp-1) and the important areas for affinity modulation (HBp-3 and Lp-2) for binding recognition at benzodiazepine (Bzs) site of GABAA receptors (GABAA-Rs). In this work ADLR method is used to rationalize the affinity data of 23 new compounds and to improve the knowledge on HBp-3 area...

  10. Novel Chemical Strategies for Labeling Small Molecule Ligands for Androgen, Progestin, and Peroxisome Proliferator-Activated Receptors for Imaging Prostate and Breast Cancer and the Heart

    International Nuclear Information System (INIS)

    Summary of Progress The specific aims of this project can be summarized as follows: Aim 1: Prepare and evaluate radiolabeled ligands for the peroxisome proliferator-activated receptor γ (PPARγ), a new nuclear hormone receptor target for tumor imaging and hormone therapy. Aim 2: Prepare steroids labeled with a cyclopentadienyl tricarbonyl technetium or rhenium unit. Aim 3: Prepare and evaluate other organometallic systems of novel design as ligand mimics and halogenated ligands for nuclear hormone receptor-based tumor imaging. As is described in detail in the report, we made excellent progress on all three of these aims; the highlights of our progress are the following: (1) we have prepared the first fluorine-18 labeled analogs of ligands for the PPARγ receptor and used these in tissue distribution studies in rats; (2) we have developed three new methods for the synthesis of cyclopentadienyltricarbonyl rhenium and technetium (CpRe(CO)3 and CpTc(CO)3) systems and we have adapted these to the synthesis of steroids labeled with these metals, as well as ligands for other receptor systems; (3) we have prepared a number of fluorine-18 labeled steroidal and non-steroidal androgens and measured their tissue distribution in rats; (4) we have prepared iodine and bromine-labeled progestins with high progesterone receptor binding affinity; and (5) we have prepared inorganic metal tricarbonyl complexes and steroid receptor ligands in which the metal tricarbonyl unit is an integral part off the ligand core

  11. Engineering the melanocortin-4 receptor to control constitutive and ligand-mediated G(S signaling in vivo.

    Directory of Open Access Journals (Sweden)

    Supriya Srinivasan

    Full Text Available The molecular and functional diversity of G protein-coupled receptors is essential to many physiological processes. However, this diversity presents a significant challenge to understanding the G protein-mediated signaling events that underlie a specific physiological response. To increase our understanding of these processes, we sought to gain control of the timing and specificity of G(s signaling in vivo. We used naturally occurring human mutations to develop two G(s-coupled engineered receptors that respond solely to a synthetic ligand (RASSLs. Our G(s-coupled RASSLs are based on the melanocortin-4 receptor, a centrally expressed receptor that plays an important role in the regulation of body weight. These RASSLs are not activated by the endogenous hormone alpha-melanocyte-stimulating hormone but respond potently to a selective synthetic ligand, tetrahydroisoquinoline. The RASSL variants reported here differ in their intrinsic basal activities, allowing the separation of the effects of basal signaling from ligand-mediated activation of the G(s pathway in vivo. These RASSLs can be used to activate G(s signaling in any tissue, but would be particularly useful for analyzing downstream events that mediate body weight regulation in mice. Our study also demonstrates the use of human genetic variation for protein engineering.

  12. Critical role of the endogenous interferon ligand-receptors in type I and type II interferons response.

    Science.gov (United States)

    Lasfar, Ahmed; Cook, Jeffry R; Cohen Solal, Karine A; Reuhl, Kenneth; Kotenko, Sergei V; Langer, Jerome A; Laskin, Debra L

    2014-07-01

    Separate ligand-receptor paradigms are commonly used for each type of interferon (IFN). However, accumulating evidence suggests that type I and type II IFNs may not be restricted to independent pathways. Using different cell types deficient in IFNAR1, IFNAR2, IFNGR1, IFNGR2 and IFN-γ, we evaluated the contribution of each element of the IFN system to the activity of type I and type II IFNs. We show that deficiency in IFNAR1 or IFNAR2 is associated with impairment of type II IFN activity. This impairment, presumably resulting from the disruption of the ligand-receptor complex, is obtained in all cell types tested. However, deficiency of IFNGR1, IFNGR2 or IFN-γ was associated with an impairment of type I IFN activity in spleen cells only, correlating with the constitutive expression of type II IFN (IFN-γ) observed on those cells. Therefore, in vitro the constitutive expression of both the receptors and the ligands of type I or type II IFN is critical for the enhancement of the IFN activity. Any IFN deficiency can totally or partially impair IFN activity, suggesting the importance of type I and type II IFN interactions. Taken together, our results suggest that type I and type II IFNs may regulate biological activities through distinct as well as common IFN receptor complexes.

  13. Identification of endogenous surrogate ligands for human P2Y12 receptors by in silico and in vitro methods

    International Nuclear Information System (INIS)

    Endogenous ligands acting on a human P2Y12 receptor, one of the G-protein coupled receptors, were searched by in silico screening against our own database, which contains more than 500 animal metabolites. The in silico screening using the docking software AutoDock resulted in selection of cysteinylleukotrienes (CysLTs) and 5-phosphoribosyl 1-pyrophosphate (PRPP), with high free energy changes, in addition to the known P2Y12 ligands such as 2MeSADP and ADP. These candidates were subjected to an in vitro Ca2+ assay using the CHO cells stably expressing P2Y12-G16α fusion proteins. We found that CysLTE4 and PRPP acted on the P2Y12 receptor as agonists with the EC50 values of 1.3 and 7.8 nM, respectively. Furthermore, we analyzed the phylogenetic relationship of the P2Y, P2Y-like, and CysLT receptors based on sequence alignment followed by evolutionary analyses. The analyses showed that the P2Y12, P2Y13, P2Y14, GPR87, CysLT-1, and CysLT-2 receptors formed a P2Y-related receptor subfamily with common sequence motifs in the transmembrane regions

  14. Homology-modeled ligand-binding domains of medaka estrogen receptors and androgen receptors: A model system for the study of reproduction

    International Nuclear Information System (INIS)

    Estrogen and androgen and their receptors play critical roles in physiological processes such as sexual differentiation and development. Using the available structural models for the human estrogen receptors alpha and beta and androgen receptor as templates, we designed in silico agonist and antagonist models of medaka estrogen receptor (meER) alpha, beta-1, and beta-2, and androgen receptor (meAR) alpha and beta. Using these models, we studied (1) the structural relationship between the ligand-binding domains (LBDs) of ERs and ARs of human and medaka, and (2) whether medaka ER and AR can be potential models for studying the ligand-binding activities of various agonists and antagonists of these receptors by docking analysis. A high level of conservation was observed between the sequences of the ligand-binding domains of meERα and huERα, meERβ1 and huERβ, meERβ2, and huERβ with 62.8%, 66.4%, and 65.1% identity, respectively. The sequence conservation between meARα and huAR, meARβ, and huAR was found with 70.1% and 61.0% of identity, respectively. Thirty-three selected endocrine disrupting chemicals (EDCs), including both agonists and antagonists, were docked into the LBD of ER and AR, and the corresponding docking score for medaka models and human templates were calculated. In order to confirm the conservation of the overall geometry and the binding pocket, the backbone root mean square deviation (RMSD) for Cα atoms was derived from the structure superposition of all 10 medaka homology models to the six human templates. Our results suggested conformational conservation between the ERs and ARs of medaka and human, Thus, medaka could be highly useful as a model system for studies involving estrogen and androgen interaction with their receptors.

  15. In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos

    International Nuclear Information System (INIS)

    We previously reported specific cross-linking of 125I-labeled insulin and 125I-labeled insulin-like growth factor I (IGF-I) to the alpha subunit of their respective receptors in chicken embryos of 20 somites and older. To achieve adequate sensitivity and localize spatially the receptors in younger embryos, we adapted an autoradiographic technique using whole-mounted chicken blastoderms. Insulin receptors and IGF-I receptors were expressed and could be localized as early as gastrulation, before the first somite is formed. Relative density was analyzed by a computer-assisted image system, revealing overall slightly higher binding of IGF-I than of insulin. Structures rich in both types of receptors were predominantly of ectodermal origin: Hensen's node in gastrulating embryos and neural folds, neural tube and optic vesicles during neurulation. The signal transduction capability of the receptors in early organogenesis was assessed by their ability to phosphorylate the exogenous substrate poly(Glu80Tyr20). Ligand-dependent tyrosine phosphorylation was demonstrable with both insulin and IGF-I in glycoprotein-enriched preparations from embryos at days 2 through 6 of embryogenesis. There was a developmentally regulated change in ligand-dependent tyrosine kinase activity, with a sharp increase from day 2 to day 4, in contrast with a small increase in the ligand binding. Binding of 125I-labeled IGF-I was, with the solubilized receptors, severalfold higher than binding of 125I-labeled insulin. However, the insulin-dependent phosphorylation was as high as the IGF-I-dependent phosphorylation at each developmental stage

  16. Assessing ligand efficiencies using template-based molecular docking and Tabu-clustering on tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) derivatives as HIV-1RT inhibitors

    Indian Academy of Sciences (India)

    Nitin S Sapre; Swagata Gupta; Neelima Sapre

    2008-07-01

    A template-based flexible docking simulation followed by `Tabu-clustering’ was performed on a series of 38 TIBO derivatives as HIV-1 reverse transcriptase (HIV-1 RT) inhibitors. Four different templates of the Cl-TIBO (1-REV) were created and used as reference templates for docking and aligning. On the basis of the optimal conformation of the ligands, when fitting to the template, the respective scoring functions were obtained; different ligand efficiencies were evaluated and analysed. Statistical modelling using artificial neural network (ANN: 2 = 0.922) and multiple linear regression method (MLR: 2 = 0.851) showed good correlation between the biological activity, binding affinity, and different ligand efficiencies of the compounds, which suggest the robustness of the template-based binding conformations of these inhibitors. Our studies suggest that, template-based docking followed by `Tabuclustering' will give a better alignment of inhibitors with respect to the crystal coordinates and enhance the docking efficiency. Also, our study indicates that scoring functions based on 3D symmetry analysis along with heavy atoms count serve as a valuable tool for estimating the efficiency of the ligands. Thus, this is a novel method based on heavy atoms count predicting the binding affinity of the TIBO group of inhibitors, so that their therapeutic utility can be enhanced.

  17. Matched sizes of activating and inhibitory receptor/ligand pairs are required for optimal signal integration by human natural killer cells.

    Directory of Open Access Journals (Sweden)

    Karsten Köhler

    Full Text Available It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions.

  18. Structural Analysis of the Ligand-Binding Domain of the Aspartate Receptor Tar from Escherichia coli.

    Science.gov (United States)

    Mise, Takeshi

    2016-07-01

    The Escherichia coli cell-surface aspartate receptor Tar mediates bacterial chemotaxis toward an attractant, aspartate (Asp), and away from a repellent, Ni(2+). These signals are transmitted from the extracellular region of Tar to the cytoplasmic region via the transmembrane domain. The mechanism by which extracellular signals are transmitted into the cell through conformational changes in Tar is predicted to involve a piston displacement of one of the α4 helices of the homodimer. To understand the molecular mechanisms underlying the induction of Tar activity by an attractant, the three-dimensional structures of the E. coli Tar periplasmic domain with and without bound aspartate, Asp-Tar and apo-Tar, respectively, were determined. Of the two ligand-binding sites, only one site was occupied, and it clearly showed the electron density of an aspartate. The slight changes in conformation and the electrostatic surface potential around the aspartate-binding site were observed. In addition, the presence of an aspartate stabilized residues Phe-150' and Arg-73. A pistonlike displacement of helix α4b' was also induced by aspartate binding as predicted by the piston model. Taken together, these small changes might be related to the induction of Tar activity and might disturb binding of the second aspartate to the second binding site in E. coli. PMID:27292793

  19. The aryl hydrocarbon receptor ligand ITE inhibits TGFβ1-induced human myofibroblast differentiation.

    Science.gov (United States)

    Lehmann, Geniece M; Xi, Xia; Kulkarni, Ajit A; Olsen, Keith C; Pollock, Stephen J; Baglole, Carolyn J; Gupta, Shikha; Casey, Ann E; Huxlin, Krystel R; Sime, Patricia J; Feldon, Steven E; Phipps, Richard P

    2011-04-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent.

  20. Immune Adjuvant Effect of Molecularly-defined Toll-Like Receptor Ligands

    Directory of Open Access Journals (Sweden)

    Deana N. Toussi

    2014-04-01

    Full Text Available Vaccine efficacy is optimized by addition of immune adjuvants. However, although adjuvants have been used for over a century, to date, only few adjuvants are approved for human use, mostly aimed at improving vaccine efficacy and antigen-specific protective antibody production. The mechanism of action of immune adjuvants is diverse, depending on their chemical and molecular nature, ranging from non-specific effects (i.e., antigen depot at the immunization site to specific activation of immune cells leading to improved host innate and adaptive responses. Although the detailed molecular mechanism of action of many adjuvants is still elusive, the discovery of Toll-like receptors (TLRs has provided new critical information on immunostimulatory effect of numerous bacterial components that engage TLRs. These ligands have been shown to improve both the quality and the quantity of host adaptive immune responses when used in vaccine formulations targeted to infectious diseases and cancer that require both humoral and cell-mediated immunity. The potential of such TLR adjuvants in improving the design and the outcomes of several vaccines is continuously evolving, as new agonists are discovered and tested in experimental and clinical models of vaccination. In this review, a summary of the recent progress in development of TLR adjuvants is presented.

  1. Multiple receptor-ligand interactions direct tissue resident gamma delta T cell activation

    Directory of Open Access Journals (Sweden)

    Deborah A. Witherden

    2014-11-01

    Full Text Available Gamma delta T cells represent a major T cell population in epithelial tissues, such as skin, intestine, and lung, where they function in maintenance of the epithelium and provide a crucial first line defense against environmental and pathogenic insults. Despite their importance, the molecular mechanisms directing their activation and function have remained elusive. Epithelial resident gamma delta T cells function through constant communication with neighboring cells, either via direct cell-to-cell contact or cell-to-matrix interactions. These intimate relationships allow gamma delta T cells to facilitate the maintenance of epithelial homeostasis, tissue repair following injury, inflammation, and protection from malignancy. Recent studies have identified a number of molecules involved in these complex interactions, under both homeostatic conditions, as well as following perturbation of these barrier tissues. These interactions are crucial to the timely production of cytokines, chemokines, growth factors and extracellular matrix proteins for restoration of homeostasis. In this review, we discuss recent advances in understanding the mechanisms directing epithelial-T cell crosstalk and the distinct roles played by individual receptor-ligand pairs of cell surface molecules in this process.

  2. Benzodiazepine metabolism: an analytical perspective.

    Science.gov (United States)

    Mandrioli, Roberto; Mercolini, Laura; Raggi, Maria Augusta

    2008-10-01

    Benzodiazepines are currently among the most frequently prescribed drugs all over the world. They act as anxiolytics, sedatives, hypnotics, amnesics, antiepileptics and muscle relaxants. Despite their common chemical scaffold, these drugs differ in their pharmacokinetic and metabolic properties. In particular, they are biotransformed by different cytochrome P450 isoforms and also by different UDP-glucuronosyltransferase subtypes. The most important studies on the metabolic characteristics of several 1,4-benzodiazepines, carried out from 1998 onwards, are reported and briefly discussed in this review. Moreover, the analytical methods related to these studies are also described and commented upon and their most important characteristics are highlighted. Most methods are based on liquid chromatography, which provides wide applicability and good analytical performance granting high precision, accuracy and feasibility. Mass spectrometry is gaining widespread acceptance, particularly if the matrix is very complex and variable, such as human or animal blood. However, spectrophotometric detection is still used for this purpose and can grant sufficient selectivity and sensitivity when coupled to suitable sample pre-treatment procedures. A monograph is included for each of the following benzodiazepines: alprazolam, bromazepam, brotizolam, clotiazepam, diazepam, etizolam, flunitrazepam, lorazepam, midazolam, oxazepam and triazolam. PMID:18855614

  3. Forced unbinding of GPR17 ligands from wild type and R255I mutant receptor models through a computational approach

    Directory of Open Access Journals (Sweden)

    Fantucci Piercarlo

    2010-03-01

    Full Text Available Abstract Background GPR17 is a hybrid G-protein-coupled receptor (GPCR activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes (cysteinyl-LTs, and involved in brain damage and repair. Its exploitment as a target for novel neuro-reparative strategies depends on the elucidation of the molecular determinants driving binding of purinergic and leukotrienic ligands. Here, we applied docking and molecular dynamics simulations (MD to analyse the binding and the forced unbinding of two GPR17 ligands (the endogenous purinergic agonist UDP and the leukotriene receptor antagonist pranlukast from both the wild-type (WT receptor and a mutant model, where a basic residue hypothesized to be crucial for nucleotide binding had been mutated (R255I to Ile. Results MD suggested that GPR17 nucleotide binding pocket is enclosed between the helical bundle and extracellular loop (EL 2. The driving interaction involves R255 and the UDP phosphate moiety. To support this hypothesis, steered MD experiments showed that the energy required to unbind UDP is higher for the WT receptor than for R255I. Three potential binding sites for pranlukast where instead found and analysed. In one of its preferential docking conformations, pranlukast tetrazole group is close to R255 and phenyl rings are placed into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No differences between the WT receptor and the R255I receptor were found for the unbinding of pranlukast. Conclusions These data thus suggest that, in contrast to which has been hypothesized for nucleotides, the lack of the R255 residue doesn't affect the binding of pranlukast a crucial role for R255 in binding of nucleotides to GPR17. Aromatic interactions are instead likely to play a predominant role in the recognition of pranlukast, suggesting that two different binding subsites are present on GPR17.

  4. Design, synthesis and structure-activity relationships of novel phenylalanine-based amino acids as kainate receptors ligands

    DEFF Research Database (Denmark)

    Szymańska, Ewa; Chałupnik, Paulina; Szczepańska, Katarzyna;

    2016-01-01

    A new series of carboxyaryl-substituted phenylalanines was designed, synthesized and pharmacologically characterized in vitro at native rat ionotropic glutamate receptors as well as at cloned homomeric kainate receptors GluK1-GluK3. Among them, six compounds bound to GluK1 receptor subtypes...... with reasonable affinity (Ki values in the range of 4.9-7.5 uM). A structure-activity relationship (SAR) for the obtained series, focused mainly on the pharmacological effect of structural modifications in the 4- and 5-position of the phenylalanine ring, was established. To illustrate the results, molecular...... docking of the synthesized series to the X-ray structure of GluK1 ligand binding core was performed. The influence of individual substituents at the phenylalanine ring for both the affinity and selectivity at AMPA, GluK1 and GluK3 receptors was analyzed, giving directions for future studies....

  5. Synthesis of (3-hydroxy-pyrazolin-5-yl)glycine based ligands interacting with ionotropic glutamate receptors.

    Science.gov (United States)

    Pinto, Andrea; Tamborini, Lucia; Mastronardi, Federica; Ettari, Roberta; Safoz, Yeliz; Bunch, Lennart; Nielsen, Birgitte; Jensen, Anders A; De Micheli, Carlo; Conti, Paola

    2014-03-21

    Following the concept that increasing the molecular complexity may enhance the receptor selectivity, we replaced the 3-hydroxy-isoxazoline ring of model compound tricholomic acid with a 3-hydroxy-pyrazoline ring, which could be variously decorated at the N1 position, inserting groups characterized by different electronic and steric properties. Binding assays on rat brain synaptic membranes showed that, depending on the nature of the substituent, some of the new synthesized ligands interacted with either AMPA or KA receptors, with affinities in the mid-micromolar range. PMID:24531228

  6. SCFHOS ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-α receptor

    OpenAIRE

    Suresh Kumar, K.G.; Tang, Weigang; Ravindranath, Abhilash K.; Clark, William A.; Croze, Ed; Fuchs, Serge Y.

    2003-01-01

    Down-regulation of activated signaling receptors in response to their ligands plays a key role in restricting the extent and duration of the signaling. Mechanisms underlying down-regulation of the type I interferon receptor consisting of IFNAR1 and IFNAR2 subunits remain largely unknown. Here we show that IFNAR1 interacts with the Homolog of Slimb (HOS) F-box protein in a phosphorylation-dependent manner, and that this interaction is promoted by interferon α (IFNα). IFNAR1 is ubiquitinated by...

  7. Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

    Science.gov (United States)

    Thompson, Andrew J; Alqazzaz, Mona; Price, Kerry L; Weston, David A; Lummis, Sarah C R

    2014-10-01

    The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.

  8. Acromegaly Clinical Trial Methodology Impact on Reported Biochemical Efficacy Rates of Somatostatin Receptor Ligand Treatments: A Meta-Analysis

    OpenAIRE

    CARMICHAEL, JOHN D.; Bonert, Vivien S.; Nuño, Miriam; Ly, Diana; Melmed, Shlomo

    2014-01-01

    Introduction: Biochemical efficacy of somatostatin receptor ligand (SRL) treatment in acromegaly is defined by metrics for GH and IGF-1 control. Since the earliest therapeutic trials, biochemical control criteria, medical formulations, and assay techniques have evolved. Materials and Methods: We searched PubMed for English-language trials published from 1974 to 2012 evaluating 10 or more patients, with a duration of more than 3 months and biochemical control as a key objective. We used a rand...

  9. Chemogenomic analysis of G-protein coupled receptors and their ligands deciphers locks and keys governing diverse aspects of signalling.

    Directory of Open Access Journals (Sweden)

    Jörg D Wichard

    Full Text Available Understanding the molecular mechanism of signalling in the important super-family of G-protein-coupled receptors (GPCRs is causally related to questions of how and where these receptors can be activated or inhibited. In this context, it is of great interest to unravel the common molecular features of GPCRs as well as those related to an active or inactive state or to subtype specific G-protein coupling. In our underlying chemogenomics study, we analyse for the first time the statistical link between the properties of G-protein-coupled receptors and GPCR ligands. The technique of mutual information (MI is able to reveal statistical inter-dependence between variations in amino acid residues on the one hand and variations in ligand molecular descriptors on the other. Although this MI analysis uses novel information that differs from the results of known site-directed mutagenesis studies or published GPCR crystal structures, the method is capable of identifying the well-known common ligand binding region of GPCRs between the upper part of the seven transmembrane helices and the second extracellular loop. The analysis shows amino acid positions that are sensitive to either stimulating (agonistic or inhibitory (antagonistic ligand effects or both. It appears that amino acid positions for antagonistic and agonistic effects are both concentrated around the extracellular region, but selective agonistic effects are cumulated between transmembrane helices (TMHs 2, 3, and ECL2, while selective residues for antagonistic effects are located at the top of helices 5 and 6. Above all, the MI analysis provides detailed indications about amino acids located in the transmembrane region of these receptors that determine G-protein signalling pathway preferences.

  10. An in Vitro and in Vivo Investigation of Bivalent Ligands That Display Preferential Binding and Functional Activity for Different Melanocortin Receptor Homodimers.

    Science.gov (United States)

    Lensing, Cody J; Freeman, Katie T; Schnell, Sathya M; Adank, Danielle N; Speth, Robert C; Haskell-Luevano, Carrie

    2016-04-14

    Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds, suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 possessed a functional profile that was unique from its monovalent counterpart providing evidence of the discrete effects of bivalent ligands. Lead compound 7 significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects. PMID:26959173

  11. Reducing Prescriptions of Long-acting Benzodiazepine Drugs in Denmark

    DEFF Research Database (Denmark)

    Eriksen, Sophie Isabel; Bjerrum, Lars

    2015-01-01

    Prolonged consumption of benzodiazepine drugs (BZD) and benzodiazepine receptor agonists (zolpidem, zaleplon, zopiclone; altogether Z drugs) is related to potential physiological and psychological dependence along with other adverse effects. This study aimed to analyse the prescribing of long......-acting BZD (half-life >10 hr), compared to short-acting BZD in Denmark during a 10-year period. Descriptive analysis of total sales data from the Danish Register of Medicinal Product Statistics, to individuals in the primary healthcare sector, of all BZD and Z-drugs in the period of 2003-2013. Prescription...... inhabitants/day in 2003 to 16.4 DDD/1000 inhabitants/day in 2013; a relative reduction of 37%. Prescription data in this study did not include information about indications for initiating treatments. In addition, due to compliance problems, some of the prescribed drugs may not have been consumed according...

  12. Neuropeptides and neuropeptide receptors: drug targets, and peptide and non-peptide ligands: a tribute to Prof. Dieter Seebach.

    Science.gov (United States)

    Hoyer, Daniel; Bartfai, Tamas

    2012-11-01

    both central and peripheral nervous system disorders. Both, receptor subtype-selective antagonists and agonists are being developed, as illustrated by the success of somatostatin agonists, angiotensin, and endothelin antagonists, and the expected clinical applications of NK-1/2/3 (substance P) receptor antagonists, CRF, vasopressin, NPY, neurotensin, orexin antagonists, or neuropeptide receptor modulators; such ligands have efficacy in preclinical or clinical models of pain and neuropsychiatric diseases, such as migraine, chronic/neuropathic pain, anxiety, sleep disorders, depression, and schizophrenia. In addition, both positive and negative allosteric modulators have been described with interesting in vivo activities (e.g., at galanin receptors). The field has become more complex now that an increasing number of heteromeric neuropeptide receptors are described, e.g., ghrelin receptors with 5-HT(2C) or dopamine D(1), D(2) receptors. At long last, structure-based drug discovery can now be envisaged with confidence, since crystal or solution structure of GPCRs and GPCR-ligand complexes, including peptide receptors, are published almost on a monthly basis. Finally, although most compounds acting at peptide receptors are still peptidomimetics, the last decade has seen the emergence of low-molecular-weight nonpeptide ligands (e.g., for orexin, ghrelin, or neurokinin receptors), and surprising progress has been made with β- and γ-peptides as very stable and potent mimetics of, e.g., somatostatin (SRIF), where the native SRIF has a half-life limited to 2-3 min. This last point will be illustrated more specifically, as we have had a long-standing collaboration with Prof. D. Seebach to whom this review is dedicated at the occasion of his 75th birthday.

  13. The relaxin family peptide receptors and their ligands: new developments and paradigms in the evolution from jawless fish to mammals.

    Science.gov (United States)

    Yegorov, Sergey; Bogerd, Jan; Good, Sara V

    2014-12-01

    Relaxin family peptide receptors (Rxfps) and their ligands, relaxin (Rln) and insulin-like (Insl) peptides, are broadly implicated in the regulation of reproductive and neuroendocrine processes in mammals. Most placental mammals harbour genes for four receptors, namely rxfp1, rxfp2, rxfp3 and rxfp4. The number and identity of rxfps in other vertebrates are immensely variable, which is probably attributable to intraspecific variation in reproductive and neuroendocrine regulation. Here, we highlight several interesting, but greatly overlooked, aspects of the rln/insl-rxfp evolutionary history: the ancient origin, recruitment of novel receptors, diverse roles of selection, differential retention and lineage-specific loss of genes over evolutionary time. The tremendous diversity of rln/insl and rxfp genes appears to have arisen from two divergent receptors and one ligand that were duplicated by whole genome duplications (WGD) in early vertebrate evolution, although several genes, notably relaxin in mammals, were also duplicated via small scale duplications. Duplication and loss of genes have varied across lineages: teleosts retained more WGD-derived genes, dominated by those thought to be involved in neuroendocrine regulation (rln3, insl5 and rxfp 3/4 genes), while eutherian mammals witnessed the diversification and rapid evolution of genes involved in reproduction (rln/insl3). Several genes that arose early in evolutionary history were lost in most mammals, but retained in teleosts and, to a lesser extent, in early diverging tetrapods. To elaborate on their evolutionary history, we provide updated phylogenies of the Rxfp1/2 and Rxfp3/4 receptors and their ligands, including new sequences from early diverging vertebrate taxa such as coelacanth, skate, spotted gar, and lamprey. We also summarize the recent progress made towards understanding the functional biology of Rxfps in non-mammalian taxa, providing a new conceptual framework for research on Rxfp signaling across

  14. Characterization and ligand identification of a membrane progesterone receptor in fungi: existence of a novel PAQR in Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    Gonzalez-Velazquez Waleska

    2012-09-01

    Full Text Available Abstract Background Adaptive responses in fungi result from the interaction of membrane receptors and extracellular ligands. Many different classes of receptors have been described in eukaryotic cells. Recently a new family of receptors classified as belonging to the progesterone-adiponectin receptor (PAQR family has been identified. These receptors have the seven transmembrane domains characteristic of G-protein coupled receptors, but their activity has not been associated directly to G proteins. They share sequence similarity to the eubacterial hemolysin III proteins. Results A new receptor, SsPAQR1 (Sporothrixschenckiiprogesterone-adiponectinQ receptor1, was identified as interacting with Sporothrix schenckii G protein alpha subunit SSG-2 in a yeast two-hybrid assay. The receptor was identified as a member of the PAQR family. The cDNA sequence revealed a predicted ORF of 1542 bp encoding a 514 amino acids protein with a calculated molecular weight of 57.8 kDa. Protein domain analysis of SsPAQR1 showed the 7 transmembrane domains (TM characteristic of G protein coupled receptors and the presence of the distinctive motifs that characterize PAQRs. A yeast-based assay specific for PAQRs identified progesterone as the agonist. S. schenckii yeast cells exposed to progesterone (0.50 mM showed an increase in intracellular levels of 3′, 5′ cyclic adenosine monophosphate (cAMP within the first min of incubation with the hormone. Different progesterone concentrations were tested for their effect on the growth of the fungus. Cultures incubated at 35°C did not grow at concentrations of progesterone of 0.05 mM or higher. Cultures incubated at 25°C grew at all concentrations tested (0.01 mM-0.50 mM with growth decreasing gradually with the increase in progesterone concentration. Conclusion This work describes a receptor associated with a G protein alpha subunit in S. schenckii belonging to the PAQR family. Progesterone was identified as the ligand

  15. Evolution of GnRH ligand precursors and GnRH receptors in protochordate and vertebrate species.

    Science.gov (United States)

    Morgan, Kevin; Millar, Robert P

    2004-12-01

    Primary structure relationships between GnRH precursors or GnRH receptors have received significant attention recently due to rapid DNA sequence determination of gene fragments and cDNAs from diverse species. Concepts concerning the evolutionary history of the GnRH system and its function in mammals, including humans, are likely to be modified as more complete sequence information becomes available. Current evidence suggests occurrence of fewer GnRH ligand and GnRH receptor genes in mammals compared to protochordates, fish and amphibians. Whilst several sequence-related GnRH decapeptide precursors and 2 or 3 separate GnRH receptors are encoded within the genomes of protochordates, fish and amphibians, only two types of GnRH (GnRH-I and GnRH-II) and two GnRH receptors occur in mammals. In addition, fish and mammalian genomes both retain inactive remnants of GnRH ligand or GnRH receptor genes. The number of distinct GnRH receptor genes in teleosts (at least five complete genes in pufferfish and three in zebrafish) partly reflects whole genome duplication during the evolution of this order of animals. Three GnRH receptor genes occur in certain frog species, consistent with the occurrence of up to three types of prepro-GnRH in amphibians. In contrast, only one functional GnRH receptor gene (the type I GnRH receptor) has been identified in humans and chimpanzees and a gene encoding a second receptor, homologous to a functional monkey receptor (the type II GnRH receptor), is either partially or completely silenced in a range of mammalian species (human, chimpanzee, sheep, cow, rat, and mouse). Further work is required to determine the significance of species-specific differences in the GnRH system to reproductive biology. For instance, recent data show that even species as closely related as humans and chimpanzees exhibit important organisational changes in the genes comprising the GnRH system. PMID:15560865

  16. Benzodiazepines and postoperative cognitive dysfunction in the elderly

    DEFF Research Database (Denmark)

    Rasmussen, L.S.; Steentoft, Anni; Rasmussen, H.;

    1999-01-01

    hypnotics benzodiazepine,diazepam,age factor,anaesthesia,geriatric,psychological responses,postoperative......hypnotics benzodiazepine,diazepam,age factor,anaesthesia,geriatric,psychological responses,postoperative...

  17. Specificity of ReceptorLigand Interactions and Their Effect on Dimerisation as Observed by Electrospray Mass Spectrometry: Bile Acids Form Stable Adducts to the RXRα

    OpenAIRE

    Lengqvist, Johan; Mata de Urquiza, Alexander; Perlmann, Thomas; Sjövall, Jan; Griffiths, William J.

    2005-01-01

    Electrospray (ES) mass spectrometry data is presented showing that agonist binding to the nuclear receptor (NR), retinoid X receptor α (RXRα), is competitive. The competitive nature of agonist binding can be used to discriminate between the specific and non-specific binding of small lipophilic molecules to NRs. Further, data is presented showing that high affinity ligand binding to the RXRα ligand binding domain (LBD) stabilises the domain homodimer. The results indicate that homodimerisation...

  18. Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding

    Energy Technology Data Exchange (ETDEWEB)

    Harada, N.; Castle, B.E.; Gorman, D.M.; Itoh, A.; Schreurs, J.; Barrett, R.L.; Howard, M.; Miyajima, A. (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA (USA))

    1990-02-01

    Interleukin 4 (IL-4) is a potent mediator of growth and differentiation for various lymphoid and myeloid cells. To isolate a cDNA encoding the murine IL-4 receptor, the authors have developed an expression cloning method that uses biotinylated ligand as a probe and that may be generally applicable to cloning of receptor genes. COS-7 cells transiently transfected with the cloned full-length cDNA bind murine IL-4 specifically with a K{sub d} = 165 pM. Crosslinking of {sup 125}I-labeled IL-4 to COS-7 cells transfected with the cDNA reveals binding to proteins of 120-140 kDa. IL-4-responsive cells also express IL-4-binding proteins of 120-140 kDa but show additional bands at 60-70 kDa; the relationship of the smaller proteins to the larger ones is unclear. The nucleotide sequence indicates that the full-length cDNA encodes 810 amino acids including the signal sequence. While no consensus sequence for protein kinases is present in the cytoplasmic domain, a sequence comparison with the erythropoietin receptor, the IL-6 receptor, and the {beta} chain of the IL-2 receptor reveals a significant homology in the extracellular domain, indicating that the IL-4 receptor is a member of a cytokine receptor family.

  19. The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate.

    Science.gov (United States)

    Kristensen, Ole; Kristensen, Lise Baadsgaard; Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm

    2016-09-01

    Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. One subfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present the X-ray structure of GluK4-LBD with kainate at 2.05 Å resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

  20. Endogenous ligands of the aryl hydrocarbon receptor regulate lung dendritic cell function.

    Science.gov (United States)

    Thatcher, Thomas H; Williams, Marc A; Pollock, Stephen J; McCarthy, Claire E; Lacy, Shannon H; Phipps, Richard P; Sime, Patricia J

    2016-01-01

    The aryl hydrocarbon receptor (AhR) is a transcription factor that has been extensively studied as a regulator of toxicant metabolism. However, recent evidence indicates that the AhR also plays an important role in immunity. We hypothesized that the AhR is a novel, immune regulator of T helper type 2 (Th2) -mediated allergic airway disease. Here, we report that AhR-deficient mice develop increased allergic responses to the model allergen ovalbumin (OVA), which are driven in part by increased dendritic cell (DC) functional activation. AhR knockout (AhR(-/-) ) mice sensitized and challenged with OVA develop an increased inflammatory response in the lung compared with wild-type controls, with greater numbers of inflammatory eosinophils and neutrophils, greater T-cell proliferation, greater production of Th2 cytokines, and higher levels of OVA-specific IgE and IgG1. Lung DCs from AhR(-/-) mice stimulated antigen-specific proliferation and Th2 cytokine production by naive T cells in vitro. Additionally, AhR(-/-) DCs produced higher levels of tumour necrosis factor-α and interleukin-6, which promote Th2 differentiation, and expressed higher cell surface levels of stimulatory MHC Class II and CD86 molecules. Overall, loss of the AhR was associated with enhanced T-cell activation by pulmonary DCs and heightened pro-inflammatory allergic responses. This suggests that endogenous AhR ligands are involved in the normal regulation of Th2-mediated immunity in the lung via a DC-dependent mechanism. Therefore, the AhR may represent an important target for therapeutic intervention in allergic airways inflammation.

  1. A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro

    Directory of Open Access Journals (Sweden)

    Han CY

    2013-04-01

    Full Text Available Cui-yan Han,1,2 Li-ling Yue,2 Ling-yu Tai,1 Li Zhou,2 Xue-yan Li,2 Gui-hua Xing,2 Xing-gang Yang,1 Ming-shuang Sun,1 Wei-san Pan1 1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China; 2Qiqihar Medical University, Qiqihar, People’s Republic of China Abstract: The epidermal growth factor receptor (EGFR serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD that were derived from three major autophosphorylation sites of the EGFR C-terminus domain in vitro. These small peptides were labeled with fluorescein isothiocyanate (FITC and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control. Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors. We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy. Keywords: EGFR, small peptide, tumor targeting, lung cancer, NLC

  2. Cross-talk between the ligand- and DNA-binding domains of estrogen receptor.

    Science.gov (United States)

    Huang, Wei; Greene, Geoffrey L; Ravikumar, Krishnakumar M; Yang, Sichun

    2013-11-01

    Estrogen receptor alpha (ERα) is a hormone-responsive transcription factor that contains several discrete functional domains, including a ligand-binding domain (LBD) and a DNA-binding domain (DBD). Despite a wealth of knowledge about the behaviors of individual domains, the molecular mechanisms of cross-talk between LBD and DBD during signal transduction from hormone to DNA-binding of ERα remain elusive. Here, we apply a multiscale approach combining coarse-grained (CG) and atomistically detailed simulations to characterize this cross-talk mechanism via an investigation of the ERα conformational landscape. First, a CG model of ERα is built based on crystal structures of individual LBDs and DBDs, with more emphasis on their interdomain interactions. Second, molecular dynamics simulations are implemented and enhanced sampling is achieved via the "push-pull-release" strategy in the search for different LBD-DBD orientations. Third, multiple energetically stable ERα conformations are identified on the landscape. A key finding is that estradiol-bound LBDs utilize the well-described activation helix H12 to pack and stabilize LBD-DBD interactions. Our results suggest that the estradiol-bound LBDs can serve as a scaffold to position and stabilize the DBD-DNA complex, consistent with experimental observations of enhanced DNA binding with the LBD. Final assessment using atomic-level simulations shows that these CG-predicted models are significantly stable within a 15-ns simulation window and that specific pairs of lysine residues in close proximity at the domain interfaces could serve as candidate sites for chemical cross-linking studies. Together, these simulation results provide a molecular view of the role of ERα domain interactions in response to hormone binding.

  3. Stoichiometry and geometry of the CXC chemokine receptor 4 complex with CXC ligand 12: Molecular modeling and experimental validation

    Science.gov (United States)

    Kufareva, Irina; Stephens, Bryan S.; Holden, Lauren G.; Qin, Ling; Zhao, Chunxia; Kawamura, Tetsuya; Abagyan, Ruben; Handel, Tracy M.

    2014-01-01

    Chemokines and their receptors regulate cell migration during development, immune system function, and in inflammatory diseases, making them important therapeutic targets. Nevertheless, the structural basis of receptor:chemokine interaction is poorly understood. Adding to the complexity of the problem is the persistently dimeric behavior of receptors observed in cell-based studies, which in combination with structural and mutagenesis data, suggest several possibilities for receptor:chemokine complex stoichiometry. In this study, a combination of computational, functional, and biophysical approaches was used to elucidate the stoichiometry and geometry of the interaction between the CXC-type chemokine receptor 4 (CXCR4) and its ligand CXCL12. First, relevance and feasibility of a 2:1 stoichiometry hypothesis was probed using functional complementation experiments with multiple pairs of complementary nonfunctional CXCR4 mutants. Next, the importance of dimers of WT CXCR4 was explored using the strategy of dimer dilution, where WT receptor dimerization is disrupted by increasing expression of nonfunctional CXCR4 mutants. The results of these experiments were supportive of a 1:1 stoichiometry, although the latter could not simultaneously reconcile existing structural and mutagenesis data. To resolve the contradiction, cysteine trapping experiments were used to derive residue proximity constraints that enabled construction of a validated 1:1 receptor:chemokine model, consistent with the paradigmatic two-site hypothesis of receptor activation. The observation of a 1:1 stoichiometry is in line with accumulating evidence supporting monomers as minimal functional units of G protein-coupled receptors, and suggests transmission of conformational changes across the dimer interface as the most probable mechanism of altered signaling by receptor heterodimers. PMID:25468967

  4. Development of small molecule non-peptide formyl peptide receptor (FPR) ligands and molecular modeling of their recognition.

    Science.gov (United States)

    Schepetkin, I A; Khlebnikov, A I; Giovannoni, M P; Kirpotina, L N; Cilibrizzi, A; Quinn, M T

    2014-01-01

    Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) expressed on a variety of cell types. These receptors play an important role in the regulation of inflammatory reactions and sensing cellular damage. They have also been implicated in the pathogenesis of various diseases, including neurodegenerative diseases, cataract formation, and atherogenesis. Thus, FPR ligands, both agonists and antagonists, may represent novel therapeutics for modulating host defense and innate immunity. A variety of molecules have been identified as receptor subtype-selective and mixed FPR agonists with potential therapeutic value during last decade. This review describes our efforts along with recent advances in the identification, optimization, biological evaluation, and structure-activity relationship (SAR) analysis of small molecule non-peptide FPR agonists and antagonists, including chiral molecules. Questions regarding the interaction at the molecular level of benzimidazoles, pyrazolones, pyridazin-3(2H)-ones, N-phenylureas and other derivatives with FPR1 and FPR2 are discussed. Application of computational models for virtual screening and design of FPR ligands is also considered. PMID:24350845

  5. The 1,4-benzodiazepine Ro5-4864 (4-chlorodiazepam) suppresses multiple pro-inflammatory mast cell effector functions

    OpenAIRE

    Yousefi, Omid Sascha; Wilhelm, Thomas; Maschke-Neuß, Karin; Kuhny, Marcel; Martin, Christian; Molderings, Gerhard J; Kratz, Felix; Hildenbrand, Bernd; Huber, Michael

    2013-01-01

    Activation of mast cells (MCs) can be achieved by the high-affinity receptor for IgE (FcεRI) as well as by additional receptors such as the lipopolysaccharide (LPS) receptor and the receptor tyrosine kinase Kit (stem cell factor [SCF] receptor). Thus, pharmacological interventions which stabilize MCs in response to different receptors would be preferable in diseases with pathological systemic MC activation such as systemic mastocytosis. 1,4-Benzodiazepines (BDZs) have been reported to suppres...

  6. Analysis of Killer Cell Immunoglobulin-like Receptor Genes and Their HLA Ligands in Iranian Patients with Ankylosing Spondylitis.

    Science.gov (United States)

    Mahmoudi, Mehdi; Jamshidi, Ahmad Reza; Karami, Jafar; Mohseni, Alireza; Amirzargar, Ali Akbar; Farhadi, Elham; Ahmadzadeh, Nooshin; Nicknam, Mohammad Hossein

    2016-02-01

    Ankylosing Spondylitis (AS) is a chronic rheumatic disease which mainly involves the axial skeleton. It seems that non-HLA genes, as well as HLA-B27 gene, are linked to the etiology of the disease. Recently, it has been documented that KIRs and their HLA ligands are contributed to the Ankylosing Spondylitis. The aim of this study was to evaluate the KIR genes and their HLA ligands in Iranian AS patients and healthy individuals. The present study includes 200 AS patient samples and 200 healthy control samples. KIR genotyping was performed using the polymerase chain reaction sequence-specific primer (PCR-SSP) method to type the presence or absence of the 16 KIR genes, 6 known specific HLA class I ligands and also, two pseudogenes. Two KIR genes (KIR-2DL3 and KIR2DL5), and among the HLA ligands, two HLA ligands (HLA-C2Lys80 and HLA-B27) genes were significantly different between case and control groups. In addition, we found some interesting KIR/HLA compound genotypes, which were associated with AS susceptibility. Our results suggest that the AS patients present more activating and less inhibitory KIR genes with combination of their HLA ligands than healthy controls. Once the balance of signal transduction between activating and inhibitory receptors is disturbed, the ability of NK cells to identify and lyse the targets in immune responses will be compromised. Accordingly, imbalance of activating and inhibitory KIR genes by up-regulating the activation and losing the inhibition of KIRs signaling or combination of both might be one of the important factors which underlying the pathogenesis of AS. PMID:26996109

  7. Peroxisome proliferator-activated receptor γ ligands induce cell cycle arrest and apoptosis in human renal carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Feng-guang YANG; Zhi-wen ZHANG; Dian-qi XIN; Chang-jin SHI; Jie-ping WU; Ying-lu GUO; You-fei GUAN

    2005-01-01

    Aim: To study the effect of peroxisome proliferator-actived receptor γ (PPARγ)ligands on cell proliferation and apoptosis in human renal carcinoma cell lines.Methods: The expression of PPARγ was investigated by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry.The effect of thiazolidinedione (TZD) PPARγ ligands on growth of renal cell carcinoma (RCC) cells was measured by MTT assay and flow cytometric analysis. Cell death ELISA, Hoechst 33342 fluorescent staining and DNA ladder assay were used to observe the effects of PPARγ ligands on apoptosis. Regulatory proteins of cell cycle and apoptosis were detected by Western blot analysis. Results:PPARγ was expressed at much higher levels in renal tumors than in the normal kidney (2.16±0.85 vs 0.90±0.73; P<0.01 ). TZD PPARγ ligands inhibited RCC cell growth in a dose-dependent manner with IC50 values of 7.08 μmol/L and 11.32 μmol/L for pioglitazone, and 5.71 μmol/L and 8.38 μmol/L for troglitazone in 786-O and A498 cells, respectively. Cell cycle analysis showed a G0/G1 arrest in human RCC cells following 24-h exposure to TZD. Analysis of cell cycle regulatory proteins revealed that TZD decreased the protein levels of proliferating cell nuclear antigen, pRb, cyclin D1, and Cdk4 but increased the levels of p21 and p27 in a timedependent manner. Furthermore, high doses of TZD induced massive apoptosis in renal cancer cells, with increased Bax expression and decreased Bcl-2 expression.Conclusion: TZD PPARγ ligands showed potent inhibitory effect on proliferation,and could induce apoptosis in RCC cells. These results suggest that ligands for PPARγ have potential antitumor effects on renal carcinoma cells.

  8. Steroid receptor coactivator 1 deficiency increases MMTV-neu mediated tumor latency and differentiation specific gene expression, decreases metastasis, and inhibits response to PPAR ligands

    International Nuclear Information System (INIS)

    The peroxisome proliferator activated receptor (PPAR) subgroup of the nuclear hormone receptor superfamily is activated by a variety of natural and synthetic ligands. PPARs can heterodimerize with retinoid X receptors, which have homology to other members of the nuclear receptor superfamily. Ligand binding to PPAR/RXRs results in recruitment of transcriptional coactivator proteins such as steroid receptor coactivator 1 (SRC-1) and CREB binding protein (CBP). Both SRC-1 and CBP are histone acetyltransferases, which by modifying nucleosomal histones, produce more open chromatin structure and increase transcriptional activity. Nuclear hormone receptors can recruit limiting amounts of coactivators from other transcription factor binding sites such as AP-1, thereby inhibiting the activity of AP-1 target genes. PPAR and RXR ligands have been used in experimental breast cancer therapy. The role of coactivator expression in mammary tumorigenesis and response to drug therapy has been the subject of recent studies. We examined the effects of loss of SRC-1 on MMTV-neu mediated mammary tumorigenesis. SRC-1 null mutation in mammary tumor prone mice increased the tumor latency period, reduced tumor proliferation index and metastasis, inhibited response to PPAR and RXR ligands, and induced genes involved in mammary gland differentiation. We also examined human breast cancer cell lines overexpressing SRC-1 or CBP. Coactivator overexpression increased cellular proliferation with resistance to PPAR and RXR ligands and remodeled chromatin of the proximal epidermal growth factor receptor promoter. These results indicate that histone acetyltransferases play key roles in mammary tumorigenesis and response to anti-proliferative therapies

  9. Aryl hydrocarbon receptor ligand effects in RBL2H3 cells

    DEFF Research Database (Denmark)

    Maaetoft-Udsen, Kristina; Shimoda, Lori M. N.; Frøkiær, Hanne;

    2012-01-01

    (KA), Resveratrol, indolmycin, and violacein, affect mast cell activation and signaling. These ligands were tested on calcium signaling, degranulation, and gene expression. The data show that AHR is present in three model mast cell lines, and that various known and putative AHR ligands regulate gene...

  10. The complex interplay between ligand binding and conformational structure of the folate binding protein (folate receptor)

    DEFF Research Database (Denmark)

    Holm, Jan; Bruun, Susanne Wrang; Hansen, Steen I.

    2015-01-01

    , and the binding induces a conformational change with formation of hydrophilic and stable holo-FBP. Holo-FBP exhibits a ligand-mediated concentration-dependent self-association into multimers of great thermal and chemical stability due to strong intermolecular forces. Both ligand and FBP are thus protected against...

  11. The high affinity ligand binding conformation of the nuclear 1,25-dihydroxyvitamin D3 receptor is functionally linked to the transactivation domain 2 (AF-2).

    OpenAIRE

    Nayeri, S; Kahlen, J P; Carlberg, C

    1996-01-01

    The nuclear receptor for 1,25-dihydroxyvitamin D3 (VD), VDR, is a transcription factor that mediates all genomic actions of the hormone. The activation of VDR by ligand induces a conformational change within its ligand binding domain (LBD). Due to the lack of a crystal structure analysis, biochemical methods have to be applied in order to investigate the details of this receptor-ligand interaction. The limited protease digestion assay can be used as a tool for the determination of a functiona...

  12. Anandamide, a natural ligand for the peripheral cannabinoid receptor is a novel synergistic growth factor for hematopoietic cells.

    Science.gov (United States)

    Valk, P; Verbakel, S; Vankan, Y; Hol, S; Mancham, S; Ploemacher, R; Mayen, A; Löwenberg, B; Delwel, R

    1997-08-15

    We recently demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) may be a proto-oncogene involved in murine myeloid leukemias. We show here that Cb2 may have a role in hematopoietic development. RNAse protection analysis showed that Cb2 is normally expressed in spleen and thymus. Cb2 mRNA is also expressed in 45 of 51 cell lines of distinct hematopoietic lineages, ie, myeloid, macrophage, mast, B-lymphoid, T-lymphoid, and erythroid cells. The effect of the fatty acid anandamide, an endogenous ligand for cannabinoid receptors, on primary murine marrow cells and hematopoietic growth factor (HGF)-dependent cell lines was then investigated. In vitro colony cultures of normal mouse bone marrow cells showed anandamide to potentiate interleukin-3 (IL-3)-induced colony growth markedly. Whereas HGFs alone stimulate proliferation of the various cell lines in serum-free culture only weakly, anandamide enhances the proliferative response of the cell lines to HGFs profoundly. This was apparent for responses induced by IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. Anandamide was already effective at concentrations as low as 0.1 to 0.3 micromol/L and plateau effects were reached at 0.3 to 3 micromol/L. The addition of anandamide as single growth factor had no effect. The costimulatory effect of anandamide was not evident when cells were cultured with fetal calf serum (FCS), suggesting that FCS contains anandamide or another ligand capable of activating the peripheral cannabinoid receptor. Other cannabinoid ligands did not enhance the proliferative responsiveness of hematopoietic cells to HGFs. Transfection experiments of Cb2 in myeloid 32D cells showed that anandamide specifically activates proliferation through activation of the peripheral cannabinoid receptor. Anandamide appears to be a novel and synergistic growth stimulator for hematopoietic cells. PMID:9269762

  13. Quantitative dissection of the binding contributions of ligand lysines of the receptor-associated protein (RAP) to the low density lipoprotein receptor-related protein (LRP1).

    Science.gov (United States)

    Dolmer, Klavs; Campos, Andres; Gettins, Peter G W

    2013-08-16

    Although lysines are known to be critical for ligand binding to LDL receptor family receptors, relatively small reductions in affinity have been found when such lysines have been mutated. To resolve this paradox, we have examined the specific binding contributions of four lysines, Lys-253, Lys-256, Lys-270, and Lys-289, in the third domain (D3) of receptor-associated protein (RAP), by eliminating all other lysine residues. Using D3 variants containing lysine subsets, we examined binding to the high affinity fragment CR56 from LRP1. With this simplification, we found that elimination of the lysine pairs Lys-253/Lys-256 and Lys-270/Lys-289 resulted in increases in Kd of 1240- and 100,000-fold, respectively. Each pair contributed additively to overall affinity, with 61% from Lys-270/Lys-289 and 39% from Lys-253/Lys-256. Furthermore, the Lys-270/Lys-289 pair alone could bind different single CR domains with similar affinity. Within the pairs, binding contributions of Lys-270 ≫ Lys-256 > Lys-253 ∼ Lys-289 were deduced. Importantly, however, Lys-289 could significantly compensate for the loss of Lys-270, thus explaining how previous studies have underestimated the importance of Lys-270. Calorimetry showed that favorable enthalpy, from Lys-256 and Lys-270, overwhelmingly drives binding, offset by unfavorable entropy. Our findings support a mode of ligand binding in which a proximal pair of lysines engages the negatively charged pocket of a CR domain, with two such pairs of interactions (requiring two CR domains), appropriately separated, being alone sufficient to provide the low nanomolar affinity found for most protein ligands of LDL receptor family members.

  14. Comparing Class A GPCRs to bitter taste receptors: Structural motifs, ligand interactions and agonist-to-antagonist ratios.

    Science.gov (United States)

    Di Pizio, Antonella; Levit, Anat; Slutzki, Michal; Behrens, Maik; Karaman, Rafik; Niv, Masha Y

    2016-01-01

    G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins that play a key role in human physiology. The GPCR superfamily comprises about 800 members, classified into several classes, with rhodopsin-like Class A being the largest and most studied thus far. A huge component of the human repertoire consists of the chemosensory GPCRs, including ∼400 odorant receptors, 25 bitter taste receptors (TAS2Rs), which are thought to guard the organism from consuming poisons, and sweet and umami TAS1R heteromers, which indicate the nutritive value of food. The location of the binding site of TAS2Rs is similar to that of Class A GPCRs. However, most of the known bitter ligands are agonists, with only a few antagonists documented thus far. The agonist-to-antagonist ratios of Class A GPCRs vary, but in general are much lower than for TAS2Rs. For a set of well-studied GPCRs, a gradual change in agonists-to-antagonists ratios is observed when comparing low (10 μM)- and high (10 nM)-affinity ligand sets from ChEMBL and the DrugBank set of drugs. This shift reflects pharmaceutical bias toward the therapeutically desirable pharmacology for each of these GPCRs, while the 10 μM sets possibly represent the native tendency of the receptors toward either agonists or antagonists. Analyzing ligand-GPCR interactions in 56 X-ray structures representative of currently available structural data, we find that the N-terminus, TM1 and TM2 are more involved in binding of antagonists than of agonists. On the other hand, ECL2 tends to be more involved in binding of agonists. This is of interest, since TAS2Rs harbor variations on the typical Class A sequence motifs, including the absence of the ECL2-TM3 disulfide bridge. This suggests an alternative mode of regulation of conformational states for TAS2Rs, with potentially less stabilized inactive state. The comparison of TAS2Rs and Class A GPCRs structural features and the pharmacology of the their ligands highlights the intricacies of

  15. Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions.

    Science.gov (United States)

    De Silva, Channa R; Vagner, Josef; Lynch, Ronald; Gillies, Robert J; Hruby, Victor J

    2010-03-01

    Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0M) prior to the luminescent enhancement step. [Nle(4),d-Phe(7)]-alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) labeled with Eu(III)-DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries. PMID:19852924

  16. Design, synthesis and evaluation of bitopic arylpiperazinephenyl-1,2,4-oxadiazoles as preferential dopamine D3 receptor ligands.

    Science.gov (United States)

    Cao, Yongkai; Min, Chengchun; Acharya, Srijan; Kim, Kyeong-Man; Cheon, Seung Hoon

    2016-01-15

    The dopamine D3 receptor (D3R) was proposed as a therapeutic target for drug development to treat drug abuse and addiction and neuropsychiatric disorders. Several D3R-selective modulators over the dopamine D2 receptor (D2R) can avoid extrapyramidal symptoms (EPS) and hyperprolactinemia. However, few biased D3R ligands were identified or showed a narrow range of selectivity at the D3R over D2R because of their high sequence homology. Herein, we designed, synthesized and evaluated the binding affinity of a series of bitopic ligands: arypiperazine-phenyl-1,2,4-oxadiazoles. Compound 9e·HCl was the most potent and selective D3R modulator among these bitopic ligands. Molecular modeling revealed that D3R selectivity depends on the divergence of secondary binding pocket (SBP) in D3R and D2R. Specifically, non-conserved Tyr36, EL1 especially non-conserved Thr92 and Gly94, and EL2 Val180, Cys181 and Ser182 of D3R may contribute to D3R specificity over D2R. PMID:26707842

  17. (99m)Tc-Cyclopentadienyl Tricarbonyl Chelate-Labeled Compounds as Selective Sigma-2 Receptor Ligands for Tumor Imaging.

    Science.gov (United States)

    Li, Dan; Chen, Yuanyuan; Wang, Xia; Deuther-Conrad, Winnie; Chen, Xin; Jia, Bing; Dong, Chengyan; Steinbach, Jörg; Brust, Peter; Liu, Boli; Jia, Hongmei

    2016-02-11

    We have designed and synthesized a series of cyclopentadienyl tricarbonyl rhenium complexes containing a 5,6-dimethoxyisoindoline or a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline pharmacophore as σ2 receptor ligands. Rhenium compound 20a possessed low nanomolar σ2 receptor affinity (K(i) = 2.97 nM) and moderate subtype selectivity (10-fold). Moreover, it showed high selectivity toward vesicular acetylcholine transporter (2374-fold), dopamine D2L receptor, NMDA receptor, opiate receptor, dopamine transporter, norepinephrine transporter, and serotonin transporter. Its corresponding radiotracer [(99m)Tc]20b showed high uptake in a time- and dose-dependent manner in DU145 prostate cells and C6 glioma cells. In addition, this tracer exhibited high tumor uptake (5.92% ID/g at 240 min) and high tumor/blood and tumor/muscle ratios (21 and 16 at 240 min, respectively) as well as specific binding to σ receptors in nude mice bearing C6 glioma xenografts. Small animal SPECT/CT imaging of [(99m)Tc]20b in the C6 glioma xenograft model demonstrated a clear visualization of the tumor at 180 min after injection.

  18. EFFECTS OF SYNTETIC CANNABINOID RECEPTOR LIGANDS WIN 55.212-2 AND ANANDAMID UPON IN VITRO ACTIVITY OF IMMUNOCOMPETENT CELLS

    Directory of Open Access Journals (Sweden)

    E. G. Lobanova

    2009-01-01

    Full Text Available Abstract. Ability of cannabinoid receptor ligands WIN 55.212-2 and anandamid to inhibit synthesis of TNFα and IL-8 was studied in healthy donors and men with allergic disorders. To establish mechanism of action for investigated substances, the selective antagonists of the СВ1-receptor (SR141716A and for СВ2 - receptor (SR144528 were applied. Studies with whole blood dilutions allowed of approximating in vivo conditions when investigating biological properties of WIN-55.212-2 and anandamid. The synthetic cannabinoids WIN - 55.212-2 and anandamid at a concentration of 3-10 μМ were capable of reducing synthesis of TNFα and IL-8 in lipopolysaccharide-stimulated blood leukocytes, both from healthy donors and subjects with allergic disorders. It was revealed that the antagonist of СВ1-receptor (SR141716A did not exert a receptor-mediated effect for WIN-55.212-2 and anandamid. Meanwhile, a СВ2-receptor antagonist (SR144528 entirely eliminated completely the blocking effect of anandamid and WIN-55.212-2.

  19. Inhibitory effect on hepatitis B virus in vitro by a peroxisome proliferator-activated receptor-{gamma} ligand, rosiglitazone

    Energy Technology Data Exchange (ETDEWEB)

    Wakui, Yuta; Inoue, Jun [Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aobaku, Sendai 980-8574 (Japan); Ueno, Yoshiyuki, E-mail: yueno@mail.tains.tohoku.ac.jp [Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aobaku, Sendai 980-8574 (Japan); Fukushima, Koji; Kondo, Yasuteru; Kakazu, Eiji; Obara, Noriyuki; Kimura, Osamu; Shimosegawa, Tooru [Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryo, Aobaku, Sendai 980-8574 (Japan)

    2010-05-28

    Although chronic infection of hepatitis B virus (HBV) is currently managed with nucleot(s)ide analogues or interferon-{alpha}, the control of HBV infection still remains a clinical challenge. Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor, that plays a role in glucose and lipid metabolism, immune reactions, and inflammation. In this study, the suppressive effect of PPAR ligands on HBV replication was examined in vitro using a PPAR{alpha} ligand, bezafibrate, and a PPAR{gamma} ligand, rosiglitazone. The effects were examined in HepG2 cells transfected with a plasmid containing 1.3-fold HBV genome. Whereas bezafibrate showed no effect against HBV replication, rosiglitazone reduced the amount of HBV DNA, hepatitis B surface antigen, and hepatitis B e antigen in the culture supernatant. Southern blot analysis showed that the replicative intermediates of HBV in the cells were also inhibited. It was confirmed that GW9662, an antagonist of PPAR{gamma}, reduced the suppressive effect of rosiglitazone on HBV. Moreover, rosiglitazone showed a synergistic effect on HBV replication with lamivudine or interferon-{alpha}-2b. In conclusion, this study showed that rosiglitazone inhibited the replication of HBV in vitro, and suggested that the combination therapy of rosiglitazone and nucleot(s)ide analogues or interferon could be a therapeutic option for chronic HBV infection.

  20. Oxytocin receptor ligand binding in embryonic tissue and postnatal brain development of the C57BL/6J mouse

    Directory of Open Access Journals (Sweden)

    Elizabeth eHammock

    2013-12-01

    Full Text Available Oxytocin (OXT has drawn increasing attention as a developmentally relevant neuropeptide given its role in the brain regulation of social behavior. It has been suggested that OXT plays an important role in the infant brain during caregiver attachment in nurturing familial contexts, but there is incomplete experimental evidence. Mouse models of OXT system genes have been particularly informative for the role of the OXT system in social behavior, however, the developing brain areas that could respond to ligand activation of the OXT receptor (OXTR have yet to be identified in this species. Here we report new data revealing dynamic ligand-binding distribution of OXTR in the developing mouse brain. Using male and female C57BL/6J mice at postnatal days (P 0, 7, 14, 21, 35, and 60 we quantified OXTR ligand binding in several brain areas which changed across development. Further, we describe OXTR ligand binding in select tissues of the near-term whole embryo at E18.5. Together, these data aid in the interpretation of findings in mouse models of the OXT system and generate new testable hypotheses for developmental roles for OXT in mammalian systems. We discuss our findings in the context of developmental disorders (including autism, attachment biology, and infant physiological regulation.