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Sample records for benzodiazepine receptor ligand

  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. Imaging of a glioma using peripheral benzodiazepine receptor ligands.

    OpenAIRE

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

    1987-01-01

    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 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 quan...

  4. 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...

  5. 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...

  6. 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

  7. [11C]vinpocetine: a prospective peripheral benzodiazepine receptor ligand for primate PET studies.

    Science.gov (United States)

    Gulyás, Balázs; Halldin, Christer; Vas, Adám; Banati, Richard B; Shchukin, Evgeny; Finnema, Sjoerd; Tarkainen, Jari; Tihanyi, Károly; Szilágyi, Géza; Farde, Lars

    2005-03-15

    Vinpocetine, a synthetic derivative of the Vinca minor alkaloid vincamine, is a widely used drug in neurological practice. We tested the hypothesis that vinpocetine binds to peripheral benzodiazepine binding sites (PBBS) and is therefore a potential ligand of PBBS. Positron emission tomography (PET) measurements in two cynomolgous monkeys showed that pretreatment with vinpocetine markedly reduced the brain uptake of [11C]PK11195, a known PBBS radioligand. On the other hand, whereas pretreatment with PK11195 increased the brain uptake of [11C]vinpocetine due to the blockade of PBBS in the periphery, it significantly reduced the binding potential (BP) values of [11C]vinpocetine in the whole brain and in individual brain structures to PK11195. These findings indicate that, whereas the two ligands have different affinities to PBBS, vinpocetine is a potent ligand of PBBS, which in turn suggests that the pharmacological activity of vinpocetine may involve the regulation of glial functions. PMID:15760643

  8. Synthesis and evaluation of [{sup 123}I] ligands for the study of the peripheral benzodiazepine receptors using SPECT

    Energy Technology Data Exchange (ETDEWEB)

    Mattner, F.; Katsifis, A.; Mardon, K.; Papazian, V.; Najdovski, L.; Dikic, B. [Australian Nuclear Science and technology Organisation, Lucas Heights, Sydney, NSW (Australia). Radiopharmaceuticals Division

    1998-06-01

    Full text: The peripheral benzodiazepine receptors (PBR), are distinct from the central benzodiazepine receptors in their pharmacology and subcellular location. PBR`s are predominantly found in the peripheral organs such as kidney, heart, adrenal cortex, as well as in the glial cells in the brain. PBR`s have been implicated in the control of cell proliferation and differentiation and shown to display increased levels in a variety of malignant tumours. Increased levels of PBR`s have also been implicated in a variety of neurodegenerative disorders. We have prepared and evaluated [{sup 123}I]N`N`-dimethyl-6-methyl-(4`iodophenyl) midazo[l,2-a]pyridine-3-acetamide (1), [{sup 123}I]N`N`-diethyl-6-chloro-(4`iodophenyl) imidazo[l,2-a]pyridine-3-acetamide (2) and [{sup 123}l]3-benzoimidomethyl-2- (4`-t-butylphenyl)-6-iodoimidazo[1,2-b]pyridazine (3) as potential probes for the study of the PBR`s in oncology and neurodegeneration using SPECT. The iodine-123 analogues 1 and 2 were prepared by iododestannylation with Na {sup 123}I in the presence of chloramine-T and HCI. Compound 3 was prepared from the bromo precursor with Na {sup 123}I using Cu{sup +} assisted halogen exchange. In vivo biodistribution of all three compounds in rodents indicated high uptake in tissues with known PBR sites. Pre-treatment of the rats with PK 11195, Ro 5-4864 and the cold material reduced significantly the uptake of activity in organs expressing the PBR`s. Other drugs including flumazenil and haloperidol, did not significantly reduce the uptake of activity in these organs. Metabolite studies on 1 and 2 indicated high in vivo stability, however significant deiodination in vivo was observed for 3. In conclusion, ligands 1, 2 and 3 indicated high and selective in vivo uptake in organs expressing the PBR whereas ligand 3 had reduced in vivo stability. Compounds 1 and 2 are therefore suitable candidates for further development as ligands for the study of the PBR`s using SPECT

  9. 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.

  10. 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

  11. 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...

  12. 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.

  13. 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....

  14. 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

  15. In vivo imaging of microglial activation using a peripheral benzodiazepine receptor ligand. [11C]PK-11195 and animal PET following ethanol injury in rat striatum

    International Nuclear Information System (INIS)

    To investigate whether [11C]PK-11195, a specific peripheral benzodiazepine receptors (PBRs) ligand for positron emission tomography (PET), can show activated microglia in a rat brain injury model. On day 1, ethanol was injected into the rat's right striatum (ST) using a stereotaxic operative procedure. On day 3, head magnetic resonance imaging (MRI) scans for surgically treated rats were performed to evaluate ethanol injury morphologically. On day 4, dynamic PET scans (17 injured rats and 7 non-injured controls) were performed for 60 min with an animal PET scanner under chloral hydrate anesthesia following a bolus injection of [11C]PK-11195 through tail vein. Because PBRs are present throughout the brain, there is no suitable receptor-free reference region. The reference tissue model may not be applicable because of low target to back-ground ratio for low affinity of [11C]PK-11195 to PBRs. We evaluated the PBRs binding with regions of interest (ROIs)-based approach to estimate total distribution volume (V). We used an integral from 0 min to 60 min (V60) as an estimate of V. On the coronal PET image, ROIs were placed on bilateral ST. Differences in right/left ST V60 ratios between lesioned and unlesioned control rats were compared using unpaired t tests. Immunohistochemical staining was performed for confirming the presence of activated microglia following decapitation on the PET experiment day. The right/left ST V60 ratios in lesioned rats (1.07±0.08) were significantly higher than those in unlesioned control rats (1.00±0.06, P11C]PK-11195 PET imaging would be a useful tool for evaluating microglial activation in a rat brain injury model. (author)

  16. 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...

  17. 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

  18. The peripheral benzodiazepine receptor ligand PK11195 binds with high affinity to the acute phase reactant α1-acid glycoprotein: implications for the use of the ligand as a CNS inflammatory marker

    International Nuclear Information System (INIS)

    The peripheral benzodiazepine receptor ligand PK11195 has been used as an in vivo marker of neuroinflammation in positron emission tomography studies in man. One of the methodological issues surrounding the use of the ligand in these studies is the highly variable kinetic behavior of [11C]PK11195 in plasma. We therefore undertook a study to measure the binding of [3H]PK11195 to whole human blood and found a low level of binding to blood cells but extensive binding to plasma proteins. Binding assays using [3H]PK11195 and purified human plasma proteins demonstrated a strong binding to α1-acid glycoprotein (AGP) and a much weaker interaction with albumin. Immunodepletion of AGP from plasma resulted in the loss of plasma [3H]PK11195 binding demonstrating: (i) the specificity of the interaction and (ii) that AGP is the major plasma protein to which PK11195 binds with high affinity. PK11195 was able to displace fluorescein-dexamethasone from AGP with IC50 of 11C]PK11195 to the brain parenchyma in diseases with blood brain barrier breakdown. Finally, local synthesis of AGP at the site of brain injury may contribute the pattern of [11C]PK11195 binding observed in neuroinflammatory diseases

  19. 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.

  20. 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

  1. 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

  2. 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...

  3. 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 ...

  4. Synthesis, structure and affinity of novel 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones for CNS central and peripheral benzodiazepine receptors.

    Science.gov (United States)

    Andronati, Sergey; Semenishyna, Ekaterina; Pavlovsky, Victor; Simonov, Yuriy; Makan, Svetlana; Boyko, Irina; Burenkova, Natalya; Gdaniec, Maria; Cardinael, Pascal; Bouillon, Jean-Philippe; Mazepa, Alexander

    2010-04-01

    A series of novel 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones (7-15) was synthesized and their in vitro affinity for both the central benzodiazepine receptor (CBR) and the peripheral benzodiazepine receptor (PBR) of rat brain was studied. Racemic mixture of 7-bromo-3-(2-methoxy)ethoxy-5-phenyl-1,2-dihydro-3H-1,4-benzodiazepin-2-one (13) was separated into enantiomers 14, 15 by chiral HPLC. Absolute configuration of R-enantiomer 15 was determined by the method of X-ray diffraction analysis. The affinity of S-enantiomer 14 for CBR ( IC50)=245 nM) is 20-fold higher than the affinity of R-enantiomer 15 (IC50)=4,930 nM). A high selectivity for CBR versus PBR (IC50) (PBR)>10,000 nM) was shown by all reported compounds. Compound 12 was revealed as a potent (IC50)=9 nM) and selective CBR ligand among the synthesized 3-alkoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-ones. PMID:20061068

  5. 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.

  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. Imidazoline receptors ligands

    Directory of Open Access Journals (Sweden)

    Agbaba Danica

    2012-01-01

    Full Text Available Extensive biochemical and pharmacological studies have determined three different subtypes of imidazoline receptors: I1-imidazoline receptors (I1-IR involved in central inhibition of sympathicus that produce hypotensive effect; I2-imidazoline receptors (I2-IR modulate monoamine oxidase B activity (MAO-B; I3-imidazoline receptors (I3-IR regulate insulin secretion from pancreatic β-cells. Therefore, the I1/I2/I3 imidazoline receptors are selected as new, interesting targets for drug design and discovery. Novel selective I1/I2/I3 agonists and antagonists have been recently developed. In the present review, we provide a brief update to the field of imidazoline research, highlighting some of the chemical diversity and progress made in the 2D-QSAR, 3D-QSAR and quantitative pharmacophore development studies of I1-IR and I2-IR imidazoline receptor ligands. Theoretical studies of I3-IR ligands are not yet performed because of insufficient number of synthesized I3-IR ligands.

  8. Glutamate receptor ligands

    DEFF Research Database (Denmark)

    Guldbrandt, Mette; Johansen, Tommy N; Frydenvang, Karla Andrea; Bräuner-Osborne, Hans; Stensbøl, Tine B; Nielsen, Birgitte; Karla, Rolf; Santi, Flavio; Krogsgaard-Larsen, Povl; Madsen, Ulf

    2002-01-01

    Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA...

  9. 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...

  10. 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.

  11. 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 ...

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Effects of vitamin B-6 nutrition on benzodiazepine (BDZ) receptor binding in the developing rat brain

    International Nuclear Information System (INIS)

    A dietary deficiency of vitamin B-6 promotes seizure activity in neonatal animals and human infants. Previous studied have shown that neonatal vitamin B-6 deprivation results in reduced levels of brain gamma-aminobutyric acid (GABA) and increased binding at the GABA site of the GABA/BDZ receptor complex. Since the GABA and BDZ receptors are allosterically linked, this study was undertaken to determine if vitamin B-6 deprivation had an effect on BDZ receptor binding. Benzodiazepine receptor binding isotherms using 3H-flunitrazepam as ligand were performed in the presence and absence of 10 μM GABA. The results indicate a significant increase in the binding affinity (Kd) in the presence of GABA in cerebellar membranes from deficient rat pups at 14 days of age with no effect on receptor number (Bmax). By 28 days of age, the increase in Kd was no longer present. No change in Kd or Bmax was observed in cortical tissue from deficient animals at 14 or 28 days of age. Preliminary studies of GABA-enhancement of 3H-flunitrazepam binding indicate that vitamin B-6 deficiency also induces alterations in the ability of GABA to enhance BZD receptor binding. In summary, these results indicate that the effects of vitamin B-6 deprivation on BDZ receptor binding are region specific and age related

  20. Flavonoids as GABAA receptor ligands: the whole story?

    Science.gov (United States)

    Wasowski, Cristina; Marder, Mariel

    2012-01-01

    Benzodiazepines are the most widely prescribed class of psychoactive drugs in current therapeutic use, despite the important unwanted side effects that they produce, such as sedation, myorelaxation, ataxia, amnesia, and ethanol and barbiturate potentiation and tolerance. They exert their therapeutic effects via binding to the benzodiazepine binding site of gamma-aminobutyric acid (GABA) type A receptors, and allosterically modulating the chloride flux through the ion channel complex. First isolated from plants used as tranquilizers in folkloric medicine, some natural flavonoids have been shown to possess selective affinity for the benzodiazepine binding site with a broad spectrum of central nervous system effects. Since the initial search for alternative benzodiazepine ligands amongst the flavonoids, a list of successful synthetic derivatives has been generated with enhanced activities. This review provides an update on research developments that have established the activity of natural and synthetic flavonoids on GABA type A receptors. Flavonoids are prominent drugs in the treatment of mental disorders, and can also be used as tools to study modulatory sites at GABA type A receptors and to develop GABA type A selective agents further.

  1. 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.

  2. 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

  3. 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

  4. Effect of chronic (-)-nicotine treatment on rat cerebral benzodiazepine receptors

    International Nuclear Information System (INIS)

    The purpose of this study was to clarify the effect of (-)-nicotine on cerebral benzodiazepine receptors (BzR) with radiotracer methods. The effect of (-)-nicotine on BzR was examined in in vitro studies using chronic (-)-nicotine-treated rats using 3H-diazepam. The in vitro radioreceptor assay showed a 14% increase in the maximum number of binding sites of BzR in chronic (-)-nicotine-treated rats in comparison with the control rats. Moreover, a convenient in vivo uptake index of 125I-iomazenil was calculated and a higher uptake of the radioactivity was observed in the chronic (-)-nicotine-treated group than in the control group. Although further studies of the mechanism of (-)-nicotine on such BzR changes are required, an increase in the amount of BzR in the cerebral cortex was found in rats that underwent chronic (-)-nicotine treatment, and this result contributed to the understanding of the effects of (-)-nicotine and smoking on neural functions

  5. Soman- or kainic acid-induced convulsions decrease muscarinic receptors but not benzodiazepine receptors

    International Nuclear Information System (INIS)

    [3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors decreased in the rat forebrain after convulsions induced by a single dose of either soman, a potent inhibitor of acetylcholinesterase, or kainic acid, an excitotoxin. A Rosenthal plot revealed that the receptors decreased in number rather than affinity. When the soman-induced convulsions were blocked, the decrease in muscarinic receptors at 3 days was less extensive than when convulsions occurred and at 10 days they approached control levels in most of the brain areas. The most prominent decrements in QNB binding were in the piriform cortex where the decline in QNB binding is probably related to the extensive convulsion-associated neuropathology. The decrements in QNB binding after convulsions suggest that the convulsive state leads to a down-regulation of muscarinic receptors in some brain areas. In contrast to the decrease in QNB binding after convulsions, [3H]flunitrazepam binding to benzodiazepine receptors did not change even in the piriform cortex where the loss in muscarinic receptors was most prominent. Thus, it appears that those neuronal processes that bear muscarinic receptors are more vulnerable to convulsion-induced change than those with benzodiazepine receptors

  6. Chronic caffeine or theophylline exposure reduces gamma-aminobutyric acid/benzodiazepine receptor site interactions.

    Science.gov (United States)

    Roca, D J; Schiller, G D; Farb, D H

    1988-05-01

    Methylxanthines, such as caffeine and theophylline, are adenosine receptor antagonists that exert dramatic effects upon the behavior of vertebrate animals by increasing attentiveness, anxiety, and convulsive activity. Benzodiazepines, such as flunitrazepam, generally exert behavioral effects that are opposite to those of methylxanthines. We report the finding that chronic exposure of embryonic brain neurons to caffeine or theophylline reduces the ability of gamma-aminobutyric acid (GABA) to potentiate the binding of [3H]flunitrazepam to the GABA/benzodiazepine receptor. This theophylline-induced "uncoupling" of GABA- and benzodiazepine-binding site allosteric interactions is blocked by chloroadenosine, an adenosine receptor agonist, indicating that the chronic effects of theophylline are mediated by a site that resembles an adenosine receptor. We speculate that adverse central nervous system effects of long-term exposure to methylxanthines such as in caffeine-containing beverages or theophylline-containing medications may be exerted by a cell-mediated modification of the GABAA receptor. PMID:2835648

  7. 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.

  8. 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

  9. 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......- and a gamma-subunit in the GABA(A) receptor, selected compounds were tested on the alpha(1)beta(2)gamma(2s), alpha(2)beta(2)gamma(2s) and alpha(3)beta(2)gamma(2s) GABA(A) receptor subtypes. The 3-acyl-4-quinolones display various degrees of selectivity for alpha(1)- versus alpha(2)- and alpha(3...

  10. Isotopically labelled benzodiazepines

    International Nuclear Information System (INIS)

    This paper reports on the benzodiazepines which are a class of therapeutic agents. Improvements in the analytical methodology in the areas of biochemistry and pharmacology were significant, particularly in the application of chromatographic and spectroscopic techniques. In addition, the discovery and subsequent development of tritium and carbon-14 as an analytical tool in the biological sciences were essentially post-world war II phenomena. Thus, as these new chemical entities were found to be biologically active, they could be prepared in labeled form for metabolic study, biological half-life determination (pharmacokinetics), tissue distribution study, etc. This use of tracer methodology has been liberally applied to the benzodiazepines and also more recently to the study of receptor-ligand interactions, in which tritium, carbon-11 or fluorine-18 isotopes have been used. The history of benzodiazepines as medicinal agents is indeed an interesting one; an integral part of that history is their use in just about every conceivable labeled form

  11. 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

  12. 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

  13. 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

  14. Binding of [3H]ethyl-β-carboline-3-carboxylate to brain benzodiazepine receptors

    International Nuclear Information System (INIS)

    It is reported that in contrast to the changes in affinity of [3H]benzodiazepines elicited by halide ions, barbiturates, and pyrazolopyridines, the apparent affinity of β-[3H]CCE (ethyl-β-carboline-3-carboxylate) is unaffected by these agents. Furthermore, Scatchard analysis of β-[3H]CCE binding to cerebral cortical and cerebellar membranes revealed a significantly greater number of binding sites than was observed with either [3H]diazepam or [3H]flunitazepam, suggesting that at low concentrations benzodiazepines selectively label a subpopulation of the receptors labelled with β-[3H]CCE. Alternatively, β-[3H]CCE may bind to sites that are distinct from those labelled with [3H]-benzodiazepines. (Auth.)

  15. 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...

  16. The benzodiazepine/GABA receptor complex during severe ethanol intoxication and withdrawal in the rat

    International Nuclear Information System (INIS)

    The benzodiazepine/GABA (gammaaminobutyric acid) receptor complex was investigated during severe ethanol intoxication and withdrawal in the rat. The intragastric intubation technique was used to establish physical ethanol dependence in the animals. Cerebral cortex from male Wistar rats was studied 1) after 31/2 days of severe ethanol intoxication, 2) during the ethanol withdrawal reaction and 3) in a control group. The effect of GABA-ergic activation by muscimol and THIP (4,5,6,7-tetrahydroisoxazole(5,4-c)pyridin-3-01) on 3H-diazepam binding was unchanged during ethanol intoxication and withdrawal, as was the affinity constant (Ksub(D)) and the maximal number of binding sites (Bsub(max)) for 3H-flunitrazepam. In conclusion, the benzodiazepine/GABA receptor complex is unlikely to play any causual part in physical ethanol dependence. (author)

  17. 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.)

  18. 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...

  19. 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.

  20. 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

  1. Studies of peripheral benzodiazepine receptors in mussels: comparison between a polluted and a nonpolluted site.

    Science.gov (United States)

    Betti, Laura; Giannaccini, Gino; Nigro, Marco; Dianda, Sabina; Gremigni, Vittorio; Lucacchini, Antonio

    2003-01-01

    The aim of this study was to investigate the peripheral benzodiazepine receptors in soft tissue membranes of the mussel Mytilus galloprovincialis from both polluted and nonpolluted seawater populations, using a radioligand specific for this receptor, [3H]PK11195. Mussels were dissected into four body parts--mantle, gills, digestive gland, and muscles-to determine the distribution of tissue-specific peripheral benzodiazepine receptors (PBRs). The specific binding was saturable and reversible. A statistically significant increase (muscle, 537% and mantle, 201%, as absolute percentages) in the maximal number of binding sites (B(max)) was found in mussels from the polluted site, compared with mussels from the nonpolluted site. By contrast, the value of the dissociation constant (K(d)) at equilibrium does not show a statistically significant variation between the two groups. In competitive experiments of the compounds clonazepam, flumazenil, flunitrazepam, Ro5-4864, PK11195, and protoporphyrin IX, only PK11195 and protoporphyrin IX displaced [3H]PK11195 specifically bound to soft tissue membranes, revealing that the binding sites of peripheral benzodiazepine receptors of mussels have pharmacological properties comparable to those of low vertebrates such as trout. M. galloprovincialis was also tested as an indicator of heavy metal exposure, and metal accumulation in the digestive gland was measured by atomic absorption spectrophotometry (AAS). The contents of Pb, Mn, and Zn in mussels collected off the polluted site were higher than those in mussels from the nonpolluted site. These data suggest that PBRs are present in the soft tissues of the mussel M. galloprovincialis. Here we report preliminary evidence of biochemical alterations in mussels from the polluted site. PMID:12547633

  2. 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.

  3. 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

  4. Application of gamma-aminobutyric acid type A-benzodiazepine receptor imaging for study of neuropsychiatric disorders

    International Nuclear Information System (INIS)

    Gamma-aminobutyric acid type A-benzodiazepine receptors are heterogeneous polypeptide pentamers widely spread in the central nervous system on the neuron membrane. Different subunit combinations educe various neuro-inhibitory pharmacological effects such as sedative, hypnosis, anticonvulsion and anxiolysis. PET can be utilized to study the binding of the receptors in vivo. PET radioligands of gamma-aminobutyric acid type A-benzodiazepine receptors can be classified into 3 types: antagonists,agonists and reverse agonists, of which antagonist radiotracer 11C-flumazenil is the most commonly applied in epilepsy, anxiety disorders, depression, vegetative state,addiction and other neuro-psychiatric disorders. (authors)

  5. 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

  6. 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.

  7. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    International Nuclear Information System (INIS)

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial [3H]diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli [3H]diazepam binding are those that are active in displacing [3H]benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed

  8. 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.

  9. 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

  10. Impact of receptor clustering on ligand binding

    Directory of Open Access Journals (Sweden)

    Caré Bertrand R

    2011-03-01

    Full Text Available Abstract Background Cellular response to changes in the concentration of different chemical species in the extracellular medium is induced by ligand binding to dedicated transmembrane receptors. Receptor density, distribution, and clustering may be key spatial features that influence effective and proper physical and biochemical cellular responses to many regulatory signals. Classical equations describing this kind of binding kinetics assume the distributions of interacting species to be homogeneous, neglecting by doing so the impact of clustering. As there is experimental evidence that receptors tend to group in clusters inside membrane domains, we investigated the effects of receptor clustering on cellular receptor ligand binding. Results We implemented a model of receptor binding using a Monte-Carlo algorithm to simulate ligand diffusion and binding. In some simple cases, analytic solutions for binding equilibrium of ligand on clusters of receptors are provided, and supported by simulation results. Our simulations show that the so-called "apparent" affinity of the ligand for the receptor decreases with clustering although the microscopic affinity remains constant. Conclusions Changing membrane receptors clustering could be a simple mechanism that allows cells to change and adapt its affinity/sensitivity toward a given stimulus.

  11. 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...

  12. Ligand-receptor Interactions by NMR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Novak. P.

    2008-04-01

    Full Text Available Today NMR spectroscopy is a method of choice for elucidation of interactions between biomolecules and the potential ligands. Knowledge on these interactions is an essential prerequisite for the rational drug design. The most important contribution of NMR to drug design a few years ago was the 3D structure determination of proteins. Besides delivering the 3D structures of the free proteins as a raw material for the modeling studies on ligand binding, NMR can directly yield valuable experimental data on the biologically important protein-ligand complexes. In addition to X-ray diffraction, NMR spectroscopy can provide information on the internal protein dynamics ordynamics of intermolecular interactions. Changes in NMR parameters allow us to detect ("SAR by NMR" and quantitatively determine binding affinities (titration, diffusion NMR experiments, etc. of potential ligands. Also, it is possible to determine the binding site and conformations of ligands, receptors and receptor-ligand complexes with the help of NMR methods such as tr-NOESY. Epitopes or functional groups responsible for binding of ligands to the receptor can be identified by employing STD or WaterLOGSY experiments. In this review are described some of the most frequent NMR methods for the characterization of the interactions between biomolecules and ligands, together with their advantages and disadvantages.

  13. [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.)

  14. 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)

  15. 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.

  16. SPECT imaging of GABAA/benzodiazepine receptors and cerebral perfusion in mild cognitive impairment

    International Nuclear Information System (INIS)

    The involvement of neocortical and limbic GABAA/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 GABAA/BZD receptors in subjects with amnestic mild cognitive impairment (MCI) and to compare possible receptor changes to those in cerebral perfusion. [123I]Iomazenil and [99mTc]HMPAO SPECT images were acquired in 16 patients with amnestic MCI and in 14 normal elderly control subjects (only [123I]iomazenil imaging in 5, only [99mTc]HMPAO imaging in 4, and both [123I]iomazenil and [99mTc]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 [123I]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 GABAA/BZD receptors are essentially preserved. They suggest that in MCI patients functional changes precede neuronal/synaptic loss in neocortical posterior regions and that [99mTc]HMPAO rCBF imaging is more sensitive than [123I]iomazenil GABAA/BZD receptor imaging in detecting prodromal AD. (orig.)

  17. Characterisation of the contribution of the GABA-benzodiazepine α1 receptor subtype to [11C]Ro15-4513 PET images

    Science.gov (United States)

    Myers, James FM; Rosso, Lula; Watson, Ben J; Wilson, Sue J; Kalk, Nicola J; Clementi, Nicoletta; Brooks, David J; Nutt, David J; Turkheimer, Federico E; Lingford-Hughes, Anne R

    2012-01-01

    This positron emission tomography (PET) study aimed to further define selectivity of [11C]Ro15-4513 binding to the GABARα5 relative to the GABARα1 benzodiazepine receptor subtype. The impact of zolpidem, a GABARα1-selective agonist, on [11C]Ro15-4513, which shows selectivity for GABARα5, and the nonselective benzodiazepine ligand [11C]flumazenil binding was assessed in humans. Compartmental modelling of the kinetics of [11C]Ro15-4513 time-activity curves was used to describe distribution volume (VT) differences in regions populated by different GABA receptor subtypes. Those with low α5 were best fitted by one-tissue compartment models; and those with high α5 required a more complex model. The heterogeneity between brain regions suggested spectral analysis as a more appropriate method to quantify binding as it does not a priori specify compartments. Spectral analysis revealed that zolpidem caused a significant VT decrease (∼10%) in [11C]flumazenil, but no decrease in [11C]Ro15-4513 binding. Further analysis of [11C]Ro15-4513 kinetics revealed additional frequency components present in regions containing both α1 and α5 subtypes compared with those containing only α1. Zolpidem reduced one component (mean±s.d.: 71%±41%), presumed to reflect α1-subtype binding, but not another (13%±22%), presumed to reflect α5. The proposed method for [11C]Ro15-4513 analysis may allow more accurate selective binding assays and estimation of drug occupancy for other nonselective ligands. PMID:22214903

  18. 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.

  19. 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

  20. 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.

  1. 125I-iomazenil-benzodiazepine receptor binding during psychological stress in rats

    International Nuclear Information System (INIS)

    We investigated the changes in 125I-iomazenil (125I-IMZ) benzodiazepine receptor (BZR) binding with psychological stress in a rat model. Six male Wistar rats were placed under psychological stress for 1 hour by using a communication box. No physical stress was not received. 1.85 MBq of 125I-IMZ was injected into the lateral tail vein and the rat was killed 3 hours later. Twenty-micormeter-thick sections of the brain were collected and % injected dose per body weight (% ID/BW) of eleven regions (frontal, parietal, temporal, occipital cortices, caudate putamen, accumubens nuclei, globus pallidus, amygdala, thalamus, hippocampus and hypothalamus) were calculated by autoradiography. The %ID/BW of rats which were placed under psychological stress was compared with that of 6 control rats. The %ID/BW of rats which were placed under psychological stress diffusely tended to show a reduction in 125I-IMZ-BZR binding. A significant decrease in BZR binding was observed in the hippocampus of the rats which were placed under psychological stress. 125I-IMZ-BZR binding tended to decrease throughout the brain. (author)

  2. Interactions of pyrethroid insecticides with GABAA and peripheral-type benzodiazepine receptors

    International Nuclear Information System (INIS)

    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 1RαS, cis cypermethrin having an ED50 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 [3H]Ro5-4864 to rat brain membranes with a significant correlation between the log EC50 values for their activities as proconvulsants and the log IC50 values for their inhibition of [3H]Ro5-4864 binding. Both Ro5-4864 and pyrethroid insecticides were found to influence specific [35S]TBPS binding in a GABA-dependent manner. PK 11195 and the Type II pyrethroid, deltamethrin antagonized the Ro5-4864-induced modulation of [35S]TBPS binding. Pyrethroid insecticides, Ro5-4864 and veratridine influenced GABA-gated 36Chloride influx. Moreover, the Type II pyrethroids elicited an increase in 36chloride influx in the absence of GABA-stimulation. Both of these actions were antagonized by PK 11195 and tetrodotoxin

  3. 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.

  4. 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.

  5. 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

  6. 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...

  7. Apparent target size of rat brain benzodiazepine receptor, acetylcholinesterase, and pyruvate kinase is highly influenced by experimental conditions

    International Nuclear Information System (INIS)

    Radiation inactivation is a method to determine the apparent target size of molecules. In this report we examined whether radiation inactivation of various enzymes and brain receptors is influenced by the preparation of samples preceding irradiation. The apparent target sizes of endogenous acetylcholinesterase and pyruvate kinase from rat brain and from rabbit muscle and benzodiazepine receptor from rat brain were investigated in some detail. In addition the target sizes of alcohol dehydrogenase (from yeast and horse liver), beta-galactosidase (from Escherichia coli), lactate dehydrogenase (endogenous from rat brain), and 5-HT2 receptors, acetylcholine muscarine receptors, and [35S] butyl bicyclophosphorothionate tertiary binding sites from rat brain were determined. The results show that apparent target sizes are highly influenced by the procedure applied for sample preparation before irradiation. The data indicate that irradiation of frozen whole tissue as opposed to lyophilized tissue or frozen tissue homogenates will estimate the smallest and most relevant functional target size of a receptor or an enzyme

  8. 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

  9. Ligand-directed trafficking of receptor stimulus.

    Science.gov (United States)

    Chilmonczyk, Zdzisław; Bojarski, Andrzej J; Sylte, Ingebrigt

    2014-12-01

    GPCRs are seven transmembrane-spanning receptors that convey specific extracellular stimuli to intracellular signalling. They represent the largest family of cell surface proteins that are therapeutically targeted. According to the traditional two-state model of receptor theory, GPCRs were considered as operating in equilibrium between two functional conformations, an active (R*) and inactive (R) state. Thus, it was assumed that a GPCR can exist either in an "off" or "on" conformation causing either no activation or equal activation of all its signalling pathways. Over the past several years it has become evident that this model is too simple and that GPCR signalling is far more complex. Different studies have presented a multistate model of receptor activation in which ligand-specific receptor conformations are able to differentiate between distinct signalling partners. Recent data show that beside G proteins numerous other proteins, such as β-arrestins and kinases, may interact with GPCRs and activate intracellular signalling pathways. GPCR activation may therefore involve receptor desensitization, coupling to multiple G proteins, Gα or Gβγ signalling, and pathway activation that is independent of G proteins. This latter effect leads to agonist "functional selectivity" (also called ligand-directed receptor trafficking, stimulus trafficking, biased agonism, biased signalling), and agonist intervention with functional selectivity may improve the therapy. Many commercially available drugs with beneficial efficacy also show various undesirable side effects. Further studies of biased signalling might facilitate our understanding of the side effects of current drugs and take us to new avenues to efficiently design pathway-specific medications. PMID:25443729

  10. 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

  11. 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.; 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...... 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...

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

    International Nuclear Information System (INIS)

    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 GABAA receptor β3 subunit (GABRB3) is located. Since the benzodiazepine receptor is constructed as a receptor-ionophore complex that contains the GABAA receptor, it is a suitable marker for GABA-ergic synapsis. To determine whether benzodiazepine receptor density, which indirectly indicates changes in GABAA receptor density, is altered in the brain in patients with AS, we investigated a 27-year-old woman with AS using 123I-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-123 iodoamphetamine. We demonstrated that benzodiazepiine 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 GABAA 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 GABAA receptor in the investigated patient. 123I-iomazenil with SPET was useful to map benzodiazepine receptors, which indicate GABAA receptor distribution and their density. (orig.)

  13. 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.)

  14. [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

  15. {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.

  16. 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

    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...

  17. 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

  18. 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...

  19. Lysine and pipecolic acid and some of their derivatives show anticonvulsant activity, and stimulation of benzodiazepine receptor activity

    International Nuclear Information System (INIS)

    Benzodiazepines are one of the most widely prescribed drugs in the treatment of anxiety, epilepsy and muscle tension. The natural products lysine and pipecolic acid known to be present in the animal, plant and microorganism, have been shown to be anticonvulsant against pentetrazol (PTZ)-induced seizures in mice. Methyl and ethyl esters of L-lysine and the N-isopropanol derivative of pipecolic acid appear to increase the anticonvulsant potency of the parent compounds, presumably due to their increase in hydrophobicity. Lysine and pipecolic acid showed significant stimulation of specific [3H]flunitrazepam (FZ) binding to mouse brain membranes. This stimulation was enhanced by chloride ions and stereospecific with L-isomer having higher effect. The dose-dependent anticonvulsant activity of lysine and pipecolic acid, and their stimulation of [3H]FZ binding appear to be correlated. The antiepileptic activity lysine, pipecolic acid and their derivatives therefore may be mediated through the γ-aminobutyric acid-benzodiazepine receptor complex

  20. 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...

  1. (/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.

  2. 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.

  3. Effects of LiCl/pilocarpine-induced status epilepticus on rat brain mu and benzodiazepine receptor binding: Regional and ontogenetic studies

    Czech Academy of Sciences Publication Activity Database

    Rocha, L.; Suchomelová, Lucie; Mareš, Pavel; Kubová, Hana

    2007-01-01

    Roč. 1181, - (2007), s. 104-117. ISSN 0006-8993 R&D Projects: GA MŠk(CZ) LC554; GA ČR GA304/05/2582 Grant ostatní: CONACyT(MX) 45943-M Institutional research plan: CEZ:AV0Z50110509 Keywords : benzodiazepine receptor * µ receptor binding * status epilepticus Subject RIV: ED - Physiology Impact factor: 2.218, year: 2007

  4. 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.

  5. 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.

  6. 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

  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. 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.

  9. 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.)

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    -(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO) in complex with the ligand-binding core of the receptor. Comparison with the only previous structure of the ligand-binding core in complex with an antagonist, 6,7-dinitro-2,3-quinoxalinedione (DNQX) (Armstrong, N.; Gouaux, E. Neuron 2000, 28, 165-181), reveals...

  11. 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

  12. Force History Dependence of Receptor-Ligand Dissociation

    OpenAIRE

    Marshall, Bryan T.; Sarangapani, Krishna K.; Lou, Jizhong; McEver, Rodger P.; Zhu, Cheng

    2004-01-01

    Receptor-ligand bonds that mediate cell adhesion are often subjected to forces that regulate their dissociation via modulating off-rates. Off-rates control how long receptor-ligand bonds last and how much force they withstand. One should therefore be able to determine off-rates from either bond lifetime or unbinding force measurements. However, substantial discrepancies exist between the force dependence of off-rates derived from the two types of measurements even for the same interactions, e...

  13. 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.)

  14. Development of 123I-labelled NNC 13-8241 as a radioligand for SPECT visualization of benzodiazepine receptor binding

    International Nuclear Information System (INIS)

    [125I]- and [123I]NNC 13-8241 were prepared from the trimethyltin precursor and radioactive iodide using the chloramine-T method. The total radiochemical yields of [125I]- and [123I]NNC 13-8241 were 60-70% and 40-50% respectively, with radiochemical purity higher than 98%. In binding studies with [125I]NNC 13-8241 in rats in vitro and in vivo a high uptake of radioactivity was demonstrated in brain regions known to have a high density of benzodiazepine (BZ) receptors such as the occipital and frontal cortex. SPECT examination with [123I]NNC 13-8241 in a Cynomolgus monkey demonstrated a high uptake of radioactivity in the occipital and frontal cortex. After displacement with flumazenil radioactivity in these brain regions was reduced to the level of a central region including the pons. Four hours after injection about 80% of the radioactivity in monkey plasma represented unchanged radioligand. This low degree of metabolism indicates that NNC 13-8241 is metabolically more stable than the radioligands hitherto developed for imaging of BZ-receptors in the primate brain

  15. 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...

  16. 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

  17. 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.

  18. The imidazoline receptors and ligands in pain modulation.

    Science.gov (United States)

    Bektas, Nurcan; Nemutlu, Dilara; Arslan, Rana

    2015-01-01

    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 I2 receptor 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 I2 receptor 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. PMID:26600633

  19. 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.)

  20. 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.)

  1. 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

  2. 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.

  3. Alterations of benzodiazepine receptor binding potential in anxiety and somatoform disorders measured by 123I-iomazenil SPECT

    International Nuclear Information System (INIS)

    123I-iomazenil (IMZ), a newly developed radioligand which acts on benzodiazepine receptors (BZR) as a partial inverse agonist, made it possible to evaluate the function of central BZR by single photon emission tomography (SPECT). To examine the alterations of the binding potential (BP) in the anxiety state, 123I-IMZ SPECT was performed in five patients with anxiety and somatoform disorders, and five epileptic patients without anxiety symptoms served as a reference. The BP of BZR was determined by using a table look-up procedure based on a three-compartment, two-parameter model in the bilateral superior frontal, inferior frontal, temporal, parietal, occipital, and cerebellar cortex. The mean BP of patients with anxiety and somatoform disorders was significantly decreased in the superior frontal, temporal, and parietal cortex, in comparison with that of epileptic patients. A significant correlation was observed between the anxiety levels scored on the Hamilton anxiety scale and BP in the right temporal cortex and left superior frontal cortex. These changes in BZR revealed by SPECT suggest the usefulness of 123I-IMZ SPECT to objectively evaluate anxiety levels in patients with anxiety symptoms. (author)

  4. 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.

  5. A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

    DEFF Research Database (Denmark)

    Kongsbak, Kristine Grønning; Bergmann, Rikke; Sørensen, Pernille Louise; Sander, Tommy; Balle, Thomas

    2013-01-01

    -gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode...... similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues a1R66, b2T202, a1T129, b2E155, b2Y205 and the backbone of b2S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy...

  6. Human locus coeruleus neurons express the GABAA receptor γ2 subunit gene and produce benzodiazepine binding

    OpenAIRE

    Hellsten, Kati S.; Sinkkonen, Saku T.; Hyde, Thomas M.; Kleinman, Joel E.; Särkioja, Terttu; Maksimow, Anu; Uusi-Oukari, Mikko; Korpi, Esa R.

    2010-01-01

    Noradrenergic neurons of the locus coeruleus project throughout the cerebral cortex and multiple subcortical structures. Alterations in the locus coeruleus firing are associated with vigilance states and with fear and anxiety disorders. Brain ionotropic type A receptors for γ-aminobutyric acid (GABA) serve as targets for anxiolytic and sedative drugs, and play an essential regulatory role in the locus coeruleus. GABAA receptors are composed of a variable array of subunits forming heteropentam...

  7. Different effects of long-term haloperidol administration on GABA/sub A/ and benzodiazepine receptors in various parts of the brain

    International Nuclear Information System (INIS)

    The data described in this paper are evidence that long-term administration of haloperidol has an opposite effect on the density of GABA/sub A/ and benzodiazepine receptors in the fore- and hindbrain. These changes are reflected at the molecular level as reversal of behavioral effect of the GABA/sub A/ agonist muscimol and the benzodiazepine agonist Ro15-1788. By means of parallel behavioral tests, binding of 3H-muscimol in the fore- and hindbrain of rats was investigated in experiments in vitro. 3H-flunitrazepam binding experiments were carried out in vivo on mice. Parallel with reversal of the behavioral effects of muscimol and Ro15-1788, the number of binding sites both for 3H-muscimol and for 3H-flunitrazepam in the forebrain was reduced; in the hindbrain the opposite process took place

  8. A response calculus for immobilized T cell receptor ligands

    DEFF Research Database (Denmark)

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

    2001-01-01

    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 affini...... affinity in solution, are of optimal two-dimensional affinity thereby allowing effective TCR binding under physiological conditions, i.e. at low ligand densities in cellular interfaces....... 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 the...

  9. Quantification of ligand bias for clinically relevant β2-adrenergic receptor ligands: implications for drug taxonomy.

    Science.gov (United States)

    van der Westhuizen, Emma T; Breton, Billy; Christopoulos, Arthur; Bouvier, Michel

    2014-03-01

    The concepts of functional selectivity and ligand bias are becoming increasingly appreciated in modern drug discovery programs, necessitating more informed approaches to compound classification and, ultimately, therapeutic candidate selection. Using the β2-adrenergic receptor as a model, we present a proof of concept study that assessed the bias of 19 β-adrenergic ligands, including many clinically used compounds, across four pathways [cAMP production, extracellular signal-regulated kinase 1/2 (ERK1/2) activation, calcium mobilization, and receptor endocytosis] in the same cell background (human embryonic kidney 293S cells). Efficacy-based clustering placed the ligands into five distinct groups with respect to signaling signatures. In some cases, apparent functional selectivity originated from off-target effects on other endogenously expressed adrenergic receptors, highlighting the importance of thoroughly assessing selectivity of the responses before concluding receptor-specific ligand-biased signaling. Eliminating the nonselective compounds did not change the clustering of the 10 remaining compounds. Some ligands exhibited large differences in potency for the different pathways, suggesting that the nature of the receptor-effector complexes influences the relative affinity of the compounds for specific receptor conformations. Calculation of relative effectiveness (within pathway) and bias factors (between pathways) for each of the compounds, using an operational model of agonism, revealed a global signaling signature for all of the compounds relative to isoproterenol. Most compounds were biased toward ERK1/2 activation over the other pathways, consistent with the notion that many proximal effectors converge on this pathway. Overall, we demonstrate a higher level of ligand texture than previously anticipated, opening perspectives for the establishment of pluridimensional correlations between signaling profiles, drug classification, therapeutic efficacy, and

  10. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    Directory of Open Access Journals (Sweden)

    A. Beaudet

    1998-11-01

    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.

  11. 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

  12. 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.

  13. 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...

  14. 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...... order to investigate the structural determinants for receptor selectivity between AMPA and the GluR5 subtype of kainate receptors. Compounds selective for either GluR5 or AMPA receptors were identified. One particular substituent position appeared to be of special importance for control of ligand...... selectivity. Using molecular modeling the observed structure-activity relationships at AMPA and GluR5 receptors were deduced....

  15. 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.

  16. 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

  17. Alterations in in-vivo benzodiazepine-receptor binding of C-11-Ro15-1788 (flumazepil)

    International Nuclear Information System (INIS)

    Alterations of the central benzodiazepine - receptor function caused by the change of physiological or psychological conditions, were recognized in both animal and human studies. Before the human study, animal experiments using tritiated Ro15-1788 were carried out. The stress was produced by forcing the mice to swim in a water-basin at 160C for 5 min. Within 3 min after the forced swimming, the tracer was injected. Brain radioactivities in stress-loaded mice increased over a period of 15 min after the intra-venous injection of tracers, while brain activities of carrier-added tracer decreased. In human study, approximately 5 mCi of C-11-Ro15-1788, which specific activity is 0.3-1.0 Ci/μmol, were intravenously injected to each case. Measurements of the brain activity were performed using positron-CT, with blood sample collection. 31 human studies were performed on. Cerebral cortex time activity curves in several volunteers in nervous and stressful state, showed the same pattern to that in the stress-loaded animal experiment. It is important that the significant different time course of cerebral activity after the injection of labelled Ro15-1788, was observed in stressful state, compared with control, in both human and animal study. From these results, it will be concluded the positron CT study using /sup 11/C-Ro15-1788 will become a new technic to detect the change of psychological conditions in human brain and to diagnose some kind of neuropsychiatric disease

  18. 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.

  19. 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.

  20. Binding affinity prediction of novel estrogen receptor ligands using receptor-based 3-D QSAR methods.

    Science.gov (United States)

    Sippl, Wolfgang

    2002-12-01

    We have recently reported the development of a 3-D QSAR model for estrogen receptor ligands showing a significant correlation between calculated molecular interaction fields and experimentally measured binding affinity. The ligand alignment obtained from docking simulations was taken as basis for a comparative field analysis applying the GRID/GOLPE program. Using the interaction field derived with a water probe and applying the smart region definition (SRD) variable selection procedure, a significant and robust model was obtained (q(2)(LOO)=0.921, SDEP=0.345). To further analyze the robustness and the predictivity of the established model several recently developed estrogen receptor ligands were selected as external test set. An excellent agreement between predicted and experimental binding data was obtained indicated by an external SDEP of 0.531. Two other traditionally used prediction techniques were applied in order to check the performance of the receptor-based 3-D QSAR procedure. The interaction energies calculated on the basis of receptor-ligand complexes were correlated with experimentally observed affinities. Also ligand-based 3-D QSAR models were generated using program FlexS. The interaction energy-based model, as well as the ligand-based 3-D QSAR models yielded models with lower predictivity. The comparison with the interaction energy-based model and with the ligand-based 3-D QSAR models, respectively, indicates that the combination of receptor-based and 3-D QSAR methods is able to improve the quality of prediction. PMID:12413831

  1. 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.

  2. 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...

  3. Opioid ligands and receptors of the joint

    OpenAIRE

    Bergström, Jonas

    2006-01-01

    The aim was to explore the occurrence of an opioid system in joints. Thus, joint tissues from rats with and without arthritis and also from patients with knee osteoarthrosis were investigated by EM, immunohistochemistry, RIA, HPLC, receptor binding assay and RT-PCR. In rat joints, EM demonstrated the occurrence of met-enkephalin (ME) in nerve fibers, but also in osteoblasts, osteocytes, endothelial, synovial and monoblastic cells. The novel finding of multiple sources of op...

  4. 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

  5. Structure and function of Toll receptors and their ligands.

    Science.gov (United States)

    Gay, Nicholas J; Gangloff, Monique

    2007-01-01

    The Toll family of class I transmembrane receptors recognizes and responds to diverse structures associated with pathogenic microorganisms. These receptors mediate initial responses in innate immunity and are required for the development of the adaptive immune response. Toll receptor signaling pathways are also implicated in serious autoimmune diseases such as endotoxic shock and thus are important therapeutic targets. In this review we discuss how microbial structures as different as nucleic acids and lipoproteins can be recognized by the extracellular domains of Toll receptors. We review recent evidence that the mechanism of signal transduction is complex and involves sequential changes in the conformation of the receptor induced by binding of the ligand. Finally, we assess the emerging area of cross talk in the Toll pathways. Recent work suggests that signaling through TLR4 in response to endotoxin is modified by inputs from at least two other pathways acting through beta2 integrins and protein kinase Cepsilon. PMID:17362201

  6. Selective effects of ligands on vitamin D3 receptor- and retinoid X receptor-mediated gene activation in vivo.

    OpenAIRE

    Lemon, B D; Freedman, L P

    1996-01-01

    Steroid/nuclear hormone receptors are ligand-regulated transcription f factors that play key roles in cell regulation, differentiation, and oncogenesis. Many nuclear receptors, including the human 1,25-dihydroxyvitamin D3 receptor (VDR), bind cooperatively to DNA either as homodimers or as heterodimers with the 9-cis retinoic acid (RA) receptor (retinoid X-receptor [RXR]). We have previously reported that the ligands for VDR and RXR can differentially modulate the affinity of the receptors' i...

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

    International Nuclear Information System (INIS)

    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 IMZ

  8. Selection of Genetic engineering peptide ligands for TNF receptor imaging

    International Nuclear Information System (INIS)

    Objective: To screen the peptide ligands of TNF receptor from phage 6-mer peptide library with the purpose of developing new peptides radiopharmaceuticals for TNF receptor imaging. Methods: The soluble protein of TNF receptor I (sTNFR I) was used to screen the TNF-specific epitopes from phage 6-mer peptide library. After four rounds of affinity screening, the peptides displayed on the selected phage were directly subjected to ELISA to determine their immunological activity to sTNFR. The amino acid sequences of the peptides with highest immunological activity were deduced through DNA sequencing. And their conserved sequences were further determined. Results: Peptides sequences mimicking TNF-specific epitopes were obtained. Conclusion: The short peptides sequences mimicking TNF -specific epitopes were successfully acquired. The method which was established in the present study may provide a feasible way in peptides radiopharmaceuticals development for TNF receptor imaging. (authors)

  9. Oxytocin receptor ligands induce changes in cytoskeleton in neuroblastoma cells.

    Science.gov (United States)

    Bakos, Jan; Strbak, Vladimir; Paulikova, Helena; Krajnakova, Lucia; Lestanova, Zuzana; Bacova, Zuzana

    2013-07-01

    Aim of the present study was to evaluate effects of ligands of oxytocin receptors on gene expression of neurofilament proteins (nestin and microtubule-associated protein 2 (MAP2)) associated with neuronal differentiation and growth factors (brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF)) related to neuronal growth. Fluorescent staining of F-actin was used to observe morphology of cells. Co-treatment with oxytocin and oxytocin receptor antagonist--atosiban--resulted in significant increase of MAP2 gene expression in SK-N-SH cells. There was no effect of oxytocin on gene expression of growth factors BDNF and NGF. Surprisingly, oxytocin with atosiban significantly increased mRNA levels for both BDNF and NGF. Gene expression of vasopressin receptor (V1aR) significantly decreased in response to vasopressin. Atosiban decreased mRNA levels for oxytocin receptor (OXTR) and V1aR. Oxytocin significantly decreased OXTR and nestin mRNA levels and increased mRNA levels for BDNF and NGF in U-87 MG cells. The densest recruitment of F-actin filaments was observed in apical parts of filopodia in SK-N-SH cells incubated in oxytocin presence. Present data demonstrate complex role of ligands of oxytocin receptors in regulation of gene expression of intermediate filaments and thus, oxytocin might be considered as a growth factor in neuronal type of cells. PMID:23335033

  10. 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...

  11. 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

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

    International Nuclear Information System (INIS)

    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

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. Structural basis for AMPA receptor activation and ligand selectivity

    DEFF Research Database (Denmark)

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

    2002-01-01

    structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex...... with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent...... the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists....

  19. 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

  20. 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)

  1. Benzodiazepine receptor and cerebral blood flow in early Alzheimer`s disease. SPECT study using {sup 123}I-Iomazenil and {sup 123}I-IMP

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, Shin; Koshi, Yasuhiko; Komiyama, Tasuku; Sakayori, Osamu; Komaba, Yuichi; Ohyama, Masashi; Mishina, Masahiro; Tsuganesawa, Toshikazu; Terashi, Akiro [Nippon Medical School, Tokyo (Japan). First Hospital

    1996-01-01

    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 {sup 123}I-Iomazenil (IMZ) and {sup 123}I-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).

  2. Imaging of neuropsychiatric disorders. The usefulness of new brain receptor radiopharmaceuticals

    International Nuclear Information System (INIS)

    Until now, imaging of brain receptors have been only possible with positron emission tomography (PET), because there have been no high specific activity, high receptor affinity tracers available for clinical use with single photon emission tomography (SPECT). During the recent years fair number of new receptor ligands have been developed. For dopaminergic system there, are ligands for studying both presynaptic and postsynaptic sites, benzodiazepine receptors can be imaged with high quality ligands and also serotonergic receptors can be imaged. Very intensive work is underway for developing muscarinic receptor ligands, as well as for many other brain receptors

  3. Differential effects of TRPV1 receptor ligands against nicotine-induced depression-like behaviors

    OpenAIRE

    2011-01-01

    Background The contributions of brain cannabinoid (CB) receptors, typically CB1 (CB type 1) receptors, to the behavioral effects of nicotine (NC) have been reported to involve brain transient receptor potential vanilloid 1 (TRPV1) receptors, and the activation of candidate endogenous TRPV1 ligands is expected to be therapeutically effective. In the present study, the effects of TRPV1 ligands with or without affinity for CB1 receptors were examined on NC-induced depression-like behavioral alte...

  4. 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)

  5. IDENTIFICATION OF VDR ANTAGONISTS AMONG NUCLEAR RECEPTOR LIGANDS USING VIRTUAL SCREENING

    OpenAIRE

    Kelly Teske; Premchendar Nandhikonda; Bogart, Jonathan W.; Belaynesh Feleke; Preetpal Sidhu; Yuan, Nina Y.; Joshua Preston; Robin Goy; Lanlan Han; Silvaggi, Nicholas R; Singh, Rakesh K.; Bikle, Daniel D.; Cook, James M.; Arnold, Leggy A.

    2014-01-01

    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 ...

  6. 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; Mariuzza, R A; Karjalainen, K

    2001-01-01

    the SEC3 variants correlated with enhanced binding without any optimum in the binding range covered by native TCR ligands. Comparable studies using anti-TCR antibodies of known affinity confirmed these observations. By comparing the biological potency of the two sets of ligands, we found a significant...... 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....

  7. Increased expression of mitochondrial benzodiazepine receptors following low-level light treatment facilitates enhanced protoporphyrin IX production in glioma-derived cells in vitro

    Science.gov (United States)

    Bisland, S. K.; Hassanali, N. S.; Johnson, C.; Wilson, B. C.

    2007-02-01

    This study investigates whether low level light treatment (LLLT) can enhance the expression of Peripheral-type mitochondrial benzodiazepine receptors (PBRs) on the glioma-derived tumour cell line, CNS-1, and by doing so promote the synthesis of protoporphyrin IX (PpIX) and increase the photodynamic therapy (PDT)-induced cell kill using 5-aminolevulinic acid (ALA). The endogenous photosensitizer, (PpIX) and related metabolites including coproporphyrin III are known to traffic via the PBRs on the outer mitochondrial membrane on their passage into or out of the mitochondria. Astrocyte-derived cells within the brain express PBRs, while neurons express the central-type of benzodiazepine receptor. CNS-1 cells were exposed to a range of differing low-level light protocols immediately prior to PDT. LLLT involved using broad-spectrum light or monochromatic laser light specific to 635 or 905 nm wavelength. Cells (5μ10 5) were exposed to a range of LLLT doses (0, 1 or 5 J/cm2) using a fixed intensity of 10 mW/cm2 and subsequently harvested for cell viability, immunofluorescence or western blot analysis of PBR expression. The amount of PpIX within the cells was determined using chemical extraction techniques. Results confirm the induction of PBR following LLLT is dependent on the dose and wavelength of light used. Broadspectrum light provided the greatest cell kill following PDT, although LLLT with 635 nm or 905 nm also increased cell kill as compared to PDT alone. All LLLT regimens increased PBR expression compared to controls with corresponding increases in PpIX production. These data suggest that by selectively increasing PBR expression in tumour cells, LLLT may facilitate enhanced cell kill using ALA-PDT without damaging surrounding normal brain.

  8. 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. PMID:26876472

  9. Using [11C]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.

    2016-01-01

    The importance of the GABA-benzodiazepine receptor complex and its subtypes are increasingly recognised in addiction. Using the α1/α5 benzodiazepine receptor PET radioligand [11C]Ro15 4513, we previously showed reduced binding in the nucleus accumbens and hippocampus in abstinent alcohol dependence. We proposed that reduced [11C]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 [11C]Ro15 4513 binding in another addiction, opiate dependence, and used spectral analysis to estimate contributions of α1 and α5 subtypes to [11C]Ro15 4513 binding in opiate and previously acquired alcohol-dependent groups. Opiate substitute maintained opiate-dependent men (n = 12) underwent an [11C]Ro15 4513 PET scan and compared with matched healthy controls (n = 13). We found a significant reduction in [11C]Ro15 4513 binding in the nucleus accumbens in the opiate-dependent compared with the healthy control group. There was no relationship between [11C]Ro15 4513 binding in the hippocampus with memory. We found that reduced [11C]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 [11C]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. PMID:26876472

  10. 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

  11. 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.

  12. 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

  13. Transport regulation of two-dimensional receptor-ligand association.

    Science.gov (United States)

    Ju, Lining; Qian, Jin; Zhu, Cheng

    2015-04-01

    The impact of flow disturbances on platelet adhesion is complex and incompletely understood. At the molecular scale, platelet glycoprotein Ibα (GPIbα) must associate with the von Willebrand factor A1 domain (VWF-A1) with a rapid on-rate under high hemodynamic forces, as occurs in arterial thrombosis, where various transport mechanisms are at work. Here, we theoretically modeled the coupled transport-reaction process of the two-dimensional (2D) receptor-ligand association kinetics in a biomembrane force probe to explicitly account for the effects of molecular length, confinement stiffness, medium viscosity, surface curvature, and separation distance. We experimentally verified the theoretical approach by visualizing association and dissociation of individual VWF-A1-GPIbα bonds in a real-time thermal fluctuation assay. The apparent on-rate, reciprocal of the average time intervals between sequential bonds, decreased with the increasing gap distance between A1- and GPIbα-bearing surfaces with an 80-nm threshold (beyond which bond formation became prohibitive) identified as the combined contour length of the receptor and ligand molecules. The biomembrane force probe spring constant and diffusivity of the protein-bearing beads also significantly influenced the apparent on-rate, in accordance with the proposed transport mechanisms. The global agreement between the experimental data and the model predictions supports the hypothesis that receptor-ligand association behaves distinctly in the transport- and reaction-limited scenarios. To our knowledge, our results represent the first detailed quantification of physical regulation of the 2D on-rate that allows platelets to sense and respond to local changes in their hemodynamic environment. In addition, they provide an approach for determining the intrinsic kinetic parameters that employs simultaneous experimental measurements and theoretical modeling of bond association in a single assay. The 2D intrinsic forward rate

  14. 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

  15. 125I-LSD: a high sensitivity ligand for serotonin receptors

    International Nuclear Information System (INIS)

    125I-labeled receptor ligands offer unique advantages over their 3H-labeled counterparts. Carrier-free 125I-labeled ligands can be synthesized with specific activities of up to 2170 Ci/mmol while (mono) tritium labeled ligands are limited to 29 Ci/mmol. Therefore, 125I-labeled ligands can be approximately 70-fold more sensitive than 3H-labeled ligands in detecting receptor sites. In addition, 125I-labeled ligands emit relatively energetic X-rays and γ-rays which are readily detected by gamma counting equipment. The authors report here the serotonergic binding properties of 125I-LSD the first reported 125I-labeled ligand for serotonin receptors. (Auth.)

  16. 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 ...

  17. Functional phylogenetics reveals contributions of pleiotropic peptide action to ligand-receptor coevolution

    Science.gov (United States)

    The evolution of peptidergic signaling has been accompanied by a significant degree of ligand-receptor coevolution. Closely related clusters of peptide signaling molecules are observed to activate related groups of receptors, implying that genes encoding these ligands may orchestrate an array of fu...

  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. 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 ...

  20. 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

  1. 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

  2. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands

    Science.gov (United States)

    Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N–H and O–H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  3. 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 m...... 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....

  4. A New Method for Ligand Docking to Flexible Receptors by Dual Alanine Scanning and Refinement (SCARE)

    OpenAIRE

    Bottegoni, Giovanni; Kufareva, Irina; Totrov, Maxim; Abagyan, Ruben

    2008-01-01

    Protein binding sites undergo ligand specific conformational changes upon ligand binding. However, most docking protocols rely on a fixed conformation of the receptor, or on the prior knowledge of multiple conformations representing the variation of the pocket, or on a known bounding box for the ligand. Here we described a general induced fit docking protocol that requires only one initial pocket conformation and identifies most of the correct ligand positions as the lowest score. We expanded...

  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. 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

  7. 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

  8. Stamping Vital Cells—a Force-Based Ligand Receptor Assay

    OpenAIRE

    Wienken, Uta; Gaub, Hermann E.

    2013-01-01

    Gaining information about receptor profiles on cells, and subsequently finding the most efficient ligands for these signaling receptors, remain challenging tasks in stem cell and cancer research as well as drug development. We introduce a live-cell method with great potential in both screening for surface receptors and analysing binding forces of different ligands. The technique is based on the molecular force assay, a parallel-format, high-throughput experiment on a single-molecule level. On...

  9. 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.

  10. 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).

  11. 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....

  12. Increase in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro5 4864, a peripheral-type (p site) benzodiazepine ligand.

    Science.gov (United States)

    Taupin, V; Toulmond, S; Serrano, A; Benavides, J; Zavala, F

    1993-02-01

    The effects of fluid percussion trauma on brain interleukin (IL)-6, IL-1 and tumor necrosis factor-alpha (TNF-alpha) levels have been studied. In the cortex and hippocampus of control and sham-operated rats, the levels of these cytokines were very low (below 4 units/mg protein) and constant. IL-6 and IL-1 levels in the ipsilateral cortex increased rapidly following trauma to reach a maximum of 350 and 16 units/mg protein, respectively, 8 h after the lesion, remained elevated until 18 h and decreased thereafter to basal values. TNF-alpha levels were maximally elevated (12 units/mg protein) at 3 h and 8 h and returned to basal values by 18 h. Qualitatively similar changes, but with 25-80-fold smaller amplitude, were seen in the contralateral cortex and in the ipsi- and contralateral hippocampus. The levels of IL-6 in the plasma of sham-operated and lesioned rats were only slightly elevated, whereas IL-1 and TNF-alpha were undetectable. Histological studies of brain tissue at early stages after trauma demonstrated an acute hemorrhage associated with neutrophil invasion. The administration of Ro5 4864 (0.5 mg/kg i.p.), a specific ligand of p (peripheral-type benzodiazepine) binding sites, did not result in any significant effect on the levels of IL-6, IL-1 or TNF-alpha in the brain of control or sham-operated animals. However, when administered 24 h before or 15 min after trauma, this benzodiazepine enhanced the increase of these cytokines by 2-4-fold in the ipsilateral cortex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8429103

  13. 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...

  14. Effects of protein restriction, melatonin administration, and short daylength on brain benzodiazepine receptors in prepubertal male rats

    International Nuclear Information System (INIS)

    The possibility that there are changes in brain benzodiazepine binding sites controlled by photoperiod was investigated in two strains of male rats. The hypothesis was tested by 3H-diazepam binding studies in various brain regions of prepubertal rats maintained in 14 or 10 h of light or treated with late-afternoon injections of melatonin (50 micrograms/day). Protein restriction was applied during the experiment to sensitize the animals to the treatments. Under the conditions employed, rats kept in short daylength throughout or kept on long photoperiod and given late-afternoon melatonin injections showed evidence of delayed puberty (seminal vesicle, ventral prostate, and testis weight decreased by 45%, 55%, and 60% respectively, compared to control rats). Binding measurements were made 1 h before and 2 and 5 h after the onset of darkness in the pubertal (42-day-old) or experimentally prepubertal rats. In the rats of the Porton strain (for which protein restriction was obligatory for the gonadal response) there was no consistent treatment or time effects on specific binding of 3H-diazepam to washed membranes of the hypothalamus, midbrain, or striatum. Similarly, there were no differences in the stimulation of 3H-diazepam binding by 100 microM GABA or the inhibition of binding by 50 microM N-acetyl 5 methoxy kynurenamine. By contrast, in Wistar rats, specific binding to midbrain membranes was reduced 5 h after dark compared to 2 h (37% saline; 20% melatonin) and the extent of stimulation by GABA in the hypothalamus was increased 5 h after darkness (35.6% to 46.7% saline; 37.4% to 50% melatonin). Melatonin treatment resulted in significantly higher specific binding in the hypothalamus 2 h after dark (10%, control fed; 20%, protein restricted) but reduced the GABA induced stimulation of binding in the midbrain (35.5% to 25%, control fed; 33.7% to 23.5%, protein restricted)

  15. 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...

  16. Receptor mapping in psychiatric patients with SPECT

    International Nuclear Information System (INIS)

    This paper summarizes some data of our studies with the single-photon-emission-computerized tomography (SPECT), focussing on the dopamine-D2- and the benzodiazepine receptor mapping. Benzodiazepine receptors: Central benzodiazepine receptors (BZr) can be visualized with iomazenil which is an analogue of the benzodiazepine antagonist flumazenil, labeled with 123-iodine. Since the involvement of the BZr system is discussed in the pathogenesis of anxiety and depression, patients with these disorders were investigated. A third study investigated the BZr-occupancy during benzodiazepine treatment (lorazepam). Results: (a) Patients with panic disorders had lower iomazenil uptake values compared to epileptic patients. (b) Depressed patients showed a positive correlation between severity of illness and frontal uptake. (c) BZr occupancy during lorazepam treatment was measurable, but not associated with lorazepam plasma levels. Dopamine-D2-receptors: With 123-I-iodobenzamide (IBZM), and iodine-labeled dopamine receptor ligand, the D2 receptor density can be measured by a semiquantitative approach (striatum/frontal cortex=ST/FC). Therefore, we investigated the D2-receptor occupancy during treatment with typical and atypical neuroleptics in relationship to dosages (normalized with different formulas of chlorpromazine equivalents), side effects, and prolactin plasma levels. Results: Dependent on the selected formula for chlorpromazine equivalents, the ST/FC ratio was correlated with dosages. Side effects and prolactin plasma levels showed a negative association with lower ST/FC ratios. (orig.)

  17. Modeling key interactions between dopamine D2 receptor second extracellular loop and arylpiperazine ligands

    Directory of Open Access Journals (Sweden)

    Šukalović Vladimir

    2012-01-01

    Full Text Available Second extracellular loop (ecl2 of dopamine (DA D2 receptor is an essential part of dopaminergic ligands binding pocket. To form a part of the ligand binding surface it has to fold down into the transmembrane domain of the DA receptor. The current study describes the modeling of the D2 DA receptor ecl2 and its interactions with arylpiperazine ligands. In order to model D2 DA receptor ecl2, the number of arylpiperazine ligands was used to propose pharmacophore model. D2 DA receptor ecl2 model was built using Accelrys Discovery Studio. To test the proposed model, docking analysis was performed and key amino acid residues were determined. Proposed receptor-ligand iteractions were rationalized and compared with measured binding affinity. It is shown that D2 DA receptor ecl2 significantly participates in receptor-ligand complex formation through aromatic, hydrophobic and polar interaction. Taking them in account would benefit GPCR molecular modeling and facilitate the design of novel active compounds.

  18. 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)

  19. Assessment of cerebral benzodiazepine receptor distribution in anxiety disorders by 123I-iomazenil-SPECT. Comparison to cerebral perfusion scintigraphy by 123I-IMP

    International Nuclear Information System (INIS)

    123I-Iomazenil (123I-IMZ) and 123I-IMP imaging were performed in 5 patients with anxiety disorder (PAD) and 6 normal volunteers (NV). On 123I-IMZ delayed imaging, the 2 PAD showed abnormally decreased findings. In anxiety disorder, decreased accumulation on 123I-IMZ delayed images was seen in left hippocampus and parahippocampal gyrus in one patient, in right frontal and temporal lobe and left occipital pole in the other. Compared with NV, PAD had lower 123I-IMZ uptake on delayed image in right upper and left lower frontal cortices, indicating the involvement of the benzodiazepine receptor complex in anxiety disorder. Compared with grading for anxiety disorder with Hamilton anxiety scale (HAS) and delayed to early count ratios of 123I-IMZ, negative correlation (R123I-IMP image, positive correlation (R>0.7) was recognized in the hippocampus, the parahippocampal gyrus, the lower outer temporal cortex and the lower frontal cortex. (author)

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

    International Nuclear Information System (INIS)

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

  1. 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.

  2. [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

  3. 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.

  4. 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.

  5. Toll-like receptors, chemokine receptors and death receptor ligands responses in SARS coronavirus infected human monocyte derived dendritic cells

    Directory of Open Access Journals (Sweden)

    Law Helen KW

    2009-06-01

    Full Text Available Abstract Background The SARS outbreak in 2003 provides a unique opportunity for the study of human responses to a novel virus. We have previously reported that dendritic cells (DCs might be involved in the immune escape mechanisms for SARS-CoV. In this study, we focussed on the gene expression of toll-like receptors (TLRs, chemokine receptors (CCRs and death receptor ligands in SARS-CoV infected DCs. We also compared adult and cord blood (CB DCs to find a possible explanation for the age-dependent severity of SARS. Results Our results demonstrates that SARS-CoV did not modulate TLR-1 to TLR-10 gene expression but significantly induced the expression of CCR-1, CCR-3, and CCR-5. There was also strong induction of TNF-related apoptosis-inducing ligand (TRAIL, but not Fas ligand gene expression in SARS-CoV infected DCs. Interestingly, the expressions of most genes studied were higher in CB DCs than adult DCs. Conclusion The upregulation of chemokines and CCRs may facilitate DC migration from the infection site to the lymph nodes, whereas the increase of TRAIL may induce lymphocyte apoptosis. These findings may explain the increased lung infiltrations and lymphoid depletion in SARS patients. Further explorations of the biological significance of these findings are warranted.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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…

  13. 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.

  14. 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...

  15. 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

  16. Ligand binding and micro-switches in 7TM receptor structures

    DEFF Research Database (Denmark)

    Nygaard, Rie; Frimurer, Thomas M; Holst, Birgitte; Rosenkilde, Mette M; Schwartz, Thue W

    2009-01-01

    The past couple of years have seen several novel X-ray structures of 7 transmembrane (7TM) receptors in complex with antagonists and even with a peptide fragment of a G protein. These structures demonstrate that the main ligand-binding pocket in 7TM receptors is like a funnel with a partial 'lid...... domains (i.e. especially TM-VI), which performs the large, global toggle switch movements connecting ligand binding with intracellular signaling....

  17. 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

  18. 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.

  19. 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.

  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. Comparative tissue distribution of conformationally restricted radioiodinated vesamicol receptor ligands

    International Nuclear Information System (INIS)

    Three conformationally restricted analogs of vesamicol, 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-spirol[1H-indene-1,4'- piperidine] (5), 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-3,4-dihydrospiro[indene-1,4'- piperidine] (6) and 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl)-3,4-dihydrospiro[naphthalene- 1(2H),4'-piperidine] (7), were labelled with iodine-125 and evaluated as potential radioligands for mapping vesamicol receptor (VR) density and cholinergic function in vivo. All compounds showed similar kinetics in most tissues. However, differences were observed in the brain. Although comparable levels of each corresponding enantiomeric pair were obtained initially in the brain, the levels of the dextrorotatory enantiomers (+)-5, (+)-6 and (+)-7 were found to decrease by 72-82% over a period of 3 h. In contrast, the brain levels of the corresponding levorotatory isomers were maintained throughout the duration of the experiment. Among the dextrorotatory isomers, (+)-6 showed the highest brain extraction, while (+)-7 showed the lowest. In tissue dissection experiments, the levels of (+)-5, (+)-6 and (+)-7 were highest in the striatum and moderate to low in the cortex and cerebellum. Co-administration of haloperidol with (+)-6 decreased the levels of the latter in the striatum by 27%, while the levels in the cortex and cerebellum were each reduced by 60%. In addition, haloperidol failed to affect the regional distribution of (+)-7 in the brain. However, both haloperidol and spiperone increased the striatal levels of (+)-5 by 67 and 76%, respectively, suggesting that the binding of this radioligand is related to cholinergic function. Furthermore, haloperidol reduced the concentration of (+)-5 in the cortex and cerebellum by 25 and 33%, respectively, thereby implicating the sigma site as a secondary target for this ligand in the cortex

  2. Assessment and Challenges of Ligand Docking into Comparative Models of G-Protein Coupled Receptors

    DEFF Research Database (Denmark)

    Nguyen, E.D.; Meiler, J.; Norn, C.; Frimurer, T.M.

    2013-01-01

    The rapidly increasing number of high-resolution X-ray structures of G-protein coupled receptors (GPCRs) creates a unique opportunity to employ comparative modeling and docking to provide valuable insight into the function and ligand binding determinants of novel receptors, to assist in virtual...... screening and to design and optimize drug candidates. However, low sequence identity between receptors, conformational flexibility, and chemical diversity of ligands present an enormous challenge to molecular modeling approaches. It is our hypothesis that rapid Monte-Carlo sampling of protein backbone and...... identification of correct ligand-receptor interactions. On average, top ranked receptor models built on template structures over 50% sequence identity are within 2.9 Å of the experimental structure, with an average root mean square deviation (RMSD) of 2.2 Å for the transmembrane region and 5 Å for the second...

  3. 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

  4. 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.

  5. 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

    signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-alpha, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking. We have compared the effect of six different ligands on endocytic...... trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-alpha and epiregulin lead to complete receptor...

  6. 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...

  7. 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.

  8. 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...

  9. Small potent ligands to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor.

    Science.gov (United States)

    Axén, Andreas; Andersson, Hanna; Lindeberg, Gunnar; Rönnholm, Harriet; Kortesmaa, Jarkko; Demaegdt, Heidi; Vauquelin, Georges; Karlén, Anders; Hallberg, Mathias

    2007-07-01

    Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor. Displacement of the C-terminal of angiotensin IV with an o-substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (K(i) = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a gamma-turn in the C-terminal of its bioactive conformation. Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT(4) receptor. PMID:17559064

  10. 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.

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

    DEFF Research Database (Denmark)

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe; van Deurs, Bo; Grøvdal, Lene Melsæther

    2013-01-01

    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...

  12. 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.

  13. 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

    This study is based on the steady state method for the calculation of Kd values recently described by Lassen (J. Cereb. Blood Flow Metab. 12 (1992), 709), in which a constant infusion of the examined nonradioactive ligand is used with a bolus injection of tracer. Eight volunteers were examined...... 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...

  14. 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

  15. 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.

  16. Heterologous production of death ligands' and death receptors' extracellular domains: structural features and efficient systems.

    Science.gov (United States)

    Muraki, Michiro

    2012-08-01

    The extracellular domains of death ligands and those of death receptors are closely related to many serious human diseases through the initiation of apoptosis. Recombinant production of the extracellular domains has been investigated due to demand for a large amount of purified samples, which are a prerequisite for their biochemical characterization and constitute the fundamentals of medical applications. This review focuses on the recombinant production of extracellular domains of the major members of death ligand and death receptor families using non-mammalian expression systems with an emphasis on Fas ligand and Fas receptor. In contrast to the efficient production of the functional extracellular domains of TRAIL, TNFα and LTα by intracellular expression systems using Escherichia coli or Pichia pastoris, that of Fas ligand requires the secretory expression systems using P. pastoris or Dictyostelium discoideum, and the productivity in P. pastoris was largely dependent on tag sequence, potential N-glycosylation site and expressed protein region. On the other hand, the exploitation of insect cell systems is generally useful for the preparation of functional extracellular domains of death receptors containing many disulfide bridges in the absence of extended secondary structure, and a Bombyx mori larvae secretion system presented a superior productivity for human Fas receptor extracellular domain. Based on the results obtained so far, further efforts should be devoted to the artificial control of death ligand - death receptor interactions in order to make a contribution to medicine, represented by the development of novel biopharmaceuticals. PMID:22762186

  17. Eph receptors and ephrin class B ligands are expressed at tissue boundaries in Hydra vulgaris.

    Science.gov (United States)

    Tischer, Susanne; Reineck, Mona; Söding, Johannes; Münder, Sandra; Böttger, Angelika

    2013-01-01

    Eph receptors and ephrins are important players in axon guidance, cell sorting and boundary formation. Both the receptors and the ligands are integrated transmembrane proteins and signalling is bidirectional. The prevalent outcome of signal transduction is repulsion of adjacent cells or cell populations. Eph/ephrins have been identified in all multicellular animals from human to sponge, their functions however appear to have been altered during evolution. Here we have identified four Eph receptors and three class B ligands in the cnidarian Hydra vulgaris, indicating that those are the evolutionary older ones. In situ hybridisation experiments revealed a striking complementarity of expression of receptors and ligands in tentacles and in developing buds. This suggests that the original function of ephrin signalling may have been in epithelial cell adhesion and the formation of tissue boundaries. PMID:24307295

  18. 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

  19. 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

  20. 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

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. Modelling studies of bitter taste, glucocorticoid and VEGFR2 receptors and their ligands

    OpenAIRE

    Capelli, Anna Maria

    2014-01-01

    Modelling studies of bitter taste receptors and their ligands The human bitter taste receptor gene family (TAS2R) belongs to the Frizzled/Taste2 subfamily of the G-protein coupled receptors (GPCR) superfamily. TAS2R are expressed on the tongue in bitter taste receptor cells co-expressing specific signal transduction components like Ggustducin and are able to detect stimuli of only one taste quality. Recently these receptors have been identified in isolated human airway smooth muscle cell...

  6. Development of novel cellular model for affinity studies of histamine H(4) receptor ligands.

    Science.gov (United States)

    Karcz, Tadeusz; Kieć-Kononowicz, Katarzyna

    2013-01-01

    The G protein-coupled histamine H4 receptor (H4R) is the last member of histamine receptors family discovered so far. Its expression pattern, together with postulated involvement in a wide variety of immunological and inflammatory processes make histamine H4 receptor an interesting target for drug development. Potential H4R ligands may provide an innovative therapies for different immuno-based diseases, including allergy, asthma, pruritus associated with allergy or autoimmune skin conditions, rheumatoid arthritis and pain. However, none of successfully developed selective and potent histamine H4 receptor ligands have been introduced to the market up to date. For that reason there is still a strong demand for pharmacological models to be used in studies on potent H4R ligands. In current work we present the development of novel mammalian cell line, stably expressing human histamine H4 receptor, with use of retroviral transduction approach. Obtained cell line was pharmacologically characterized in radioligand binding studies and its utility for affinity testing of potent receptor ligands was confirmed in comparative studies with the use of relevant insect cells expression model. Obtained results allow for statement that developed cellular model may be successfully employed in search for new compounds active at histamine H4 receptor. PMID:24432340

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. Occurrence of xenobiotic ligands for retinoid X receptors and thyroid hormone receptors in the aquatic environment of Taiwan

    International Nuclear Information System (INIS)

    Highlights: • Xenobiotic RXR agonists and antagonists were found in river systems in Taiwan. • Potential TR antagonists were mainly detected in water extracts. • Samples collected near the river mouths exhibited high RXR disrupting activity. • RXR/TR antagonist activity in water became stronger after HPLC fractionation. • Bioassays are useful tools to investigate xenobiotic ligands for nuclear receptors. - Abstract: Various synthetic compounds are frequently discharged into the environment via human activities. Among them, certain contaminants may disrupt normal physiological functions of wildlife and humans via interactions with nuclear receptors. To protect human health and the environment, it is important to detect environmental ligands for human nuclear receptors. In this study, yeast-based reporter gene assays were used to investigate the occurrence of xenobiotic ligands for retinoid X receptors (RXR) and thyroid hormone receptors (TR) in the aquatic environment of Taiwan. Experimental results revealed that RXR agonist/antagonist activity was detected in river water and sediment samples. In particular, high RXR agonist/antagonist activity was found in the samples collected near river mouths. Additionally, few samples also elicited significant TR antagonist activity. Our findings show that the aquatic environment of Taiwan was contaminated with RXR and TR ligands. Further study is necessary to identify these xenobiotic RXR and TR agonists and antagonists

  14. 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...

  15. 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

  16. 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...

  17. Toll-like receptor 2 ligands regulate monocyte Fcγ receptor expression and function.

    Science.gov (United States)

    Shah, Prexy; Fatehchand, Kavin; Patel, Hemal; Fang, Huiqing; Justiniano, Steven E; Mo, Xiaokui; Jarjoura, David; Tridandapani, Susheela; Butchar, Jonathan P

    2013-04-26

    Fcγ receptor (FcγR) clustering on monocytes/macrophages results in phagocytosis and inflammatory cytokine production, which serve to eliminate antibody-opsonized targets and activate neighboring immune cells. Toll-like receptor 2 (TLR2), which recognizes a range of both bacterial and fungal components, elicits strong proinflammatory responses in these cells when stimulated by ligands, either natural or synthetic. Thus, we explored the possibility that TLR2 agonists could strengthen FcγR activity within the context of antibody therapy. Human peripheral blood monocytes treated with the TLR2 agonist Pam2CSK4 showed significantly enhanced FcγR-mediated cytokine production as well as phagocytic ability. An examination of the molecular mechanism behind this enhancement revealed increased expression of both FcγRIIa and the common γ subunit following Pam2CSK4 treatment. Interestingly however, expression of the inhibitory receptor FcγRIIb was also modestly increased. Further investigation revealed that Pam2CSK4 also dramatically decreased the expression of SHIP, the major mediator of FcγRIIb inhibitory activity. Using a murine Her2/neu solid tumor model of antibody therapy, we found that Pam2CSK4 significantly enhanced the ability of anti-Her2 antibody to reduce the rate of tumor growth. To verify that the FcγR enhancement was not unique to the diacylated Pam2CSK4, we also tested Pam3CSK4, a related triacylated TLR2 agonist. Results showed significant enhancement in FcγR function and expression. Taken together, these findings indicate that TLR2 activation can positively modulate FcγR and suggest that TLR2 agonists should be considered for testing as adjuvants for antitumor antibody therapy. PMID:23504312

  18. 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...

  19. Human TSH receptor ligands as pharmacological probes with potential clinical application

    OpenAIRE

    Neumann, Susanne; Raaka, Bruce M.; Gershengorn, Marvin C.

    2009-01-01

    The biologic role of thyroid-stimulating hormone (TSH; thyrotropin) as an activator (agonist) of the TSH receptor (TSHR) in the hypothalamic–pituitary–thyroid axis is well known and activation of TSHR by recombinant human TSH is used clinically in patients with thyroid cancer. TSHR ligands other than TSH could be used to probe TSHR biology in thyroidal and extrathyroidal tissues, and potentially be employed in patients. A number of different TSHR ligands have been reported, including TSH anal...

  20. 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...

  1. 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...

  2. 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...

  3. Caged vanilloid ligands for activation of TRPV1 receptors by 1- and 2-photon excitation†

    OpenAIRE

    Zhao, Jun; Gover, Tony D; Muralidharan, Sukumaran; Auston, Darryl A.; Weinreich, Daniel; Kao, Joseph P.Y.

    2006-01-01

    Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV1, a non-selective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids—biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged...

  4. Nonsteroidal Bivalent Estrogen Ligands - An Application of the Bivalent Concept to the Estrogen Receptor

    Science.gov (United States)

    Shan, Min; Carlson, Kathryn E.; Bujotzek, Alexander; Wellner, Anja; Gust, Ronald; Weber, Marcus; Katzenellenbogen, John A.; Haag, Rainer

    2013-01-01

    The estrogen receptor (ER) is a hormone-regulated transcription factor that binds, as a dimer, to estrogens and to specific DNA sequences. To explore at a fundamental level the geometric and topological features of bivalent-ligand binding to the ER dimer, dimeric ER crystal structures were used to rationally design nonsteroidal bivalent estrogen ligands. Guided by this structure-based ligand design, we prepared two series of bivalent ligands (agonists and antagonists) tethered by flexible spacers of varying lengths (7–47Å) and evaluated their ER-binding affinities for the two ER subtypes and their biological activities in cell lines. Bivalent ligands based on the agonist diethylstilbestrol (DES) proved to be poor candidates, but bivalent ligands based on the antagonist hydroxytamoxifen (OHT) were well suited for intensive study. Binding affinities of the OHT-based bivalent ligands were related to spacer length in a distinctive fashion, reaching two maximum values at 14 and 29Å in both ER subtypes. These results demonstrate that the bivalent concept can operate in determining ER-ligand binding affinity and suggest that two distinct modes operate for the binding of bivalent estrogen ligands to the ER dimers, an intermolecular as well as an intramolecular mode. Our insights, particularly the possibility of intramolecular bivalent binding on a single ER monomer, may provide an alternative strategy to prepare more selective and active ER antagonists for endocrine therapy of breast cancer. PMID:23312071

  5. Comparison of benzodiazepine receptor SPECT and 18F-FDG PET using a coincidence detection camera in patients with temporal lobe epilepsy: preliminary results

    International Nuclear Information System (INIS)

    Full text: The aim of this preliminary study was to compare the results of benzodiazepine receptor (BDR) SPECT using 123I-Iomazenil with those of 18F-FDG (FDG) PET obtained on a double-headed gamma camera with a coincidence detection system in patients with temporal lobe epilepsy (TLE). We evaluated 6 patients (4 female, 2 male; age range 26-54 years, average 43.5 years) with therapy-refractory TLE due to mesiotemporal sclerosis or other focal brain anomalies. To delineate the epileptogenic zone, clinical evaluation, ictal and interictal surface EEG using the international 10-20 system, brain MRI, interictal CBF SPECT using 99mTc-ECD, BDR SPECT and FDG coincidence PET were performed. The CBF SPECT, BDR SPECT and coincidence PET scans were viewed independently by 2 observers considering the regional cerebral blood flow, BDR density and FDG uptake asymmetry in the temporal lobe visually as none (0), low (1), moderate (2) and high (3). Ictal and interictal EEG recordings located the epileptogenic focus in all patients in the temporal region. Both the BDR SPECT and the FDG coincidence PET located the epileptogenic focus correctly in circumscribed areas of the temporal lobe in all patients, whereas brain MRI revealed focal anomalies only in 5 of 6 cases . The lateralization to the right (n=4) and left hemisphere (n=2) by interictal CBF SPECT, BDR SPECT and FDG coincidence PET corresponded to the EEG findings in all patients. The visual consideration of the asymmetry revealed a slightly but not statistically significant higher value for the FDG coincidence PET (observer 1: mean 2.333, SD 0.516; observer 2: mean 2.000, SD 0.632) than for the BDR SPECT (observer 1: mean 1.667, SD 1.033; observer 2: mean 1.833, SD 0.753). Visual consideration of the interictal CBF SPECT revealed mean values of 2.000 for both observers. The inter-observer variability was higher in the BDR SPECT than in the FDG coincidence PET and the interictal CBF SPECT, but the difference was not

  6. Unnatural agrochemical ligands for engineered abscisic acid receptors.

    Science.gov (United States)

    Rodriguez, Pedro L; Lozano-Juste, Jorge

    2015-06-01

    Existing agrochemicals can be endowed with new applications through protein engineering of plant receptors. A recent study shows an engineered PYR1 ABA receptor can be activated by mandipropamid. Plants engineered with such PYR1 variant are responsive to this agrochemical, which confers protection against drought through activation of ABA signaling. PMID:25891067

  7. 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.

  8. Evolution of gonadotropin-inhibitory hormone receptor and its ligand.

    Science.gov (United States)

    Ubuka, Takayoshi; Tsutsui, Kazuyoshi

    2014-12-01

    Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide inhibitor of gonadotropin secretion, which was first identified in the Japanese quail hypothalamus. GnIH peptides share a C-terminal LPXRFamide (X=L or Q) motif in most vertebrates. The receptor for GnIH (GnIHR) is the seven-transmembrane G protein-coupled receptor 147 (GPR147) that inhibits cAMP production. GPR147 is also named neuropeptide FF (NPFF) receptor 1 (NPFFR1), because it also binds NPFF that has a C-terminal PQRFamide motif. To understand the evolutionary history of the GnIH system in the animal kingdom, we searched for receptors structurally similar to GnIHR in the genome of six mammals (human, mouse, rat, cattle, cat, and rabbit), five birds (pigeon, chicken, turkey, budgerigar, and zebra finch), one reptile (green anole), one amphibian (Western clawed flog), six fishes (zebrafish, Nile tilapia, Fugu, coelacanth, spotted gar, and lamprey), one hemichordate (acorn worm), one echinoderm (purple sea urchin), one mollusk (California sea hare), seven insects (pea aphid, African malaria mosquito, honey bee, buff-tailed bumblebee, fruit fly, jewel wasp, and red flour beetle), one cnidarian (hydra), and constructed phylogenetic trees by neighbor joining (NJ) and maximum likelihood (ML) methods. A multiple sequence alignment of the receptors showed highly conserved seven-transmembrane domains as well as disulfide bridge sites between the first and second extracellular loops, including the receptor of hydra. Both NJ and ML analyses grouped the receptors of vertebrates into NPFFR1 and NPFFR2 (GPR74), and the receptors of insects into the receptor for SIFamide peptides that share a C-terminal YRKPPFNGSIFamide motif. Although human, quail and zebrafish GnIHR (NPFFR1) were most structurally similar to SIFamide receptor of fruit fly in the Famide peptide (FMRFamide, neuropeptide F, short neuropeptide F, drosulfakinin, myosuppressin, SIFamide) receptor families, the amino acid sequences and the peptide coding

  9. 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...

  10. Cinnamamides, Novel Liver X Receptor Antagonists that Inhibit Ligand-Induced Lipogenesis and Fatty Liver.

    Science.gov (United States)

    Sim, Woo-Cheol; Kim, Dong Gwang; Lee, Kyeong Jin; Choi, You-Jin; Choi, Yeon Jae; Shin, Kye Jung; Jun, Dae Won; Park, So-Jung; Park, Hyun-Ju; Kim, Jiwon; Oh, Won Keun; Lee, Byung-Hoon

    2015-12-01

    Liver X receptor (LXR) is a member of the nuclear receptor superfamily, and it regulates various biologic processes, including de novo lipogenesis, cholesterol metabolism, and inflammation. Selective inhibition of LXR may aid the treatment of nonalcoholic fatty liver diseases. In the present study, we evaluated the effects of three cinnamamide derivatives on ligand-induced LXRα activation and explored whether these derivatives could attenuate steatosis in mice. N-(4-trifluoromethylphenyl) 3,4-dimethoxycinnamamide (TFCA) decreased the luciferase activity in LXRE-tk-Luc-transfected cells and also suppressed ligand-induced lipid accumulation and expression of the lipogenic genes in murine hepatocytes. Furthermore, it significantly attenuated hepatic neutral lipid accumulation in a ligand-induced fatty liver mouse system. Modeling study indicated that TFCA inhibited activation of the LXRα ligand-binding domain by hydrogen bonding to Arg305 in the H5 region of that domain. It regulated the transcriptional control exerted by LXRα by influencing coregulator exchange; this process involves dissociation of the thyroid hormone receptor-associated proteins (TRAP)/DRIP coactivator and recruitment of the nuclear receptor corepressor. These results show that TFCA has the potential to attenuate ligand-induced lipogenesis and fatty liver by selectively inhibiting LXRα in the liver. PMID:26384859

  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. PMID:25367076

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

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud; Lundstrom, Kenneth; Vogel, Horst

    2003-01-01

    of receptor function and in turn for the design and development of novel therapeutic compound. Here we show how ligand-receptor interaction can be investigated in situ with high sensitivity on sensor surfaces by total internal reflection fluorescence (TIRF) measurements. A generally applicable method...... streptavidin. TIRF measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without...

  13. Ligand Binding Sensitivity of the Extracellular Loop Two of the Cannabinoid Receptor 1

    OpenAIRE

    Bertalovitz, Alexander C.; Ahn, Kwang H.; Kendall, Debra A.

    2010-01-01

    The cannabinoid receptor one (CB1) is a class A G-protein-coupled receptor thought to bind ligands primarily within its helical bundle. Evidence suggests, however, that the extracellular domain may also play a role. We have previously shown that the C-terminus of the extracellular loop 2 of CB1 is important in binding some compounds; receptors with mutations in this region (F268W, P269A, H270A, and I271A) bound some agonists with severely reduced affinity relative to the wild-type receptor. I...

  14. 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...

  15. Ionotropic excitatory amino acid receptor ligands. Synthesis and pharmacology of a new amino acid AMPA antagonist

    DEFF Research Database (Denmark)

    Madsen, U; Sløk, F A; Stensbøl, T B;

    2000-01-01

    We have previously described the potent and selective (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), and the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4...... excitatory amino acid (EAA) receptors using receptor binding and electrophysiological techniques, and for activity at metabotropic EAA receptors using second messenger assays. Compounds 1 and 4 were essentially inactive. (RS)-2-Amino-3-[3-(2-carboxyethyl)-5-methyl-4-isoxazolyl]propionic acid (ACMP, 2), on......-isoxazolyl]propionic acid (AMOA). Using these AMPA receptor ligands as leads, a series of compounds have been developed as tools for further elucidation of the structural requirements for activation and blockade of AMPA receptors. The synthesized compounds have been tested for activity at ionotropic...

  16. 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

  17. 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.

  18. 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.

  19. 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)

  20. 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.

  1. 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:...

  2. 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

    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...... ligands and that the two peripheral chemical moieties of the ligands from this central point in the receptor structure explore each of the two halves of the main ligand binding pocket. It is envisioned that knowledge of this binding mode can be exploited in structure-based discovery and design of novel...

  3. Post-docking optimization and analysis of protein-ligand interactions of estrogen receptor alpha using AMMOS software.

    Science.gov (United States)

    Pencheva, Tania; Jereva, Dessislava; Miteva, Maria A; Pajeva, Ilza

    2013-03-01

    Understanding protein-ligand interactions is a critical step in rational drug design/virtual ligand screening. In this work we applied the AMMOS_ProtLig software for post-docking optimization of estrogen receptor alpha complexes generated after virtual ligand screening protocol. Using MOE software we identified the ligand-receptor interactions in the optimized complexes at different levels of protein flexibility and compared them to the experimentally observed interactions. We analyzed in details the binding sites of three X-ray complexes of the same receptor and identified the key residues for the protein-ligand interactions. The complexes were further processed with AMMOS_ProtLig and the interactions in the predicted poses were compared to those observed in the X-ray structures. The effect of employing different levels of flexibility was analyzed. The results confirmed the AMMOS_ProtLig applicability as a helpful postdocking optimization tool for virtual ligand screening of estrogen receptors. PMID:23106778

  4. LiCABEDS II. Modeling of ligand selectivity for G-protein-coupled cannabinoid receptors.

    Science.gov (United States)

    Ma, Chao; Wang, Lirong; Yang, Peng; Myint, Kyaw Z; Xie, Xiang-Qun

    2013-01-28

    The cannabinoid receptor subtype 2 (CB2) is a promising therapeutic target for blood cancer, pain relief, osteoporosis, and immune system disease. The recent withdrawal of Rimonabant, which targets another closely related cannabinoid receptor (CB1), accentuates the importance of selectivity for the development of CB2 ligands in order to minimize their effects on the CB1 receptor. In our previous study, LiCABEDS (Ligand Classifier of Adaptively Boosting Ensemble Decision Stumps) was reported as a generic ligand classification algorithm for the prediction of categorical molecular properties. Here, we report extension of the application of LiCABEDS to the modeling of cannabinoid ligand selectivity with molecular fingerprints as descriptors. The performance of LiCABEDS was systematically compared with another popular classification algorithm, support vector machine (SVM), according to prediction precision and recall rate. In addition, the examination of LiCABEDS models revealed the difference in structure diversity of CB1 and CB2 selective ligands. The structure determination from data mining could be useful for the design of novel cannabinoid lead compounds. More importantly, the potential of LiCABEDS was demonstrated through successful identification of newly synthesized CB2 selective compounds. PMID:23278450

  5. 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

  6. 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 lung...ors, Notch ligands, and cytokines drive the chronicity of lunginflammation. Authors Raymond T, Schaller M, H...2007 Dec;4(8):635-41. (.png) (.svg) (.html) (.csml) Show Toll-like receptors, Notch ligands, and cytokines d...rive the chronicity of lunginflammation. PubmedID 18073395 Title Toll-like recept

  7. Receptor-based 3D QSAR analysis of estrogen receptor ligands--merging the accuracy of receptor-based alignments with the computational efficiency of ligand-based methods.

    Science.gov (United States)

    Sippl, W

    2000-08-01

    One of the major challenges in computational approaches to drug design is the accurate prediction of binding affinity of biomolecules. In the present study several prediction methods for a published set of estrogen receptor ligands are investigated and compared. The binding modes of 30 ligands were determined using the docking program AutoDock and were compared with available X-ray structures of estrogen receptor-ligand complexes. On the basis of the docking results an interaction energy-based model, which uses the information of the whole ligand-receptor complex, was generated. Several parameters were modified in order to analyze their influence onto the correlation between binding affinities and calculated ligand-receptor interaction energies. The highest correlation coefficient (r2 = 0.617, q2Loo = 0.570) was obtained considering protein flexibility during the interaction energy evaluation. The second prediction method uses a combination of receptor-based and 3D quantitative structure-activity relationships (3D QSAR) methods. The ligand alignment obtained from the docking simulations was taken as basis for a comparative field analysis applying the GRID/GOLPE program. Using the interaction field derived with a water probe and applying the smart region definition (SRD) variable selection, a significant and robust model was obtained (r2 = 0.991, q2LOO = 0.921). The predictive ability of the established model was further evaluated by using a test set of six additional compounds. The comparison with the generated interaction energy-based model and with a traditional CoMFA model obtained using a ligand-based alignment (r2 = 0.951, q2L00 = 0.796) indicates that the combination of receptor-based and 3D QSAR methods is able to improve the quality of the underlying model. PMID:10921772

  8. Imaging GABAc Receptors with Ligand-Conjugated Quantum Dots

    Directory of Open Access Journals (Sweden)

    Sandra J. Rosenthal

    2008-04-01

    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.

  9. Structure-Based Virtual Screening for Dopamine D2 Receptor Ligands as Potential Antipsychotics.

    Science.gov (United States)

    Kaczor, Agnieszka A; Silva, Andrea G; Loza, María I; Kolb, Peter; Castro, Marián; Poso, Antti

    2016-04-01

    Structure-based virtual screening using a D2 receptor homology model was performed to identify dopamine D2 receptor ligands as potential antipsychotics. From screening a library of 6.5 million compounds, 21 were selected and were subjected to experimental validation. From these 21 compounds tested, ten D2 ligands were identified (47.6 % success rate, among them D2 receptor antagonists, as expected) that have additional affinity for other receptors tested, in particular 5-HT2A receptors. The affinity (Ki values) of the compounds ranged from 58 nm to about 24 μm. Similarity and fragment analysis indicated a significant degree of structural novelty among the identified compounds. We found one D2 receptor antagonist that did not have a protonatable nitrogen atom, which is a key structural element of the classical D2 pharmacophore model necessary for interaction with the conserved Asp(3.32) residue. This compound exhibited greater than 20-fold binding selectivity for the D2 receptor over the D3 receptor. We provide additional evidence that the amide hydrogen atom of this compound forms a hydrogen bond with Asp(3.32), as determined by tests of its derivatives that cannot maintain this interaction. PMID:26990027

  10. 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...

  11. 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.

  12. 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...

  13. 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 of...

  14. Ligand Binding Pathways of Clozapine and Haloperidol in the Dopamine D2 and D3 Receptors.

    Science.gov (United States)

    Thomas, Trayder; Fang, Yu; Yuriev, Elizabeth; Chalmers, David K

    2016-02-22

    The binding of a small molecule ligand to its protein target is most often characterized by binding affinity and is typically viewed as an on/off switch. The more complex reality is that binding involves the ligand passing through a series of intermediate states between the solution phase and the fully bound pose. We have performed a set of 29 unbiased molecular dynamics simulations to model the binding pathways of the dopamine receptor antagonists clozapine and haloperidol binding to the D2 and D3 dopamine receptors. Through these simulations we have captured the binding pathways of clozapine and haloperidol from the extracellular vestibule to the orthosteric binding site and thereby, we also predict the bound pose of each ligand. These are the first long time scale simulations of haloperidol or clozapine binding to dopamine receptors. From these simulations, we have identified several important stages in the binding pathway, including the involvement of Tyr7.35 in a "handover" mechanism that transfers the ligand between the extracellular vestibule and Asp3.32. We have also performed interaction and cluster analyses to determine differences in binding pathways between the D2 and D3 receptors and identified metastable states that may be of use in drug design. PMID:26690887

  15. The ligand-binding domain of the cell surface receptor for urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Behrendt, N; Ploug, M; Patthy, L;

    1991-01-01

    The purified urokinase plasminogen activator receptor (u-PAR) was cleaved into two fragments by mild chymotrypsin treatment. The smaller fragment (apparent Mr 16,000) possessed the ligand-binding capability, as shown by chemical cross-linking analysis. This fragment constituted the NH2-terminal p...

  16. 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

  17. 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

  18. 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

  19. 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

  20. Labeling of receptor ligands with bromine radionuclides. Progress report, March 1, 1981-February 29, 1984

    International Nuclear Information System (INIS)

    We have developed techniques to label several types of organic molecules with radiobromine. We have emphasized techniques to label ligands for the estrogen receptor and have studied two brominated compounds in rat models. One of these compounds has been studied in a limited number of patients and estrogen containing tumors visualized by nuclear medicine imaging. We have recently expanded the size of the work to label receptor ligands with fluorine-18 and have carried out preliminary animal studies which suggest that a clinically useful compound can be prepared. In addition to the receptor studies, we have collaborated in assessing 77Br-labeled compounds as therapeutic agents and in studying 77Br-labeled compounds using perturbed angular correlation techniques

  1. How does oxygen rise drive evolution? Clues from oxygen-dependent biosynthesis of nuclear receptor ligands

    International Nuclear Information System (INIS)

    It is well known that oxygen rise greatly facilitated biological evolution. However, the underlying mechanisms remain elusive. Recently, Raymond and Segre revealed that molecular oxygen allows 1000 more metabolic reactions than can occur in anoxic conditions. From the novel metabolites produced in aerobic metabolism, we serendipitously found that some of the metabolites are signaling molecules that target nuclear receptors. Since nuclear signaling systems are indispensable to superior organisms, we speculated that aerobic metabolism may facilitate biological evolution through promoting the establishment of nuclear signaling systems. This hypothesis is validated by the observation that most (97.5%) nuclear receptor ligands are produced by aerobic metabolism, which is further explained in terms of the chemical criteria (appropriate volume and rather high hydrophobicity) of nuclear receptor ligands that aerobic metabolites are more ready than anaerobic counterparts to satisfy these criteria.

  2. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    International Nuclear Information System (INIS)

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed

  3. Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin.

    Science.gov (United States)

    Christoffers, Keith H; Khokhar, Arshia; Chaturvedi, Kirti; Howells, Richard D

    2002-04-15

    We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions. PMID:11853866

  4. 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...

  5. 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.

  6. Ligand modulates the conversion of DNA-bound vitamin D3 receptor (VDR) homodimers into VDR-retinoid X receptor heterodimers.

    OpenAIRE

    Cheskis, B; Freedman, L P

    1994-01-01

    Protein dimerization facilitates cooperative, high-affinity interactions with DNA. Nuclear hormone receptors, for example, bind either as homodimers or as heterodimers with retinoid X receptors (RXR) to half-site repeats that are stabilized by protein-protein interactions mediated by residues within both the DNA- and ligand-binding domains. In vivo, ligand binding among the subfamily of steroid receptors unmasks the nuclear localization and DNA-binding domains from a complex with auxiliary fa...

  7. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Biswas, Arunima; Pasquel, Danielle [Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Tyagi, Rakesh Kumar [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India); Mani, Sridhar, E-mail: sridhar.mani@einstein.yu.edu [Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461 (United States)

    2011-03-18

    Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} 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.

  9. 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.

  10. Differential utilization of binding loop flexibility in T cell receptor ligand selection and cross-reactivity.

    Science.gov (United States)

    Ayres, Cory M; Scott, Daniel R; Corcelli, Steven A; Baker, Brian M

    2016-01-01

    Complementarity determining region (CDR) loop flexibility has been suggested to play an important role in the selection and binding of ligands by T cell receptors (TCRs) of the cellular immune system. However, questions remain regarding the role of loop motion in TCR binding, and crystallographic structures have raised questions about the extent to which generalizations can be made. Here we studied the flexibility of two structurally well characterized αβ TCRs, A6 and DMF5. We found that the two receptors utilize loop motion very differently in ligand binding and cross-reactivity. While the loops of A6 move rapidly in an uncorrelated fashion, those of DMF5 are substantially less mobile. Accordingly, the mechanisms of binding and cross-reactivity are very different between the two TCRs: whereas A6 relies on conformational selection to select and bind different ligands, DMF5 uses a more rigid, permissive architecture with greater reliance on slower motions or induced-fit. In addition to binding site flexibility, we also explored whether ligand-binding resulted in common dynamical changes in A6 and DMF5 that could contribute to TCR triggering. Although binding-linked motional changes propagated throughout both receptors, no common features were observed, suggesting that changes in nanosecond-level TCR structural dynamics do not contribute to T cell signaling. PMID:27118724

  11. 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

  12. 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

  13. 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...

  14. Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor CD148

    DEFF Research Database (Denmark)

    Whiteford, James R; Xian, Xiaojie; Chaussade, Claire; Vanhaesebroeck, Bart; Nourshargh, Sussan; Couchman, John R

    2011-01-01

    Syndecan-2 is a heparan sulfate proteoglycan that has a cell adhesion regulatory domain contained within its extracellular core protein. Cell adhesion to the syndecan-2 extracellular domain (S2ED) is ß1 integrin dependent; however, syndecan-2 is not an integrin ligand. Here the protein tyrosine...... phosphatase receptor CD148 is shown to be a key intermediary in cell adhesion to S2ED, with downstream ß1 integrin-mediated adhesion and cytoskeletal organization. We show that S2ED is a novel ligand for CD148 and identify the region proximal to the transmembrane domain of syndecan-2 as the site of...

  15. 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...

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

    OpenAIRE

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

    2014-01-01

    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 intra...

  17. A Vitamin D Receptor Selectively Activated by Gemini Analogs Reveals Ligand Dependent and Independent Effects

    OpenAIRE

    Tiphaine Huet; Gilles Laverny; Fabrice Ciesielski; Ferdinand Molnár; Thanuja Gali Ramamoorthy; Anna Y. Belorusova; Pierre Antony; Noelle Potier; Daniel Metzger; Dino Moras; Natacha Rochel

    2015-01-01

    The bioactive form of vitamin D [1,25(OH)2D3] regulates mineral and bone homeostasis and exerts potent anti-inflammatory and antiproliferative properties through binding to the vitamin D receptor (VDR). The 3D structures of the VDR ligand-binding domain with 1,25(OH)2D3 or gemini analogs unveiled the molecular mechanism underlying ligand recognition. On the basis of structure-function correlations, we generated a point-mutated VDR (VDRgem) that is unresponsive to 1,25(OH)2D3, but the activity...

  18. 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

  19. Synthesis and carbon-11-labeling of p-MeO-SSR180575, a novel indoleacetamide-based candidate for PET imaging of the peripheral benzodiazepine receptor (TSPO 18 kDa)

    International Nuclear Information System (INIS)

    Complete text of publication follows: Objectives: The 3-iso-quinolinecarboxamide [11C]PK11195, despite its low brain uptake and high level of nonspecific binding, is still the most widely used PET-radioligand for the in vivo imaging of the peripheral benzodiazepine receptor (PBR or TSPO 18 kDa). Several new PBR radioligands are currently developed to replace [11C]PK11195, e.g the pyrazolo[1, 5-a]pyrimidine-acetamides [11C]DPA-713 and [18F]DPA-714, the imidazo[1, 2-a]pyridine-acetamides [11C]CLINME and [18F]PBR111 and the N-benzyl-N-(2-phenoxy-aryl)- acetamides [11C]PBR28 and [18F]FEDAA1106. Another attractive newly identified chemical class of structures are the indole-acetamides and notably compounds derived from the lead compound SSR180575. Herein are reported the synthesis and the labelling with the positron-emitter carbon-11 (half-life: 20.38 min) of a novel derivative of SSR180575, bearing a para methoxy function on its phenyl ring. Methods: p-MeO-SSR180575 (1) was synthesized from commercially available 4-chloro-2-nitrotoluene in 10 steps. O-demethylation, performed with a boron tribromide solution in dichloromethane at low temperature, afforded the free phenol derivative 2. Carbon-11 labeling of p-MeO-SSR180575 (1) was performed using a TRACERLab FX-C Pro synthesizer (GEMS) and comprised (1) trapping of [11C]MeOTf at -10 C in acetone (0.3 mL) containing the nor-derivative 2 (O-demethylated, 0.6-0.9 mg) and aq. 3N NaOH (8 μL); (2) heating at 110 C for 2 min; (3) concentration to dryness and taking up the residue in 1.0 mL of the HPLC mobile phase; (4) purification using semi-preparative reversed-phase HPLC (Waters Symmetry C-18 - eluent: CH3CN / H2O / TFA: 50 / 50 / 0.1 (v:v:v) - flow rate: 5 mL/min - detection at 254 nm) and (5) SepPakR Plus C-18-based formulation for i.v. injection. Results: p-MeO-SSR180575 (1) was obtained in 10% overall yield. The tricky and low-yielding step in our approach was the pyridazine ring formation reaction that proceeded

  20. 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.

  1. 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.

  2. Investigation on critical structural motifs of ligands for triggering glucocorticoid receptor nuclear migration through molecular docking simulations.

    Science.gov (United States)

    Liu, Ya-Lin; Jang, Soonmin; Wang, Shih-Min; Chen, Chiu-Hao; Li, Feng-Yin

    2016-06-01

    The glucocorticoid receptor (GR), a transcription factor regulating gene expression in a ligand-dependent fashion, is known for flexibility in adapting various ligands with their structures ranging from steroid to non-steroid. However, in our previous study, GR shows a stringent discrimination against a set of steroid ligands with highly similar structures for triggering its nuclear migration. In order to resolve this puzzle, we employed molecular docking simulations to investigate the origin of this structural discrimination. By analyzing the docking orientations and the related ligand-GR interaction patterns, we found that the hydrophilicity mismatch between the docking ligand and the GR ligand-binding site is the main cause combined with the steric hindrance and structural rigidness of these steroid ligands. Furthermore, we utilized this knowledge to rationalize how the structure-binding interaction of non-steroid ligands triggers GR nuclear migration with their structures available in Protein Data Bank. PMID:26198481

  3. WScore: A Flexible and Accurate Treatment of Explicit Water Molecules in Ligand-Receptor Docking.

    Science.gov (United States)

    Murphy, Robert B; Repasky, Matthew P; Greenwood, Jeremy R; Tubert-Brohman, Ivan; Jerome, Steven; Annabhimoju, Ramakrishna; Boyles, Nicholas A; Schmitz, Christopher D; Abel, Robert; Farid, Ramy; Friesner, Richard A

    2016-05-12

    We have developed a new methodology for protein-ligand docking and scoring, WScore, incorporating a flexible description of explicit water molecules. The locations and thermodynamics of the waters are derived from a WaterMap molecular dynamics simulation. The water structure is employed to provide an atomic level description of ligand and protein desolvation. WScore also contains a detailed model for localized ligand and protein strain energy and integrates an MM-GBSA scoring component with these terms to assess delocalized strain of the complex. Ensemble docking is used to take into account induced fit effects on the receptor conformation, and protein reorganization free energies are assigned via fitting to experimental data. The performance of the method is evaluated for pose prediction, rank ordering of self-docked complexes, and enrichment in virtual screening, using a large data set of PDB complexes and compared with the Glide SP and Glide XP models; significant improvements are obtained. PMID:27054459

  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. Structure of the ligand-binding domain of the EphB2 receptor at 2 Å resolution

    International Nuclear Information System (INIS)

    The crystal structure of the ligand-binding domain of a receptor tyrosine kinase EphB2, an important mediator of cell-cell communication, has been determined at a resolution of 2 Å. The structure confirms the induced-fit mechanism for the binding of ligands to EphB receptors. Eph tyrosine kinase receptors, the largest group of receptor tyrosine kinases, and their ephrin ligands are important mediators of cell–cell communication regulating cell attachment, shape and mobility. Recently, several Eph receptors and ephrins have also been found to play important roles in the progression of cancer. Structural and biophysical studies have established detailed information on the binding and recognition of Eph receptors and ephrins. The initial high-affinity binding of Eph receptors to ephrin occurs through the penetration of an extended G–H loop of the ligand into a hydrophobic channel on the surface of the receptor. Consequently, the G–H loop-binding channel of Eph receptors is the main target in the search for Eph antagonists that could be used in the development of anticancer drugs and several peptides have been shown to specifically bind Eph receptors and compete with the cognate ephrin ligands. However, the molecular details of the conformational changes upon Eph/ephrin binding have remained speculative, since two of the loops were unstructured in the original model of the free EphB2 structure and their conformational changes upon ligand binding could consequently not be analyzed in detail. In this study, the X-ray structure of unbound EphB2 is reported at a considerably higher 2 Å resolution, the conformational changes that the important receptor loops undergo upon ligand binding are described and the consequences that these findings have for the development of Eph antagonists are discussed

  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. 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

  9. Somatostatin receptor subtype 2 sensitizes human pancreatic cancer cells to death ligand-induced apoptosis.

    Science.gov (United States)

    Guillermet, Julie; Saint-Laurent, Nathalie; Rochaix, Philippe; Cuvillier, Olivier; Levade, Thierry; Schally, Andrew V; Pradayrol, Lucien; Buscail, Louis; Susini, Christiane; Bousquet, Corinne

    2003-01-01

    Somatostatin receptor subtype 2 (sst2) gene expression is lost in 90% of human pancreatic adenocarcinomas. We previously demonstrated that stable sst2 transfection of human pancreatic BxPC-3 cells, which do not endogenously express sst2, inhibits cell proliferation, tumorigenicity, and metastasis. These sst2 effects occur as a consequence of an autocrine sst2-dependent loop, whereby sst2 induces expression of its own ligand, somatostatin. Here we investigated whether sst2 induces apoptosis in sst2-transfected BxPC-3 cells. Expression of sst2 induced a 4.4- +/- 0.05-fold stimulation of apoptosis in BxPC-3 through the activation of tyrosine phosphatase SHP-1. sst2 also sensitized these cells to apoptosis induced by tumor necrosis factor alpha (TNFalpha), enhancing it 4.1- +/- 1.5-fold. Apoptosis in BxPC-3 cells mediated by TNF-related apoptosis-inducing ligand (TRAIL) and CD95L was likewise increased 2.3- +/- 0.5-fold and 7.4- +/- 2.5-fold, respectively. sst2-dependent activation and cell sensitization to death ligand-induced apoptosis involved activation of the executioner caspases, key factors in both death ligand- or mitochondria-mediated apoptosis. sst2 affected both pathways: first, by up-regulating expression of TRAIL and TNFalpha receptors, DR4 and TNFRI, respectively, and sensitizing the cells to death ligand-induced initiator capase-8 activation, and, second, by down-regulating expression of the antiapoptotic mitochondrial Bcl-2 protein. These results are of interest for the clinical management of chemoresistant pancreatic adenocarcinoma by using a combined gene therapy based on the cotransfer of genes for both the sst2 and a nontoxic death ligand. PMID:12490654

  10. 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.

  11. 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

  12. 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...

  13. Synthesis and preliminary pharmacological evaluation of a new putative radioiodinated AMPA receptor ligand for molecular imaging

    International Nuclear Information System (INIS)

    A new (radio)iodinated AMPA receptor ligand has been developed and pharmacologically evaluated in vitro and ex vivo using rodents. The new radioligand was directly labeled by electrophilic radioiodo-destannylation with iodine-131 in high radiochemical yields of 97% within 2 min. The new radioligand showed an excellent initial brain uptake of 2.1%ID/g at 10 min post injection, but a fast wash-out reduced the uptake by about 10-fold at 60 min post injection. Due to high nonspecific binding accompanied with a uniform distribution in brain tissue, however, the new radiotracer appears not suitable for AMPA receptor imaging in vivo.

  14. 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

  15. 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

  16. Characterization of host responses induced by Toll-like receptor ligands in chicken cecal tonsil cells.

    Science.gov (United States)

    Taha-Abdelaziz, Khaled; Alkie, Tamiru Negash; Hodgins, Douglas C; Shojadoost, Bahram; Sharif, Shayan

    2016-06-01

    The innate responses of cecal tonsils against invading microorganisms are mediated by conserved pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs). TLRs expressed by mammalian and avian immune system cells have the capability to recognize pathogen-associated molecular patterns (PAMPs). Although, the role of TLR ligands in innate and adaptive responses in chickens has been characterized in spleen and bursa of Fabricius, considerably less is known about responses in cecal tonsils. The aim of the current study was to assess responses of mononuclear cells from cecal tonsils to treatment with the TLR2, TLR4 and TLR21 ligands, Pam3CSK4, lipopolysaccharide (LPS), and CpG oligodeoxynucleotide (ODN), respectively. All three ligands induced significant up-regulation of interferon (IFN)-γ, interleukin (IL)-1β, IL-6 and CxCLi2/IL-8, whereas no significant changes were observed in expression of IL-13 or the antimicrobial peptides, avian β-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). In general, CpG ODN elicited the highest cytokine responses by cecal tonsil mononuclear cells, inducing significantly higher expression compared to LPS and Pam3CSK4, for IFNγ, IL-1β, IL-6 and CxCLi2 at various time points. These findings suggest the potential use of TLR21 ligands as mucosal vaccine adjuvants, especially in the context of pathogens of the intestinal tract. PMID:27185259

  17. 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

  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. Dimeric Arrangement of the Parathyroid Hormone Receptor and a Structural Mechanism for Ligand-induced Dissociation

    Energy Technology Data Exchange (ETDEWEB)

    Pioszak, Augen A.; Harikumar, Kaleeckal G.; Parker, Naomi R.; Miller, Laurence J.; Xu, H. Eric (Van Andel); (Mayo)

    2010-06-25

    The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an {alpha}-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure, PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.

  20. 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

  1. 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.

  2. NO EFFECT OF DIFFERENT ESTROGEN RECEPTOR LIGANDS ON COGNITION IN ADULT FEMALE MONKEYS

    OpenAIRE

    Lacreuse, Agnès; Wilson, Mark E.; Herndon, James G.

    2008-01-01

    Many studies in women and animal models suggest that estrogens affect cognitive function. Yet, the mechanisms by which estrogens may impact cognition remain unclear. The goal of the present study was to assess the effects of different estrogen receptor (ER) ligands on cognitive function in adult ovariectomized female rhesus monkeys. The monkeys were tested for 6 weeks on a battery of memory and attentional tasks administered on a touchscreen: the object, face, and spatial versions of the Dela...

  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. Microchemical synthesis of the serotonin receptor ligand, 125I-LSD

    International Nuclear Information System (INIS)

    The synthesis and properties of 2-[125I]-lysergic acid diethylamide, the first 125I-labeled serotonin receptor ligand, are described. A novel microsynthesis apparatus was developed for this synthesis. The apparatus employs a micromanipulator and glass micro tools to handle microliter to nanoliter volumes on a microscope stage. This apparatus should be generally useful for the synthesis of radioligands and other compounds when limited amounts of material must be handled in small volumes

  5. The actions of some cannabinoid receptor ligands in the rat isolated mesenteric artery

    OpenAIRE

    White, Richard; Robin Hiley, C

    1998-01-01

    The actions of a number of cannabinoid receptor ligands were investigated using the myograph-mounted rat isolated mesenteric artery. Anandamide, CP 55,940, HU-210, palmitoylethanolamide and WIN 55,212-2 all caused concentration-dependent relaxations of methoxamine-precontracted vessels which were not affected by removal of the endothelium.Precontracting vessels with 60 mM KCl instead of methoxamine greatly reduced the vasorelaxant effects of anandamide and palmitoylethanolamide. High K+ solut...

  6. 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

  7. Recombinant T Cell Receptor Ligands Improve Outcome After Experimental Cerebral Ischemia

    OpenAIRE

    Akiyoshi, Kozaburo; Dziennis, Suzan; Palmateer, Julie; Ren, Xuefang; Vandenbark, Arthur A.; Offner, Halina; Herson, Paco S; Hurn, Patricia D.

    2011-01-01

    A key target for novel stroke therapy is the regulation of post-ischemic inflammatory mechanisms. Recent evidence emphasizes the role of T lymphocytes of differing subtypes in the evolution is ischemic brain damage. We have recently demonstrated the benefit of myelin antigen-specific immunodulatory agents known as recombinant T cell receptor ligands (RTLs) in a standard murine model of focal stroke. The aim of the current study was to extend this initial observation to RTL treatment in a ther...

  8. Characterization of human platelet binding of recombinant T cell receptor ligand

    OpenAIRE

    Meza-Romero Roberto; Patel Ishan A; White-Adams Tara C; Sinha Sushmita; Aslan Joseph E; Itakura Asako; Vandenbark Arthur A; Burrows Gregory G; Offner Halina; McCarty Owen JT

    2010-01-01

    Abstract Background Recombinant T cell receptor ligands (RTLs) are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS). RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE). The mechanisms by which RTLs impede loca...

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. Initial receptor-ligand interactions modulate gene expression and phagosomal properties during both early and late stages of phagocytosis.

    Science.gov (United States)

    Hoffmann, Eik; Marion, Sabrina; Mishra, Bibhuti Bhusan; John, Mathias; Kratzke, Ramona; Ahmad, Syed Furquan; Holzer, Daniela; Anand, Paras Kumar; Weiss, Dieter G; Griffiths, Gareth; Kuznetsov, Sergei A

    2010-09-01

    The receptors engaged during recognition and phagocytic uptake of microorganisms and particles influence signaling events and diverse subcellular responses that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands on their surface, making it difficult to dissect the roles of individual receptors during phagocytosis. Moreover, it remains elusive to which extent receptor-ligand interactions and early binding events define the subsequent intracellular fate of phagosomes. Here, we used latex beads coupled to single ligands, focusing on immunoglobulin G, mannan, bacterial lipopolysaccharides and avidin, and monitored: (1) phagocytic uptake rates, (2) fusion of phagosomes with lysosomal compartments, (3) the gene expression profile during phagocytosis, (4) the protein composition of mature phagosomes and (5) time-dependent dynamics of protein association with phagosomes in J774.A1 mouse macrophages. The differently coated latex beads were internalized at different rates and exhibited different kinetics of phagolysosomal fusion events dependent on their specific ligand. Furthermore, less than 60% of identified phagosomal proteins and only 10-15% of changes in gene expression were common to all investigated ligands. These findings demonstrate that each single ligand induced a distinct pattern of genes and a different protein composition of phagosomes. Taken together, our data argue that phagocytic receptor-specific programs of signaling events direct phagosomes to different physiological states and support the existence of a specific receptor-ligand 'signature' during the whole process of phagocytosis. PMID:20579766

  1. Ring Substituents on Substituted Benzamide Ligands Indirectly Mediate Interactions with Position 7.39 of Transmembrane Helix 7 of the D4 Dopamine Receptor

    OpenAIRE

    Ericksen, Spencer S.; Cummings, David F.; Teer, Michael E.; Amdani, Shahnawaz; Schetz, John A.

    2012-01-01

    In an effort to delineate how specific molecular interactions of dopamine receptor ligand classes vary between D2-like dopamine receptor subtypes, a conserved threonine in transmembrane (TM) helix 7 (Thr7.39), implicated as a key ligand interaction site with biogenic amine G protein-coupled receptors, was substituted with alanine in D2 and D4 receptors. Interrogation of different ligand chemotypes for sensitivity to this substitution revealed enhanced affinity in the D4, but not the D2 recept...

  2. 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.

  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. 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

  5. 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)

  6. 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.

  7. 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

  8. 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

  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. Ligand-Receptor Interaction-Mediated Transmembrane Transport of Dendrimer-like Soft Nanoparticles: Mechanisms and Complicated Diffusive Dynamics.

    Science.gov (United States)

    Liang, Junshi; Chen, Pengyu; Dong, Bojun; Huang, Zihan; Zhao, Kongyin; Yan, Li-Tang

    2016-05-01

    Nearly all nanomedical applications of dendrimer-like soft nanoparticles rely on the functionality of attached ligands. Understanding how the ligands interact with the receptors in cell membrane and its further effect on the cellular uptake of dendrimer-like soft nanoparticles is thereby a key issue for their better application in nanomedicine. However, the essential mechanism and detailed kinetics for the ligand-receptor interaction-mediated transmembrane transport of such unconventional nanoparticles remain poorly elucidated. Here, using coarse-grained simulations, we present the very first study of molecular mechanism and kinetics behaviors for the transmembrane transport of dendrimer-like soft nanoparticles conjugated with ligands. A phase diagram of interaction states is constructed through examining ligand densities and membrane tensions that allows us to identify novel endocytosis mechanisms featured by the direct wrapping and the penetration-extraction vesiculation. The results provide an in-depth insight into the diffusivity of receptors and dendrimer in the membrane plane and demonstrate how the ligand density influences receptor diffusion and uptake kinetics. It is interesting to find that the ligand-conjugated dendrimers present superdiffusive behaviors on a membrane, which is revealed to be driven by the random fluctuation dynamics of the membrane. The findings facilitate our understanding of some recent experimental observations and could establish fundamental principles for the future development of such important nanomaterials for widespread nanomedical applications. PMID:27049403

  11. Synthesis and biological activity of small peptides as NOP and opioid receptors' ligands: view on current developments.

    Science.gov (United States)

    Naydenova, Emilia; Todorov, Petar; Zamfirova, Rositza

    2015-01-01

    The heptadecapeptide nociceptin, also called orphanin FQ (N/OFQ), is the endogenous agonist of the N/OFQ peptide receptor (NOP receptor) and is involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents overview of the several recently reported NOP ligands (agonists and antagonists), with an emphasis of the structural features that may be important for modulating the intrinsic activity of these ligands. In addition, a brief account on the characterization of newly synthesized ligands of NOP receptor with aminophosphonate moiety and β-tryptophan analogues will be presented. PMID:25677770

  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. Discontinuing benzodiazepines: best practices.

    Science.gov (United States)

    Guaiana, G; Barbui, C

    2016-06-01

    In July 2015, the Canadian Agency for Drugs and Technologies in Health (CADTH) released a Rapid Response report summary, with a critical appraisal, on discontinuation strategies for patients with long-term benzodiazepines (BDZ) use. The CADTH document is a review of the literature. It includes studies whose intervention is BDZ discontinuation. Also, clinical guidelines, systematic reviews and meta-analyses are included. What emerges from the CADTH guidelines is that the best strategy remains gradual tapering of BDZ with little evidence for the use of adjunctive medications. The results show that simple interventions such as discontinuation letters from clinicians, self-help information and support in general, added to gradual tapering may be associated with a two- to three-fold higher chance of successful withdrawal, compared with treatment as usual. We suggest possible implications for day-to-day clinical practice. PMID:26818890

  14. 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...

  15. 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

  16. 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

  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 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.

  19. Synthesis and evaluation of [18F] labeled benzamides: High affinity sigma receptor ligands for PET imaging

    International Nuclear Information System (INIS)

    We have synthesized and characterized four new fluorinated halobenzamides as sigma receptor ligands for use with positron emission tomography (PET). All the compounds were found to have high sigma-1 affinities (Ki = 0.38-0.98 nM), and the 4-fluoro-substituted benzamides were found to be more potent sigma-2 ligands (Ki = 3.77-4.02 nM) than their corresponding 2-fluoro analogs (Ki = 20.3-22.8 nM). The [18F] radiochemical syntheses of two of the analogs gave overall yields between 3-10% (EOS), radiochemical purities >99%, and specific activities between 800-1200 Ci/mmol (29.6-44.4 TBq/mmol). Rat biodistribution and blocking experiments were performed with 2-[18F](N-fluorobenzylpiperidin-4yl)-4-iodobenzamide, the analog with the best Ki value for sigma-1 sites (0.38 nM). Results of these experiments demonstrate specific uptake of the compound in tissues believed to contain sigma receptors, such as lungs, kidneys, heart, brain, and spleen and indicate its potential as a candidate for use in PET imaging of tissues containing these receptors

  20. 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

  1. 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

  2. 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.

  3. 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 DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results. PMID:26537635

  4. 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

  5. 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

  6. Dopaminergic receptor-ligand binding assays based on molecularly imprinted polymers on quartz crystal microbalance sensors.

    Science.gov (United States)

    Naklua, Wanpen; Suedee, Roongnapa; Lieberzeit, Peter A

    2016-07-15

    Molecularly imprinted polymers (MIPs) have been successfully applied as selective materials for assessing the binding activity of agonist and antagonist of dopamine D1 receptor (D1R) by using quartz crystal microbalance (QCM). In this study, D1R derived from rat hypothalamus was used as a template and thus self-organized on stamps. Those were pressed into an oligomer film consisting of acrylic acid: N-vinylpyrrolidone: N,N'-(1,2-dihydroxyethylene) bis-acrylamide in a ratio of 2:3:12 spin coated onto a dual electrode QCM. Such we obtained one D1R-MIP-QCM electrode, whereas the other electrode carried the non-imprinted control polymer (NIP) that had remained untreated. Successful imprinting of D1R was confirmed by AFM. The polymer can re-incorporate D1R leading to frequency responses of 100-1200Hz in a concentration range of 5.9-47.2µM. In a further step such frequency changes proved inherently useful for examining the binding properties of test ligands to D1R. The resulting mass-sensitive measurements revealed Kd of dopamine∙HCl, haloperidol, and (+)-SCH23390 at 0.874, 25.6, and 0.004nM, respectively. These results correlate well with the values determined in radio ligand binding assays. Our experiments revealed that D1R-MIP sensors are useful for estimating the strength of ligand binding to the active single site. Therefore, we have developed a biomimetic surface imprinting strategy for QCM studies of D1R-ligand binding and presented a new method to ligand binding assay for D1R. PMID:26926593

  7. 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...

  8. Vitamin D-influenced gene expression via a ligand-independent, receptor-DNA complex intermediate.

    OpenAIRE

    Ross, T K; Darwish, H M; Moss, V E; DeLuca, H F

    1993-01-01

    A lingering question regarding the regulation of target gene expression by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has been the delineation of vitamin D receptor (VDR)-DNA binding and transactivation. This report confirms that initial VDR-DNA interaction occurs in a ligand-independent fashion. An electrophoretic mobility-shift analysis demonstrated that VDR, derived from extracts of the small intestines of vitamin D-deficient rats, is capable of binding a vitamin D response element (DRE). Add...

  9. 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...

  10. 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...

  11. 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...

  12. 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

  13. The ligand specificities of the insulin receptor and the insulin-like growth factor I receptor reside in different regions of a common binding site

    Energy Technology Data Exchange (ETDEWEB)

    Kjeldsen, T.; Andersen, A.S.; Wiberg, F.C.; Rasmussen, J.S.; Schaeffer, L.; Balschmidt, P.; Moller, K.B.; Moller, N.P.H. (Novo Nordisk, Bagsvaerd (Denmark))

    1991-05-15

    To identify the region(s) of the insulin receptor and the insulin-like growth factor I (IGF-I) receptor responsible for ligand specificity (high-affinity binding), expression vectors encoding soluble chimeric insulin/IGF-I receptors were prepared. The chimeric receptors were expressed in mammalian cells and partially purified. Binding studies revealed that a construct comprising an IGF-I receptor in which the 68 N-terminal amino acids of the insulin receptor {alpha}-subunit had replaced the equivalent IGF-I receptor segment displayed a markedly increased affinity for insulin. In contrast, the corresponding IGF-I receptor sequence is not critical for high-affinity IGF-I binding. It is shown that part of the cysteine-rich domain determines IGF-I specificity. The authors have previously shown that exchanging exons 1, 2, and 3 of the insulin receptor with the corresponding IGF-I receptor sequence results in loss of high affinity for insulin and gain of high affinity for IGF-I. Consequently, it is suggested that the ligand specificities of the two receptors (i.e., the sequences that discriminate between insulin and IGF-I) reside in different regions of a binding site with common features present in both receptors.

  14. 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.

  15. 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-...

  16. 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.

  17. Fish genomes provide novel insights into the evolution of vertebrate secretin receptors and their ligand.

    Science.gov (United States)

    Cardoso, João C R; Félix, Rute C; Trindade, Marlene; Power, Deborah M

    2014-12-01

    The secretin receptor (SCTR) is a member of Class 2 subfamily B1 GPCRs and part of the PAC1/VPAC receptor subfamily. This receptor has long been known in mammals but has only recently been identified in other vertebrates including teleosts, from which it was previously considered to be absent. The ligand for SCTR in mammals is secretin (SCT), an important gastrointestinal peptide, which in teleosts has not yet been isolated, or the gene identified. This study revises the evolutionary model previously proposed for the secretin-GPCRs in metazoan by analysing in detail the fishes, the most successful of the extant vertebrates. All the Actinopterygii genomes analysed and the Chondrichthyes and Sarcopterygii fish possess a SCTR gene that shares conserved sequence, structure and synteny with the tetrapod homologue. Phylogenetic clustering and gene environment comparisons revealed that fish and tetrapod SCTR shared a common origin and diverged early from the PAC1/VPAC subfamily group. In teleosts SCTR duplicated as a result of the fish specific whole genome duplication but in all the teleost genomes analysed, with the exception of tilapia (Oreochromis niloticus), one of the duplicates was lost. The function of SCTR in teleosts is unknown but quantitative PCR revealed that in both sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) transcript abundance is high in the gastrointestinal tract suggesting it may intervene in similar processes to those in mammals. In contrast, no gene encoding the ligand SCT was identified in the ray-finned fishes (Actinopterygii) although it was present in the coelacanth (lobe finned fish, Sarcopterygii) and in the elephant shark (holocephalian). The genes in linkage with SCT in tetrapods and coelacanth were also identified in ray-finned fishes supporting the idea that it was lost from their genome. At present SCTR remains an orphan receptor in ray-finned fishes and it will be of interest in the future to establish why SCT was

  18. 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

  19. Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

    DEFF Research Database (Denmark)

    Wang, Qian; Pless, Stephan Alexander; Lynch, Joseph W

    2010-01-01

    from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting...

  20. In vivo evaluation of [11C]SA4503 as a PET ligand for mapping CNS sigma1 receptors

    International Nuclear Information System (INIS)

    The potential of the 11C-labeled selective sigma1 receptor ligand 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine ([11C]SA4503) was evaluated in vivo as a positron emission tomography (PET) ligand for mapping sigma1 receptors in rats. SA4503 is known to have a high affinity (IC50 17.4 nM) and a higher selectivity (sigma1/sigma2=103) for the sigma1 receptor. A high and increasing brain uptake of [11C]SA4503 was found. Pre-, co- and postinjection of cold SA4503 significantly decreased uptake of [11C]SA4503 in the brain, spleen, heart, lung, and kidney in which sigma receptors are present as well as in the skeletal muscle. In the blocking study with one of four sigma receptor ligands including haloperidol, (+)-pentazocine, SA4503, and (-)-pentazocine (in the order of their affinity for sigma1 receptor subtype), SA4503 and haloperidol significantly reduced the brain uptake of [11C]SA4503 to approximately 30% of the control, but the other two benzomorphans did not. A high specific uptake of [11C]SA4503 by the brain was also confirmed by ex vivo autoradiography (ARG) and PET. Ex vivo ARG showed a higher uptake in the vestibular nucleus, temporal cortex, cingulate cortex, inferior colliculus, thalamus, and frontal cortex, and a moderate uptake in the parietal cortex and caudate putamen. Peripherally, the blocking effects of the four ligands depended on their affinity for sigma1 receptors. No 11C-labeled metabolite was detected in the brain 30 min postinjection, whereas approximately 20% of the radioactivity was found as 11C-labeled metabolites in plasma. These results have demonstrated that the 11C-labeled sigma1 receptor ligand [11C]SA4503 has a potential for mapping sigma1 receptors in the central nervous system and peripheral organs

  1. Caged vanilloid ligands for activation of TRPV1 receptors by 1- and 2-photon excitation†

    Science.gov (United States)

    Zhao, Jun; Gover, Tony D.; Muralidharan, Sukumaran; Auston, Darryl A.; Weinreich, Daniel; Kao, Joseph P. Y.

    2008-01-01

    Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV1, a non-selective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids—biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged vanilloids, Nb-VNA and Nv-VNA, are photoreleased with quantum efficiency of 0.13 and 0.041, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in ∼40 μs and ∼125 μs, respectively. Through 1-photon excitation with ultraviolet light (360 nm), or 2-photon excitation with red light (720 nm), the caged vanilloids can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons. The consequent increase in intracellular free Ca2+ concentration ([Ca2+]i) can be visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca2+ indicator, fluo-3. Stimulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca2+-free extracellular medium, photoreleased vanilloid can still elevate [Ca2+]i, which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca2+ release from intracellular stores. Notably, whole-cell voltage clamp measurements showed that flash photorelease of vanilloid can activate TRPV1 channels in < 4 msec at 22°C. In combination with 1- or 2-photon excitation, caged vanilloids are a powerful tool for probing morphologically distinct structures of nociceptive sensory neurons with high spatial and temporal precision. PMID:16605259

  2. Analysis of ligand-receptor cross-linked fragments by mass spectrometry.

    Science.gov (United States)

    Son, C D; Sargsyan, H; Hurst, G B; Naider, F; Becker, J M

    2005-03-01

    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 alpha-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 alpha-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to cross-linked fragments of Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an alpha-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 alpha-factor and a region of Ste2p covering residues 251-294. PMID:15787972

  3. 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.

  4. Modulation of anxiety behavior in the elevated plus maze using peptidic oxytocin and vasopressin receptor ligands in the rat.

    Science.gov (United States)

    Mak, Plato; Broussard, Christina; Vacy, Kristina; Broadbear, Jillian H

    2012-04-01

    Oxytocin (OT) and arginine vasopressin (AVP), in their capacities as neuromodulators, are believed to play an important role in mood control, including regulation of the anxiety response. In the present study, the contributions of oxytocin and vasopressin receptor modulation to anxiety-like behaviors were examined in male Sprague-Dawley rats. The behavioral effects of the OT receptor agonist, carbetocin (intracerebroventricular, intravenous and intraperitoneal routes), the AVP receptor agonist desmopressin (intravenous route), and the OT/AVP(1A) receptor antagonist atosiban (intravenous route) were evaluated in the elevated plus maze. The benzodiazepine diazepam was included as a positive control. Central but not systemic administration of carbetocin produced pronounced anxiolytic-like behavioral changes comparable to those measured following systemic diazepam treatment. The anxiolytic efficacy of carbetocin was maintained following 10 days of once-daily treatment, contrasting with the effects of diazepam which were no longer distinguishable from saline treatment. Systemic administration of desmopressin produced anxiogenic-like effects whereas systemic atosiban produced anxiolytic-like effects. Co-administration of desmopressin with atosiban resulted in saline-like behavioral responses, implicating an AVP(1A) receptor mechanism in the anxiolytic and anxiogenic effects of these neuropeptides following systemic administration. A peripherally-mediated antidiuretic effect of desmopressin on water consumption was also demonstrated. These results highlight the potential therapeutic utility of AVP(1A) receptor blockade in the modulation of anxiety-related behaviors; AVP(1A) receptor blockade appears to be a more promising pharmacological target than does OT receptor activation following systemic drug administration. PMID:21890582

  5. 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.

  6. 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

  7. 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.

  8. 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. PMID:15481976

  9. Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella

    Directory of Open Access Journals (Sweden)

    Tang Baozhen

    2012-10-01

    Full Text Available Abstract Background The diamondback moth, Plutella xylostella (L. (Lepidoptera: Plutellidae, is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. Result In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM. In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. Conclusions Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists to their targets (ecdysone receptors leads to an adaptive response (resistance, is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella.

  10. Catalposide is a natural agonistic ligand of peroxisome proliferator-activated receptor

    International Nuclear Information System (INIS)

    Highlights: ► Catalposide is a novel ligand for PPARα. ► Cell stimulated with catalposide improved fatty acid uptake, regulated target genes in fatty acid β-oxidation and synthesis. ► Catalposdie reduces hepatic triacylglycerides. ► Theses demonstrate catalposide could ameliorate hyperlipidemia and hepatic steatosis. -- Abstract: Peroxisome proliferator-activated receptor-alpha (PPARα) is a nuclear receptor that regulates the expression of genes related to cellular lipid uptake and oxidation. Thus, PPARα agonists may be important in the treatment of hypertriglyceridemia and hepatic steatosis. In this study, we demonstrated that catalposide is a novel natural PPARα 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 PPARα. 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 PPARα via a ligand-mediated mechanism that modulates the expression of in lipid metabolism genes in hepatocytes.

  11. 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

  12. 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.

  13. 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...

  14. 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

  15. Development of sigma-1 (σ1) receptor fluorescent ligands as versatile tools to study σ1 receptors✩

    Science.gov (United States)

    Abate, Carmen; Riganti, Chiara; Pati, Maria Laura; Ghigo, Dario; Berardi, Francesco; Mavlyutov, Timur; Guo, Lian-Wang; Ruoho, Arnold

    2016-01-01

    Despite their controversial physiology, sigma-1 (σ1) receptors are intriguing targets for the development of therapeutic agents for central nervous system diseases. With the aim of providing versatile pharmacological tools to study σ1 receptors, we developed three σ1 fluorescent tracers by functionalizing three well characterized σ1 ligands with a fluorescent tag. A good compromise between σ1 binding affinity and fluorescent properties was reached, and the σ1 specific targeting of the novel tracers was demonstrated by confocal microscopy and flow cytometry. These novel ligands were also successfully used in competition binding studies by flow cytometry, showing their utility in nonradioactive binding assays as an alternative strategy to the more classical radioligand binding assays. To the best of our knowledge these are the first σ1 fluorescent ligands to be developed and successfully employed in living cells, representing promising tools to strengthen σ1 receptors related studies. PMID:26717207

  16. Nuclear localization and function of polypeptide ligands and their receptors: a new paradigm for hormone specificity within the mammary gland?

    International Nuclear Information System (INIS)

    The specific effects triggered by polypeptide hormone/growth factor stimulation of mammary cells were considered mediated solely by receptor-associated signaling networks. A compelling body of new data, however, clearly indicates that polypeptide ligands and/or their receptors are transported into the nucleus, where they function directly to regulate the expression of specific transcription factors and gene loci. The intranuclear function of these complexes may contribute to the explicit functions associated with a given ligand, and may serve as new targets for pharmacologic intervention

  17. Lipid domain formation and ligand-receptor distribution in lipid bilayer membranes investigated by atomic force microscopy

    DEFF Research Database (Denmark)

    Kaasgaard, Thomas; Mouritsen, O.G.; Jørgensen, K.

    A novel experimental technique, based on atomic force microscopy (AFM), is proposed to visualize the lateral organization of membrane systems in the nanometer range. The technique involves the use of a ligand-receptor pair, biotin-avidin, which introduces a height variation on a solid-supported l......A novel experimental technique, based on atomic force microscopy (AFM), is proposed to visualize the lateral organization of membrane systems in the nanometer range. The technique involves the use of a ligand-receptor pair, biotin-avidin, which introduces a height variation on a solid...

  18. 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.

  19. 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...

  20. (/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.

  1. 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.

  2. No effect of different estrogen receptor ligands on cognition in adult female monkeys.

    Science.gov (United States)

    Lacreuse, Agnès; Wilson, Mark E; Herndon, James G

    2009-03-01

    Many studies in women and animal models suggest that estrogens affect cognitive function. Yet, the mechanisms by which estrogens may impact cognition remain unclear. The goal of the present study was to assess the effects of different estrogen receptor (ER) ligands on cognitive function in adult ovariectomized female rhesus monkeys. The monkeys were tested for 6 weeks on a battery of memory and attentional tasks administered on a touchscreen: the object, face, and spatial versions of the Delayed Recognition Span Test (DRST) and a Visual Search task. Following a 2-week baseline period with oil vehicle treatment, monkeys were randomly assigned to one of 3 treatment groups: estradiol benzoate (EB), selective ERbeta agonist (diarylpropionitrile DPN) or selective ER modulator tamoxifen (TAM). In each treatment group, monkeys received oil vehicle for 2 weeks and the drug for 2 weeks, in a cross-over design. After a 4-week washout, a subset of monkeys was re-tested on the battery when treated with a selective ERalpha agonist (propyl-pyrazole-triol, PPT) or oil vehicle. Overall, drug treatments had no or negligible effects on cognitive performance. These results support the contention that exogenous estrogens and selective estrogen receptor modulators (SERMS) do not significantly affect cognition in young adult female macaques. Additional studies are needed to determine whether the cognitive effects of estrogens in monkeys of more advanced age are mediated by ERbeta, ERalpha or complex interactions between the two receptors. PMID:19101578

  3. 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.

  4. /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.

  5. 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).

  6. Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

    Science.gov (United States)

    Fotticchia, Iolanda; Guarnieri, Daniela; Fotticchia, Teresa; Falanga, Andrea Patrizia; Vecchione, Raffaele; Giancola, Concetta; Netti, Paolo Antonio

    2016-08-01

    Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier. PMID:27100851

  7. Protein dynamics at Eph receptor-ligand interfaces as revealed by crystallography, NMR and MD simulations

    International Nuclear Information System (INIS)

    The role of dynamics in protein functions including signal transduction is just starting to be deciphered. Eph receptors with 16 members divided into A- and B- subclasses are respectively activated by 9 A- and B-ephrin ligands. EphA4 is the only receptor capable of binding to all 9 ephrins and small molecules with overlapped interfaces. We first determined the structures of the EphA4 ligand binding domain (LBD) in two crystals of P1 space group. Noticeably, 8 EphA4 molecules were found in one asymmetric unit and consequently from two crystals we obtained 16 structures, which show significant conformational variations over the functionally critical A-C, D-E, G-H and J-K loops. The 16 new structures, together with previous 9 ones, can be categorized into two groups: closed and open forms which resemble the uncomplexed and complexed structures of the EphA4 LBD respectively. To assess whether the conformational diversity over the loops primarily results from the intrinsic dynamics, we initiated 30-ns molecular dynamics (MD) simulations for both closed and open forms. The results indicate that the loops do have much higher intrinsic dynamics, which is further unravelled by NMR H/D exchange experiments. During simulations, the open form has the RMS deviations slightly larger than those of the closed one, suggesting the open form may be less stable in the absence of external contacts. Furthermore, no obvious exchange between two forms is observed within 30 ns, implying that they are dynamically separated. Our study provides the first experimental and computational result revealing that the intrinsic dynamics are most likely underlying the conformational diversity observed for the EphA4 LBD loops mediating the binding affinity and specificity. Interestingly, the open conformation of the EphA4 LBD is slightly unstable in the absence of it natural ligand ephrins, implying that the conformational transition from the closed to open has to be driven by the high

  8. 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

  9. Cell-free synthesis of isotopically labelled peptide ligands for the functional characterization of G protein-coupled receptors.

    Science.gov (United States)

    Joedicke, Lisa; Trenker, Raphael; Langer, Julian D; Michel, Hartmut; Preu, Julia

    2016-01-01

    Cell-free systems exploit the transcription and translation machinery of cells from different origins to produce proteins in a defined chemical environment. Due to its open nature, cell-free protein production is a versatile tool to introduce specific labels such as heavy isotopes, non-natural amino acids and tags into the protein while avoiding cell toxicity. In particular, radiolabelled peptides and proteins are valuable tools for the functional characterization of protein-protein interactions and for studying binding kinetics. In this study we evaluated cell-free protein production for the generation of radiolabelled ligands for G protein-coupled receptors (GPCRs). These receptors are seven-transmembrane-domain receptors activated by a plethora of extracellular stimuli including peptide ligands. Many GPCR peptide ligands contain disulphide bonds and are thus inherently difficult to produce in bacterial expression hosts or in Escherichia coli-based cell-free systems. Here, we established an adapted E. coli-based cell-free translation system for the production of disulphide bond-containing GPCR peptide ligands and specifically introduce tritium labels for detection. The bacterial oxidoreductase DsbA is used as a chaperone to favour the formation of disulphide bonds and to enhance the yield of correctly folded proteins and peptides. We demonstrate the correct folding and formation of disulphide bonds and show high-affinity ligand binding of the produced radio peptide ligands to the respective receptors. Thus, our system allows the fast, cost-effective and reliable synthesis of custom GPCR peptide ligands for functional and structural studies. PMID:27047736

  10. 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...

  11. 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...

  12. 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.

  13. Synthesis of the 11C-labelled β-adrenergic receptor ligands atenolol, metoprolol and propanolol

    International Nuclear Information System (INIS)

    The 11C-labelled β-adrenergic receptor ligands atenolol 1, metoprolol 2 and propranolol 3 have been synthesized by an N-alkylation reaction using [2-11C]isopropyl iodide. The labelled isopropyl iodide was prepared in a one-pot reactor system from [11C]carbon dioxide and obtained in 40% radiochemical yield within 14 min reaction time. The total reaction times for compounds 1-3, counted from the start of the isopropyl iodide synthesis and including purification were 45-55 min. The products were obtained in 5-15% radiochemical yields and with radiochemical purities higher than 98%. The specific activity ranged from 0.4 to 4 GBq/μmol. In a typical experiment starting with 4 GBq around 75 MBq of product was obtained. (author)

  14. Synthesis of the sup 11 C-labelled. beta. -adrenergic receptor ligands atenolol, metoprolol and propanolol

    Energy Technology Data Exchange (ETDEWEB)

    Antoni, G.; Ulin, J.; Laangstroem, B. (Uppsala Univ. (Sweden). Dept. of Organic Chemistry)

    1989-01-01

    The {sup 11}C-labelled {beta}-adrenergic receptor ligands atenolol 1, metoprolol 2 and propranolol 3 have been synthesized by an N-alkylation reaction using (2-{sup 11}C)isopropyl iodide. The labelled isopropyl iodide was prepared in a one-pot reactor system from ({sup 11}C)carbon dioxide and obtained in 40% radiochemical yield within 14 min reaction time. The total reaction times for compounds 1-3, counted from the start of the isopropyl iodide synthesis and including purification were 45-55 min. The products were obtained in 5-15% radiochemical yields and with radiochemical purities higher than 98%. The specific activity ranged from 0.4 to 4 GBq/{mu}mol. In a typical experiment starting with 4 GBq around 75 MBq of product was obtained. (author).

  15. 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

  16. 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.

  17. 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

  18. Ligand independent aryl hydrocarbon receptor inhibits lung cancer cell invasion by degradation of Smad4.

    Science.gov (United States)

    Lee, Chen-Chen; Yang, Wen-Hao; Li, Ching-Hao; Cheng, Yu-Wen; Tsai, Chi-Hao; Kang, Jaw-Jou

    2016-07-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Although AhR plays a crucial role in air toxicant-induced carcinogenesis, AhR expression was shown to negatively regulate tumorigenesis. Therefore, in the present study, we investigated the effect of AhR without ligand treatment on cancer invasion in lung cancer cell lines. Lung cancer cells expressing lower levels of AhR showed higher invasion ability (H1299 cells) compared with cells expressing higher levels of AhR (A549 cells). Overexpression of AhR in H1299 cells inhibited the invasion ability. We found that vimentin expression was inhibited in AhR-overexpressing H1299 cells. Additionally, the expression of EMT-related transcriptional factors Snail and ID-1 decreased. Interestingly, we found that Smad4 degradation was induced in AhR-overexpressing H1299 cells. Our data showed that AhR could interact with Jun-activation domain binding protein (Jab1) and Smad4, which may cause degradation of Smad4 by the proteasome. Our data suggest that AhR affects the transforming growth factor-β signaling pathway by inducing Smad4 degradation by the proteasome and suppressing tumor metastasis via epithelial to mesenchymal transition reduction in lung cancer cells. PMID:27060206

  19. Genetic functions of the NAIP family of inflammasome receptors for bacterial ligands in mice.

    Science.gov (United States)

    Zhao, Yue; Shi, Jianjin; Shi, Xuyan; Wang, Yupeng; Wang, Fengchao; Shao, Feng

    2016-05-01

    Biochemical studies suggest that the NAIP family of NLR proteins are cytosolic innate receptors that directly recognize bacterial ligands and trigger NLRC4 inflammasome activation. In this study, we generated Naip5(-/-), Naip1(-/-), and Naip2(-/-) mice and showed that bone marrow macrophages derived from these knockout mice are specifically deficient in detecting bacterial flagellin, the type III secretion system needle, and the rod protein, respectively. Naip1(-/-), Naip2(-/-), and Naip5(-/-) mice also resist lethal inflammasome activation by the corresponding ligand. Furthermore, infections performed in the Naip-deficient macrophages have helped to define the major signal in Legionella pneumophila, Salmonella Typhimurium and Shigella flexneri that is detected by the NAIP/NLRC4 inflammasome. Using an engineered S. Typhimurium infection model, we demonstrate the critical role of NAIPs in clearing bacterial infection and protecting mice from bacterial virulence-induced lethality. These results provide definitive genetic evidence for the important physiological function of NAIPs in antibacterial defense and inflammatory damage-induced lethality in mice. PMID:27114610

  20. 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

  1. 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

  2. Comparison of monoclonal antibodies and tritiated ligands for estrogen receptor assays in 241 breast cancer cytosols

    International Nuclear Information System (INIS)

    Estrogen receptor determinations have been performed on 241 cytosols from 160 breast cancer tumors using both radioactive ligands ([3H]-estradiol, [3H]R2858) and monoclonal antibodies (Abbott ER-EIA Kit) to compare the two methods and to evaluate the clinical usefulness of the new immunological, simplified assay. Intra- and interassay reproducibility of the enzyme immunoassay (EIA) method was studied during a 6-month period on 35 standard curves with 4 different batches of monoclonal antibodies. Intraassay coefficients of variation studied on duplicates were smaller than 5% in most cases and reproducibility of the curves showed coefficients of variation lower than 10% except for standard 0 and 5 fmol/ml. Pooled cytosols used as control for the dextran coated charcoal method had interassay variation coefficients between 3.8 and 11.4%. Reproducibility has been studied on clinical specimens assayed twice at two different periods with either EIA or dextran coated charcoal methods. Slopes obtained were 1.05 and 0.96, respectively. A good stability of EIA results was obtained with protein concentrations in the range 4-0.15 mg/ml cytosol. No significant effects of dithiothreitol or monothioglycerol (1 mM) on EIA and dextran coated charcoal assay were observed. Eighty breast cancer cytosols were assayed with both EIA and Scatchard analysis. The slope of the regression curve obtained was 1.04 (r = 0.963). Cytosols were assayed by EIA and by a saturating concentration of tritiated ligand (5 nM). With 153 cytosols the EIA/5 nM slope was 1.34 (r = 0.978). This slope can be compared with the slope Scatchard/5 nM obtained with 90 cytosols: 1.29 (r = 0.985). Absence of cross-reactivity of monoclonal ER antibodies with progesterone receptor was observed

  3. Macrophage Membrane Potential Changes Associated with γ 2b/γ 1 Fc Receptor-Ligand Binding

    Science.gov (United States)

    Young, John Ding-E; Unkeless, Jay C.; Kaback, H. Ronald; Cohn, Zanvil A.

    1983-03-01

    We have studied the effects of specific ligands of the receptor for the IgG Fc fragment (FcR) on the membrane potential (Δ Psi ) of the macrophage cell line J774 by the [3H]tetraphenylphosphonium ion equilibration technique. We observe a membrane depolarization with binding of FcR ligands that is dependent on the degree of receptor crosslinking. Binding of the FcR by monovalent ligands is not sufficient to induce a significant drop in Δ Psi , but a sustained depolarization lasting ≈ 20 min occurs with insoluble multivalent ligands. This FcR-mediated depolarization can be inhibited by substitution of Na+ from the cell incubation medium with monovalent choline cation, indicating that depolarization is due to Na+ influx into the cell. The extracellular Ca2+ does not play a significant role in membrane depolarization. The depolarization response is not triggered by monoclonal antibodies directed against three other major macrophage surface antigens. The cell depolarization mediated by FcR ligands is followed by a prolonged hyperpolarization that can be partially blocked by ouabain and quinine, indicating that the hyperpolarization response is a result of a combination of a Na+, K+-ATPase activity and a Ca2+-activated K+ conductance. These data support our hypothesis that the mouse macrophage IgG FcR is a ligand-dependent ion channel.

  4. 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

  5. Substituted benzamides as ligands for visualization of dopamine receptor binding in the human brain by positron emission tomography

    International Nuclear Information System (INIS)

    Two substituted benzamides, FLB 524 and raclopride, were labeled with 11C and examined for their possible use as ligands for positron emission tomography (PET) scan studies on dopamine-2 (D-2) receptors in the brains of monkeys and healthy human subjects. Both ligands allowed the in vivo visualization of D-2 receptor binding in the corpus striatum caudate nucleus/putamen complex in PET-scan images. [11C]Raclopride showed a high ratio of specific striatal to nonspecific cerebellar binding, and the kinetics of binding of this ligand made it optimal for PET studies. The in vivo binding of [11C]raclopride in the striatum of cynomolgus monkeys was markedly reduced by displacement with haloperidol. In healthy human subjects, [11C]raclopride binding in the caudate nucleus/putamen was 4- to 5-fold greater than nonspecific binding in the cerebellum. In comparison with previously available ligands for PET-scan studies on central dopamine receptors in man, [11C]raclopride appears to be advantageous with regard to (i) specificity of binding to D-2 receptors, (ii) the high ratio between binding in dopamine-rich (caudate, putamen) and dopamine-poor (cerebellum) human brain regions, and (iii) rapid association and reversibility of specific binding

  6. 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...

  7. 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...

  8. Design, synthesis and biological evaluation of ligands selective for the melanocortin-3 receptor.

    Science.gov (United States)

    Hruby, Victor J; Cai, Minying; Cain, James P; Mayorov, Alexander V; Dedek, Matthew M; Trivedi, Devendra

    2007-01-01

    The processed products of the proopiomelanocortin gene (ACTH, alpha-MSH, beta-MSH, gamma-MSH, etc.) interact with five melanocortin receptors, the MC1R, MC2R, MC3R, MC4R, and MC5R to modulate and control many important biological functions crucial for good health both peripherally (as hormones) and centrally (as neurotransmitters). Pivotal biological functions include pigmentation, adrenal function, response to stress, fear/flight, energy homeostasis, feeding behavior, sexual function and motivation, pain, immune response, and many others, and are believed to be involved in many disease states including pigmentary disorders, adrenal disorders, obesity, anorexia, prolonged and neuropathic pain, inflammatory response, etc. The melanocortin-3 receptor (MC3R) is found primarily in the brain and spinal cord and also in the periphery, and its biological functions are still not well understood. Here we review some of the biological functions attributed to the MC3R, and then examine in more detail efforts to design and synthesize ligands that are potent and selective for the MC3R, which might help resolve the many questions still remaining about its function. Though some progress has been made, there is still much to be done in this critical area. PMID:17584128

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. Characterization of human platelet binding of recombinant T cell receptor ligand

    Directory of Open Access Journals (Sweden)

    Meza-Romero Roberto

    2010-11-01

    Full Text Available Abstract Background Recombinant T cell receptor ligands (RTLs are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS. RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE. The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood. Methods We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function. Results Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen. Conclusions Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.

  15. 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...

  16. 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.

  17. 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...

  18. 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...

  19. 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

  20. Synthesis and in vivo evaluation of [11C]SA6298 as a PET sigma1 receptor ligand

    International Nuclear Information System (INIS)

    The potential of a 11C-labeled selective sigma1 receptor ligand, 1-(3,4-dimethoxyphenethyl)-4-[3-(3,4-dichlorophenyl)propyl]piperazine ([11C]SA6298), was evaluated as a positron emission tomography (PET) ligand for mapping sigma1 receptors in the central nervous system and peripheral organs. [11C]SA6298 was synthesized by methylation of the desmethyl SA6298 with [11C]CH3I, with the decay-corrected radiochemical yield of 39±5% based on [11C]CH3I and with the specific activity of 53±17 TBq/mmol within 20 min from end of bombardment (EOB). In mice, the uptake of [11C]SA6298 was significantly decreased by carrier loading in the brain, liver, spleen, heart, lung, small intestine, and kidney in which sigma receptors are present as well as in the skeletal muscle. Pretreatment with SA6298 also blocked the uptake of [11C]SA6298 by these organs except for the small intestine, but significant displacement of [11C]SA6298 by posttreatment with SA6298 was observed only in the heart, lung, and muscle. In the blocking study with one of the eight sigma receptor ligands, including haloperidol, SA6298, NE-100, (+)-pentazocine, SA4503, (-)-pentazocine, (+)-3-PPP, and (+)-SKF 10,047 (in the order of the affinity for sigma1 receptor subtype), only SA6298 and an analog SA4503 significantly reduced the brain uptake of [11C]SA6298 to approximately 80% of the control, but the other six ligands did not. Peripherally, the uptake of [11C]SA6298 by the organs described above was decreased predominantly by SA6298 or SA4503, but the blocking effects of the other five ligands except for NE-100 depended on their affinity for sigma1 receptors. The saturable brain uptake of [11C]SA6298, approximately 20%, was also observed by tissue dissection method in rats and by PET in a cat. Ex vivo autoradiography of the rat brain showed a high uptake in the cortex and thalamus. In the cat brain a relatively high uptake was found in the cortex, thalamus, striatum, and cerebellum. These results have indicated a

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. Experiment K-7-18: Effects of Spaceflight in the Muscle Adductor Longus of Rats Flown in the Soviet Biosatellite Cosmos 2044. Part 2; Quantitative Autoradiographic Analysis of Gaba (Benzodiazepine) and Muscarinic (Cholinergic) Receptors in the Forebrain of Rats Flown on Cosmos 2044

    Science.gov (United States)

    Wu, L.; Daunton, N. G.; Krasnov, I. B.; DAmelio, F.; Hyde, T. M.; Sigworth, S. K.

    1994-01-01

    Quantitative autoradiographic analysis of receptors for GABA and acetylcholine in the forebrain of rats flown on COSMOS 2044 was undertaken as part of a joint US-Soviet study to determine the effects of microgravity on the central nervous system, and in particular on the sensory and motor portions of the forebrain. Changes in binding of these receptors in tissue from animals exposed to microgravity would provide evidence for possible changes in neural processing as a result of exposure to microgravity. Tritium-labelled diazepam and Quinuclidinyl-benzilate (QNB) were used to visualize GABA (benzodiazepine) and muscarinic (cholinergic) receptors, respectively. The density of tritium-labelled radioligands bound to various regions in the forebrain of both flight and control animals were measured from autoradiograms. Data from rats flown in space and from ground-based control animals that were not exposed to microgravity were compared.

  6. 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.

  7. 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

  8. 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

  9. Recombinant T cell receptor ligands improve outcome after experimental cerebral ischemia.

    Science.gov (United States)

    Akiyoshi, Kozaburo; Dziennis, Suzan; Palmateer, Julie; Ren, Xuefang; Vandenbark, Arthur A; Offner, Halina; Herson, Paco S; Hurn, Patricia D

    2011-09-01

    A key target for novel stroke therapy is the regulation of post-ischemic inflammatory mechanisms. Recent evidence emphasizes the role of T lymphocytes of differing subtypes in the evolution is ischemic brain damage. We have recently demonstrated the benefit of myelin antigen-specific immunodulatory agents known as recombinant T cell receptor ligands (RTLs) in a standard murine model of focal stroke. The aim of the current study was to extend this initial observation to RTL treatment in a therapeutically relevant timing after middle cerebral artery occlusion (MCAO) and verify functional benefit to complement histological outcome measures. We observed that the administration of mouse-specific RTL551 reduced infarct size and improved sensorimotor outcome when administered within a 3 h post-ischemic therapeutic window. RTL551 treatment reduced cortical, caudate putamen, and total infarct volume as compared to vehicle-treated mice. Using a standard behavioral testing repertoire, we observed that RTL551 reduced sensorimotor impairment 3 days after MCAO. Humanized RTL1000 (HLA-DR2 moiety linked to hMOG-35-55 peptide) also reduced infarct size in HLA-DR2 transgenic mice. These data indicate that this neuroantigen-specific immunomodulatory agent reduces damage when administered in a therapeutically relevant reperfusion timeframe. PMID:21961027

  10. 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.

  11. Specific binding of a ligand of σ-opioid receptors - N-allylnormetazocine (SKF 10047) - with liver membranes

    International Nuclear Information System (INIS)

    A ligand of the σ-opioid receptors - N-allylnormetazocine (SKF 10047) -binds specifically and reversible with rat liver membranes. In relation to a number of properties, the sites binding SKF 10047 in the liver are similar to the σ-opioid receptors of the central nervous system. They do not interact with classical opiates (morphine, naloxone) and with opioid peptides, but bind well benzomorphans (bremazocine, SKF 10047) and a number of compounds of different chemical structures with a pronounced psychtropic action (haloperidol, imipramine, phencyclidine, etc.)

  12. 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...

  13. Specific binding of a ligand of sigma-opioid receptors - N-allylnormetazocine (SKF 10047) - with liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Samovilova, N.N.; Yarygin, K.N.; Vinogradov, V.A.

    1986-08-01

    A ligand of the sigma-opioid receptors - N-allylnormetazocine (SKF 10047) -binds specifically and reversible with rat liver membranes. In relation to a number of properties, the sites binding SKF 10047 in the liver are similar to the sigma-opioid receptors of the central nervous system. They do not interact with classical opiates (morphine, naloxone) and with opioid peptides, but bind well benzomorphans (bremazocine, SKF 10047) and a number of compounds of different chemical structures with a pronounced psychtropic action (haloperidol, imipramine, phencyclidine, etc.).

  14. Fibrinogen, an endogenous ligand of Toll-like receptor 4, activates monocytes in pre-eclamptic patients.

    Science.gov (United States)

    Al-ofi, Ebtisam; Coffelt, Seth B; Anumba, Dilly O

    2014-06-01

    Pre-eclampsia (PE) remains the leading cause of pregnancy-associated mortality and morbidity, urging the need for a better understanding of its aetiology and pathophysiological progression. A key characteristic of PE is a systemic, exaggerated, inflammatory condition involving abnormal cytokine levels in serum, altered immune cell phenotype and Th1/Th2-type immunological imbalance. However, it is unknown how this heightened inflammatory condition manifests. We previously reported increased expression of the lipopolysaccharide receptor, Toll-like receptor 4 (TLR4), on monocytes from PE patients compared with normotensive, pregnant patients (NP). This upregulation of TLR4 on PE monocytes was accompanied by a hyper-responsiveness to bacterial TLR4 ligands. To determine whether non-microbial, endogenous TLR4 ligands also activate monocytes from PE patients, we investigated the expression of host-derived TLR4 ligands and the response of monocytes to these endogenous ligands. Plasma levels of fibrinogen - but not fibronectin or heparan sulphate - were higher in PE patients than in NP. Exposure to fibrinogen was associated with significantly increased production of inflammatory cytokines by monocytes from PE patients. Interestingly, this effect was not observed with NP monocytes. Our findings suggest that the fibrinogen-TLR4 axis might play an important role in the atypical activation of monocytes observed in PE patients that may contribute to the exaggerated inflammatory condition. PMID:24661950

  15. Crystallographic analysis of murine constitutive androstane receptor ligand-binding domain complexed with 5α-androst-16-en-3α-ol

    International Nuclear Information System (INIS)

    The purification and structure determination of the murine constitutive androstane receptor bound to its inverse agonist/antagonist androstenol is described. The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily. In contrast to classical nuclear receptors, which possess small-molecule ligand-inducible activity, CAR exhibits constitutive transcriptional activity in the apparent absence of ligand. CAR is among the most important transcription factors; it coordinately regulates the expression of microsomal cytochrome P450 genes and other drug-metabolizing enzymes. The murine CAR ligand-binding domain (LBD) was coexpressed with the steroid receptor coactivator protein (SRC-1) receptor-interacting domain (RID) in Escherichia coli. The mCAR LBD subunit was purified away from SRC-1 by affinity, anion-exchange and size-exclusion chromatography, crystallized with androstenol and the structure of the complex determined by molecular replacement

  16. Receptor activator of nuclear factor-κB ligand and osteoprotegerin: maintaining the balance to prevent bone loss

    OpenAIRE

    Anne-Priscille Trouvin; Vincent Goëb

    2010-01-01

    Anne-Priscille Trouvin, Vincent GoëbDepartment of Rheumatology, Rouen University Hospital, Rouen, FranceAbstract: Bone remodeling requires a precise balance between resorption and formation. It is a complex process that involves numerous factors: hormones, growth factors, vitamins, and cytokines, and notably osteoprotegerin (OPG) and receptor activator for nuclear factor-κB (RANK) ligand. The signaling pathway OPG/RANK/RANKL is key to regulation for maintaining the balance ...

  17. Receptor activator of nuclear factor-κB ligand and osteoprotegerin: maintaining the balance to prevent bone loss

    OpenAIRE

    Trouvin, Anne-Priscille; Goëb, Vincent

    2010-01-01

    Bone remodeling requires a precise balance between resorption and formation. It is a complex process that involves numerous factors: hormones, growth factors, vitamins, and cytokines, and notably osteoprotegerin (OPG) and receptor activator for nuclear factor-κB (RANK) ligand. The signaling pathway OPG/RANK/RANKL is key to regulation for maintaining the balance between the activity of osteoblasts and osteoclasts in order to prevent bone loss and ensure a normal bone turnover. In this review, ...

  18. 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...

  19. 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.

  20. CM156, a Sigma Receptor Ligand, Reverses Cocaine-Induced Place Conditioning and Transcriptional Responses in the Brain

    OpenAIRE

    Xu, Yan-Tong; Robson, Matthew J.; Szeszel-Fedorowicz, Wioletta; Patel, Divyen; Rooney, Robert; Christopher R. McCurdy; Matsumoto, Rae R.

    2011-01-01

    Repeated exposure to cocaine induces neuroadaptations which contribute to the rewarding properties of cocaine. Using cocaine-induced conditioned place preference (CPP) as an animal model of reward, earlier studies have shown that sigma (σ) receptor ligands can attenuate the acquisition, expression and reactivation of CPP. However, the underlying molecular mechanisms that are associated with these changes are not yet understood. In the present study, CM156, a novel antagonist with high selecti...

  1. Poke Weed Mitogen Requires Toll-Like Receptor Ligands for Proliferative Activity in Human and Murine B Lymphocytes

    OpenAIRE

    Bekeredjian-Ding, Isabelle; Foermer, Sandra; Kirschning, Carsten J.; Parcina, Marijo; Heeg, Klaus

    2012-01-01

    Poke weed mitogen (PWM), a lectin purified from Phytolacca americana is frequently used as a B cell-specific stimulus to trigger proliferation and immunoglobulin secretion. In the present study we investigated the mechanisms underlying the B cell stimulatory capacity of PWM. Strikingly, we observed that highly purified PWM preparations failed to induce B cell proliferation. By contrast, commercially available PWM preparations with B cell activity contained Toll-like receptor (TLR) ligands suc...

  2. 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

  3. Identification, characterization, and developmental regulation of embryonic benzodiazepine binding sites

    International Nuclear Information System (INIS)

    We report the identification and characterization of 2 classes of benzodiazepine binding sites in the embryonic chick CNS. Binding was examined by competition and saturation binding experiments, using as radioligands 3H-flunitrazepam, a classical benzodiazepine anxiolytic, and 3H-Ro5-4864, a convulsant benzodiazepine. The results demonstrate that high-affinity (KD = 2.3 nM) 3H-flunitrazepam binding sites (site-A) are present by embryonic day 5 (Hamburger and Hamilton stage 27) and increase throughout development (Bmax = 0.3 and 1.3 pmol/mg protein in 7 and 20 d brain membranes, respectively). When 7 or 20 d brain membranes are photoaffinity-labeled with 3H-flunitrazepam and ultraviolet light, the radioactivity migrates as 2 bands on SDS-PAGE, consistent with Mrs of 48,000 and 51,000. GABA potentiates 3H-flunitrazepam binding at both 7 and 20 d of development, indicating that site-A is coupled to receptors for GABA early in development. Importantly, we have also identified a novel site (site-B) that binds classical benzodiazepine agonists with low affinity (micromolar) but displays high affinity for Ro5-4864 (KD = 41 nM). Site-B displays characteristics expected for a functional receptor, including stereospecificity and sensitivity to inactivation by heat and protease treatment. Saturation binding studies employing 3H-Ro5-4864 indicate that the levels of site-B are similar in 7 and 20 d brain (ca. 2.5 pmol/mg protein). The function of site-B is not known, but its preponderance in 7 d brain, relative to site-A, suggests that it might be important during early embryonic development

  4. T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice.

    Science.gov (United States)

    VanSeggelen, Heather; Hammill, Joanne A; Dvorkin-Gheva, Anna; Tantalo, Daniela G M; Kwiecien, Jacek M; Denisova, Galina F; Rabinovich, Brian; Wan, Yonghong; Bramson, Jonathan L

    2015-10-01

    Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution. PMID:26122933

  5. Forced Homo- and Heterodimerization of All gp130-Type Receptor Complexes Leads to Constitutive Ligand-independent Signaling and Cytokine-independent Growth

    OpenAIRE

    Suthaus, Jan; Tillmann, Anna; Lorenzen, Inken; Bulanova, Elena; Rose-John, Stefan; Scheller, Jürgen

    2010-01-01

    Naturally ligand independent constitutively active gp130 variants were described to be responsible for inflammatory hepatocellular adenomas. Recently, we genetically engineered a ligand-independent constitutively active gp130 variant based on homodimerization of Jun leucine zippers. Because also heterodimeric complexes within the gp130 family may have tumorigenic potential, we seek to generate ligand-independent constitutively active heterodimers for all known gp130-receptor complexes based o...

  6. Benzodiazepines and postoperative cognitive dysfunction in the elderly

    DEFF Research Database (Denmark)

    Rasmussen, L.S.; Steentoft, Anni; Rasmussen, H.; Kristensen, P.A.; Møller, J.T.

    1999-01-01

    hypnotics benzodiazepine,diazepam,age factor,anaesthesia,geriatric,psychological responses,postoperative......hypnotics benzodiazepine,diazepam,age factor,anaesthesia,geriatric,psychological responses,postoperative...

  7. Acyl-CoA esters antagonize the effects of ligands on peroxisome proliferator-activated receptor alpha conformation, DNA binding, and interaction with Co-factors

    DEFF Research Database (Denmark)

    Elholm, M; Dam, I; Jorgensen, C;

    2001-01-01

    The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARalpha. Here we demonstrate that S-hexadecyl-CoA, a nonhydrolyzable...

  8. 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

  9. 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

  10. 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...

  11. 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...

  12. 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.

  13. Ligand similarity guided receptor selection enhances docking accuracy and recall for non-nucleoside HIV reverse transcriptase inhibitors.

    Science.gov (United States)

    Stanton, Richard A; Nettles, James H; Schinazi, Raymond F

    2015-11-01

    Non-nucleoside reverse transcriptase inhibitors (NNRTI) are allosteric inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a viral polymerase essential to infection. Despite the availability of >150 NNRTI-bound RT crystal structures, rational design of new NNRTI remains challenging because of the variability of their induced fit, hydrophobic binding patterns. Docking NNRTI yields inconsistent results that vary markedly depending on the receptor structure used, as only 27% of the >20k cross-docking calculations we performed using known NNRTI were accurate. In order to determine if a hospitable receptor for docking could be selected a priori, we evaluated more than 40 chemical descriptors for their ability to pre-select a best receptor for NNRTI cross-docking. The receptor selection was based on similarity scores between the bound- and target-ligands generated by each descriptor. The top descriptors were able to double the probability of cross-docking accuracy over random receptor selection. Additionally, recall of known NNRTI from a large library of similar decoys was increased using the same approach. The results demonstrate the utility of pre-selecting receptors when docking into difficult targets. Graphical Abstract Cross-docking accuracy increases when using chemical descriptors to determine the NNRTI with maximum similarity to the new compound and then docking into its respective receptor. PMID:26450349

  14. Structure-function relationships of peptides forming the calcin family of ryanodine receptor ligands.

    Science.gov (United States)

    Xiao, Liang; Gurrola, Georgina B; Zhang, Jing; Valdivia, Carmen R; SanMartin, Mario; Zamudio, Fernando Z; Zhang, Liming; Possani, Lourival D; Valdivia, Héctor H

    2016-05-01

    Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure-function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [(3)H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin > urocalcin. All calcins significantly augment the bell-shaped [Ca(2+)]-[(3)H]ryanodine binding curve with variable effects on the affinity constants for Ca(2+) activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca(2+) release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca(2+) release and deplete Ca(2+) load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency. PMID:27114612

  15. Serine Lipids of Porphyromonas gingivalis Are Human and Mouse Toll-Like Receptor 2 Ligands

    Science.gov (United States)

    Clark, Robert B.; Cervantes, Jorge L.; Maciejewski, Mark W.; Farrokhi, Vahid; Nemati, Reza; Yao, Xudong; Anstadt, Emily; Fujiwara, Mai; Wright, Kyle T.; Riddle, Caroline; La Vake, Carson J.; Salazar, Juan C.; Finegold, Sydney

    2013-01-01

    The total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2−/−) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2−/− mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate. PMID:23836823

  16. 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

  17. 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

  18. Relationship between Structure and Conformational Change of the Vitamin D Receptor Ligand Binding Domain in 1α,25-Dihydroxyvitamin D3 Signaling

    OpenAIRE

    Lin-Yan Wan; Yan-Qiong Zhang; Meng-Di Chen; You-Qin Du; Chang-Bai Liu; Jiang-Feng Wu

    2015-01-01

    Vitamin D Receptor (VDR) belongs to the nuclear receptor (NR) superfamily. Whereas the structure of the ligand binding domain (LBD) of VDR has been determined in great detail, the role of its amino acid residues in stabilizing the structure and ligand triggering conformational change is still under debate. There are 13 α-helices and one β-sheet in the VDR LBD and they form a three-layer sandwich structure stabilized by 10 residues. Thirty-six amino acid residues line the ligand binding pocket...

  19. 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...

  20. 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.