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1.
Biochem Pharmacol ; 76(9): 1134-41, 2008 Oct 30.
Article in English | MEDLINE | ID: mdl-18761325

ABSTRACT

Peptides with agonist activity at the vasopressin V(2) receptor are used clinically to treat fluid homeostasis disorders such as polyuria and central diabetes insipidus. Of these peptides, the most commonly used is desmopressin, which displays poor bioavailability as well as potent activity at the V(1b) receptor, with possible stress-related adverse effects. Thus, there is a strong need for the development of small molecule chemistries with selective V(2) receptor agonist activity. Using the functional cell-based assay Receptor Selection and Amplification Technology (R-SAT((R))), a screening effort identified three small molecule chemotypes (AC-94544, AC-88324, and AC-110484) with selective agonist activity at the V(2) receptor. One of these compounds, AC-94544, displayed over 180-fold selectivity at the V(2) receptor compared to related vasopressin and oxytocin receptors and no activity at 28 other G protein-coupled receptors (GPCRs). All three compounds also showed partial agonist activity at the V(2) receptor in a cAMP accumulation assay. In addition, in a rat model of central diabetes insipidus, AC-94544 was able to significantly reduce urine output in a dose-dependent manner. Thus, AC-94544, AC-88324, and AC-110484 represent novel opportunities for the treatment of disorders associated with V(2) receptor agonist deficiency.


Subject(s)
Pharmaceutical Preparations/chemical synthesis , Pharmaceutical Preparations/metabolism , Receptors, Vasopressin/agonists , Receptors, Vasopressin/metabolism , Animals , Antidiuretic Agents/administration & dosage , Antidiuretic Agents/chemical synthesis , Deamino Arginine Vasopressin/administration & dosage , Deamino Arginine Vasopressin/chemistry , Deamino Arginine Vasopressin/metabolism , Deamino Arginine Vasopressin/therapeutic use , Diabetes Insipidus/prevention & control , Diabetes Insipidus/urine , Dose-Response Relationship, Drug , Humans , Male , Mice , NIH 3T3 Cells , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacology , Peptides/therapeutic use , Pharmaceutical Preparations/administration & dosage , Rats , Rats, Brattleboro , Vasopressins/deficiency , Vasopressins/genetics , Vasopressins/metabolism , Vasopressins/therapeutic use
2.
Eur J Pharmacol ; 592(1-3): 158-9, 2008 Sep 11.
Article in English | MEDLINE | ID: mdl-18644363

ABSTRACT

To understand the contribution of the estrogen receptor beta, the potent and selective agonist ERb-131 was evaluated in animal models of inflammatory pain. In paradigms of acute and persistent inflammatory pain, ERb-131 did not alleviate the nociception induced by either carrageenan or formalin. However, in the chronic complete Freund's adjuvant model, ERb-131 resolved both inflammatory and hyperalgesic components. Thus, ERb-131 is sufficient to alleviate chronic but not acute inflammatory pain states.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal , Estrogen Receptor beta/agonists , Inflammation/drug therapy , Pain/drug therapy , Acute Disease , Animals , Carrageenan , Chronic Disease , Formaldehyde , Freund's Adjuvant , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Inflammation/chemically induced , Inflammation/etiology , Male , Pain/chemically induced , Pain/etiology , Pain Measurement/drug effects , Rats , Rats, Sprague-Dawley
3.
Eur J Pharmacol ; 590(1-3): 423-9, 2008 Aug 20.
Article in English | MEDLINE | ID: mdl-18559275

ABSTRACT

The effects of estrogens on pain perception remain controversial. In animal models, both beneficial and detrimental effects of non-selective estrogens have been reported. ERb-131 a non-steroidal estrogen receptor beta ligand was evaluated in several pain animal models involving nerve injury or sensitization. Using functional and binding assays, ERb-131 was characterized as a potent and selective estrogen receptor beta agonist. In vivo, ERb-131 was devoid of estrogen receptor alpha activity as assessed in a rat uterotrophic assay. ERb-131 alleviated tactile hyperalgesia induced by capsaicin, and reversed tactile allodynia caused by spinal nerve ligation and various chemical insults. Moreover, ERb-131 did not influence the pain threshold of normal healthy animals. Thus, estrogen receptor beta agonism is a critical effector in attenuating a broad range of anti-nociceptive states.


Subject(s)
Estrogen Receptor beta/agonists , Pain/drug therapy , Peripheral Nervous System Diseases/drug therapy , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Estradiol/analogs & derivatives , Estradiol/pharmacology , Female , Fulvestrant , Humans , Male , Mice , Mice, Inbred BALB C , Pain Threshold/drug effects , Rats , Rats, Sprague-Dawley , Uterus/drug effects
4.
Mol Pharmacol ; 73(1): 94-103, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17959710

ABSTRACT

The aim of this study was to create and characterize constitutively active mutant (CAM) histamine H(1) receptors (H(1)R) using random mutagenesis methods to further investigate the activation process of the rhodopsin-like family of G protein-coupled receptors (GPCRs). This approach identified position 6.40 in TM 6 as a "hot spot" because mutation of Ile6.40(420) either to Glu, Gly, Ala, Arg, Lys, or Ser resulted in highly active CAM H(1)Rs, for which almost no histamine-induced receptor activation response could be detected. The highly conserved hydrophobic amino acid at position 6.40 defines, in a computational model of the H(1)R, the asparagine cage motif that restrains the side chain of Asn7.49 of the NPxxY motif toward transmembrane domain (TM 6) in the inactive state of the receptor. Mutation of the asparagine cage into Ala or Gly, removing the interfering bulky constraints, increases the constitutive activity of the receptor. The fact that the Ile6.40(420)Arg/Lys/Glu mutant receptors are highly active CAM H(1)Rs leads us to suggest that a positively charged residue, presumably the highly conserved Arg3.50 from the DRY motif, interacts in a direct or an indirect (through other side chains or/and internal water molecules) manner with the acidic Asp2.50..Asn7.49 pair for receptor activation.


Subject(s)
Asparagine/chemistry , Receptors, G-Protein-Coupled/metabolism , Receptors, Histamine H1/metabolism , Mutagenesis , Receptors, Histamine H1/chemistry , Receptors, Histamine H1/genetics
5.
Int J Neuropsychopharmacol ; 11(2): 163-71, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17708779

ABSTRACT

The mechanisms underlying the clinical properties of atypical antipsychotics have been postulated to be mediated, in part, by interactions with the 5-HT2A receptor. Recently, it has been recognized that clinically effective antipsychotic drugs are 5-HT2A receptor inverse agonists rather than neutral antagonists. In the present study, which is part of the clinical development of the novel, selective 5-HT2A receptor inverse agonist ACP-103, we applied positron emission tomography (PET) with the radioligand [11C]N-methylspiperone ([11C]NMSP) to study the relationship between oral dose, plasma level, and uptake of ACP-103 in living human brain. The safety of drug administration was also assessed. Four healthy volunteers were examined by PET at baseline, and after the oral administration of various single doses of ACP-103. Two subjects each received 1, 5, and 20 mg doses, and two subjects each received 2, 10, and 100 mg doses, respectively. ACP-103 was well tolerated. Detectable receptor binding was observed at very low ACP-103 serum levels. Cortical [11C]NMSP binding was found to be dose-dependent and fitted well to the law of mass action. A reduction in binding was detectable after an oral dose of ACP-103 as low as 1 mg, and reached near maximal displacement following the 10-20 mg dose. In conclusion, administration of ACP-103 to healthy volunteers was found to be safe and well tolerated, and single oral doses as low as 10 mg were found to fully saturate 5-HT2A receptors in human brain as determined by PET.


Subject(s)
Antipsychotic Agents/pharmacokinetics , Brain/diagnostic imaging , Brain/metabolism , Piperidines/pharmacokinetics , Positron-Emission Tomography , Receptor, Serotonin, 5-HT2A/metabolism , Urea/analogs & derivatives , Administration, Oral , Adult , Antipsychotic Agents/administration & dosage , Antipsychotic Agents/adverse effects , Binding, Competitive , Carbon Radioisotopes , Dose-Response Relationship, Drug , Drug Inverse Agonism , Humans , Male , Piperidines/administration & dosage , Piperidines/adverse effects , Radiopharmaceuticals/metabolism , Spiperone/analogs & derivatives , Spiperone/metabolism , Urea/administration & dosage , Urea/adverse effects , Urea/pharmacokinetics
6.
Mol Pharmacol ; 72(6): 1440-6, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17715395

ABSTRACT

A limited number of whole-cell assays allow monitoring of receptor tyrosine kinase (RTK) activity in a signaling pathway-specific manner. We present the general use of the bioluminescence resonance energy transfer (BRET) technology to quantitatively study the pharmacology and signaling properties of the receptor tyrosine kinase (RTK) superfamily. RTK BRET-2 assays monitor, in living cells, the specific interaction between RTKs and their effector proteins, which control the activation of specific downstream signaling pathways. A total of 22 BRET assays have been established for nine RTKs derived from four subfamilies [erythroblastic leukemia viral (v-erb-b) oncogene homolog (ErbB), platelet-derived growth factor (PDGF), neurotrophic tyrosine kinase receptor (TRK), vascular endothelial growth factor (VEGF)] monitoring the interactions with five effectors (Grb2, p85, Stat5a, Shc46, PLCgamma1). These interactions are dependent on the RTK kinase activity and autophosphorylation of specific tyrosine residues in the carboxyl terminus. RTK BRET assays are highly sensitive for quantifying ligand-independent (constitutive), agonist-induced, or antagonist-inhibited RTK activity levels. We studied the signaling properties of the PDGF receptor, alpha polypeptide (PDGFRA) isoforms (V561D; D842V and delta842-845) carrying activating mutations identified in gastrointestinal stromal tumors (GIST). All three PDGFRA isoforms are fully constitutively activated, insensitive to the growth factor PDGF-BB, but show differential sensitivity of their constitutive activity to be inhibited by the inhibitor imatinib (Gleevec). Epidermal growth factor receptor (EGFR) BRET structure-function studies identify the tyrosine residues 1068, 1114, and 1148 as the main residues mediating the interaction of EGFR with the adapter protein Grb2. The BRET technology provides an assay platform to study signaling pathway-specific RTK structure-function and will facilitate drug discovery efforts for the identification of novel RTK modulators.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Fluorescence Resonance Energy Transfer/methods , Luminescent Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Adaptor Proteins, Signal Transducing/analysis , Animals , Cell Line , Humans , Luminescent Proteins/analysis , Protein Binding/physiology , Receptor Protein-Tyrosine Kinases/analysis , Renilla
7.
J Pharmacol Exp Ther ; 322(2): 862-70, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17519387

ABSTRACT

Dopamine D(2) receptor antagonism contributes to the therapeutic action of antipsychotic drugs (APDs) but also produces undesirable side effects, including extrapyramidal motor deficits, cognitive dulling, and prolactinemia. The introduction of atypical APDs was a significant advancement in the treatment of schizophrenia. Whereas these agents are D(2) receptor antagonists, they are also potent 5-hydroxytryptamine (5-HT)(2A) receptor inverse agonists, a feature that may explain their improved efficacy and tolerability. Recently, we reported that N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl) carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103), a novel selective 5-HT(2A) receptor inverse agonist that fails to bind D(2) receptors, is active in several models predictive of antipsychotic activity. Using ACP-103, we tested the hypothesis that combining high levels of 5-HT(2A) inverse agonism with low levels of D(2) antagonism would result in a favorable interaction, such that antipsychotic efficacy could be achieved with reduced D(2) receptor-related adverse effects. Here we show that ACP-103 1) potently inhibited head-twitching produced by the 5-HT(2A/2C) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine, 2) increased the potency of haloperidol against amphetamine-induced hyperactivity, 3) interacted synergistically with haloperidol or risperidone to suppress hyperactivity induced by the N-methyl-d-aspartate receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and, by contrast, 4) attenuated haloperido-l- or risperidone-induced prolactinemia. ACP-103 also attenuated catalepsy produced by haloperidol or risperidone. However, the doses that were required for this effect were higher than would be expected for a 5-HT(2A) receptor-mediated mechanism. These data indicate that utilizing ACP-103 as an adjunctive therapy to currently used APDs may result in enhanced antipsychotic efficacy while reducing adverse effects including those attributable to D(2) receptor antagonism.


Subject(s)
Haloperidol/pharmacology , Motor Activity/drug effects , Piperidines/pharmacology , Risperidone/pharmacology , Serotonin 5-HT2 Receptor Agonists , Urea/analogs & derivatives , Amphetamine/pharmacology , Amphetamines/pharmacology , Animals , Antipsychotic Agents/pharmacology , Antipsychotic Agents/toxicity , Behavior, Animal/drug effects , Brain Chemistry , Catalepsy/chemically induced , Catalepsy/prevention & control , Dizocilpine Maleate/pharmacology , Dopamine D2 Receptor Antagonists , Dose-Response Relationship, Drug , Drug Interactions , Drug Synergism , Haloperidol/toxicity , Head Movements/drug effects , Male , Mice , Mice, Inbred Strains , Prolactin/blood , Rats , Rats, Sprague-Dawley , Risperidone/toxicity , Serotonin Receptor Agonists/pharmacology , Urea/pharmacology
8.
Mol Pharmacol ; 72(2): 380-6, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17475811

ABSTRACT

Using a high-throughput functional screen, the atypical L-type Ca2+ channel blocker diltiazem was discovered to be an agonist at the human ghrelin (GHSR1a) receptor. In cellular proliferation, Ca2+ mobilization, and bioluminescence resonance energy transfer (BRET-2) assays, diltiazem was a partial agonist at GHSR1a receptors, with 50 to 80% relative efficacy compared with the GHSR1a peptide agonist GHRP-6, and high nanomolar to low micromolar potency, depending upon the assay. Seven of the known primary metabolites of diltiazem were synthesized, and three of them (MA, M1, and M2) were more efficacious and/or more potent than diltiazem at GHSR1a receptors, with a rank order of agonist activity of M2 > M1 > MA > diltiazem, whereas M4 and M6 metabolites displayed weak agonist activity, and the M8 and M9 metabolites were inactive. Binding affinities of diltiazem and these metabolites to GHSR1a receptors followed a similar rank order. In vivo tests showed that diltiazem and M2 each stimulated growth hormone release in male Sprague-Dawley neonatal rats, although to a lesser degree than GHRP-6. Thus, diltiazem and chemical analogs of diltiazem represent a new class of GHSR1a receptor agonists. The possible contributions of GHSR1a receptor activation to the clinical actions of diltiazem are discussed in the context of the known beneficial cardiovascular effects of ghrelin.


Subject(s)
Calcium Channels, L-Type/drug effects , Diltiazem/pharmacology , Receptors, G-Protein-Coupled/agonists , Animals , Calcium/metabolism , Diltiazem/metabolism , Growth Hormone/metabolism , Humans , Luminescent Measurements , Male , Mice , NIH 3T3 Cells , Oligopeptides/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Ghrelin
9.
Mol Pharmacol ; 71(2): 508-18, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16968809

ABSTRACT

We have developed a new assay for measuring epidermal growth factor receptor (EGFR) activation using the bioluminescence resonance energy transfer (BRET) technology, which directly measures the recruitment of signaling proteins to activated EGFR. Our results demonstrate that EGFR BRET assays precisely measure the pharmacology and signaling properties of EGFR expressed in human embryonic kidney 293T cells. EGFR BRET assays are highly sensitive to known EGFR ligands [pEC50 of epidermal growth factor (EGF)=10.1+/-0.09], consistent with previous pharmacological methods for measuring EGFR activation. We applied EGFR BRET assays to study the characteristics of somatic EGFR mutations that were recently identified in lung cancer. In agreement with recent reports, we detected constitutively active mutant EGFR isoforms, which predominantly signal through the phosphatidylinositol-3-kinase/Akt pathway. The EGFR inhibitors Iressa or Tarceva are severalfold more potent in inhibiting constitutive activity of mutant EGFR isoforms compared with wild-type EGFR. Notable, our results reveal that most of the mutant EGFR isoforms tested were significantly impaired in their response to EGF. The highest level of constitutive activity and nearly complete loss of epidermal growth factor responsiveness was detected in isoforms that carry the activating mutation L858R and the secondary resistance mutation T790M. In summary, our study reveals that somatic mutations in EGFR quantitatively differ in pharmacology and signaling properties, which suggest the possibility of differential clinical responsiveness to treatment with EGFR inhibitors. Furthermore, we demonstrate that the EGFR BRET assays are a useful tool to study the pharmacology of ligand-induced interaction between EGFR and signaling pathway-specifying adapter proteins.


Subject(s)
ErbB Receptors/metabolism , Luminescent Measurements/methods , Signal Transduction , Cell Line , Drug Resistance/genetics , ErbB Receptors/analysis , ErbB Receptors/genetics , Fluorescence Resonance Energy Transfer , Humans , Luminescent Proteins , Lung Neoplasms/genetics , Mutation , Phosphatidylinositol 3-Kinases/metabolism , Protein Isoforms , Proto-Oncogene Proteins c-akt/metabolism
10.
Mol Pharmacol ; 70(6): 1974-83, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16959945

ABSTRACT

Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M(1) muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M(1). In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M(1) but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M(1) receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.


Subject(s)
Benzoxazines/pharmacology , Clozapine/analogs & derivatives , Clozapine/pharmacology , Muscarinic Agonists/pharmacology , Piperidines/pharmacology , Receptor, Muscarinic M1/agonists , Cell Line , Humans , Protein Conformation , Radioligand Assay , Receptor, Muscarinic M1/chemistry
11.
Curr Pharm Des ; 12(14): 1717-29, 2006.
Article in English | MEDLINE | ID: mdl-16712484

ABSTRACT

Chemical genomics is a drug discovery strategy that relies heavily on high-throughput screening (HTS) and therefore benefits from functional assay platforms that allow HTS against all relevant genomic targets. Receptor Selection and Amplification Technology (R-SAT) is a cell-based, high-throughput functional assay where the receptor stimulus is translated into a measurable cellular response through an extensive signaling cascade occurring over several days. The large biological and chronological separation of stimulus from response provides numerous opportunities for enabling assays and increasing assay sensitivity. Here we review strategies for building homogeneous assay platforms across large gene families by redirecting and/or amplifying signal transduction pathways.


Subject(s)
Genomics , Signal Transduction , Animals , Humans , Receptors, G-Protein-Coupled/metabolism
12.
J Pharmacol Exp Ther ; 317(2): 910-8, 2006 May.
Article in English | MEDLINE | ID: mdl-16469866

ABSTRACT

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)(2A) receptor inverse agonist ACP-103 competitively antagonized the binding of [(3)H]ketanserin to heterologously expressed human 5-HT(2A) receptors with a mean pK(i) of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC(50) of 8.7. ACP-103 demonstrated lesser affinity (mean pK(i) of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC(50) 7.1 in R-SAT) at human 5-HT(2C) receptors, and lacked affinity and functional activity at 5-HT(2B) receptors, dopamine D(2) receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT(2A) receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT(2A) receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.


Subject(s)
Behavior, Animal/drug effects , Piperidines/pharmacology , Serotonin 5-HT2 Receptor Antagonists , Serotonin Antagonists/pharmacology , Urea/analogs & derivatives , Animals , Biological Availability , Cloning, Molecular , Humans , Male , Mice , NIH 3T3 Cells , Piperidines/pharmacokinetics , Radioligand Assay , Rats , Rats, Sprague-Dawley , Serotonin Antagonists/pharmacokinetics , Urea/pharmacokinetics , Urea/pharmacology
13.
Br J Pharmacol ; 147(1): 73-82, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16284629

ABSTRACT

Recently, a large family of G-protein-coupled receptors called Mas-related genes (Mrgs), which is selectively expressed in small-diameter sensory neurons of dorsal root ganglia, was described. A subgroup of human Mrg receptors (MrgX1-X4) is not found in rodents and this has hampered efforts to define the physiological roles of these receptors. MrgX receptors were cloned from rhesus monkey and functionally characterized alongside their human orthologs. Most of the human and rhesus MrgX receptors displayed high constitutive activity in a cellular proliferation assay. Proliferative responses mediated by human or rhesus MrgX1, or rhesus MrgX2 were partially blocked by pertussis toxin (PTX). Proliferative responses mediated by rhesus MrgX3 and both human and rhesus MrgX4 were PTX insensitive. These results indicate that human and rhesus MrgX1 and MrgX2 receptors activate both Gq- and Gi-regulated pathways, while MrgX3 and MrgX4 receptors primarily stimulate Gq-regulated pathways. Peptides known to activate human MrgX1 and MrgX2 receptors activated the corresponding rhesus receptors in cellular proliferation assays, Ca(2+)-mobilization assays, and GTP-gammaS-binding assays. Cortistatin-14 was selective for human and rhesus MrgX2 receptors over human and rhesus MrgX1 receptors. BAM22 and related peptides strongly activated human MrgX1 receptors, but weakly activated rhesus MrgX1, human MrgX2, and rhesus MrgX2 receptors. These data suggest that the rhesus monkey may be a suitable animal model for exploring the physiological roles of the MrgX receptors.


Subject(s)
Macaca mulatta/genetics , Multigene Family , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Transcription Factors/chemistry , Transcription Factors/genetics , Amino Acid Sequence , Animals , Conserved Sequence , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , Proto-Oncogene Mas , Proto-Oncogene Proteins/physiology , Receptors, G-Protein-Coupled/physiology , Transcription Factors/physiology
14.
Biochem Pharmacol ; 71(1-2): 156-62, 2005 Dec 19.
Article in English | MEDLINE | ID: mdl-16303118

ABSTRACT

Drugs targeting retinoid receptors have been developed to treat a variety of therapeutic indications, but their success has been limited in part due to lack of selectivity. A novel functional cell-based assay R-SATtrade mark was employed to screen a small molecule chemical library and identify a variety of novel RAR agonists with various subtype selectivities, including RARbeta/gamma and RARgamma selective agonists. A novel class of synthetic compounds that distinguishes between the different RARbeta isoforms is described. This pharmacophore displays anti-proliferative activity and induces differentiation in a neuronal cell line, consistent with a classical retinoid mechanism of action while providing unique subtype selectivity. These novel subtype selective RAR agonists could serve as powerful tools to probe into subtype and isoform-specific retinoid function.


Subject(s)
Receptors, Retinoic Acid/agonists , Animals , Cell Line , Cell Proliferation , Humans , Mice , Neurites
15.
J Med Chem ; 48(24): 7517-9, 2005 Dec 01.
Article in English | MEDLINE | ID: mdl-16302793

ABSTRACT

4'-Octyl-4-biphenylcarboxylic acid (1g, AC-55649) was identified as a highly isoform-selective agonist at the human RARbeta2 receptor in a functional intact cell-based screening assay. The subsequent hit to lead optimization transformed the lipophilic, poorly soluble hit into a more potent and orally available compound (2, AC-261066) with retained beta2 selectivity and greatly improved physiochemical properties. Being an isoform-selective RARbeta2 receptor agonist that discriminates between nuclear receptor isoforms having identical ligand binding domains, 2 will be useful as a pharmacological research tool but also a valuable starting point for drug development.


Subject(s)
Benzoates/chemical synthesis , Biphenyl Compounds/chemical synthesis , Receptors, Retinoic Acid/agonists , Thiazoles/chemical synthesis , Administration, Oral , Animals , Benzoates/chemistry , Benzoates/pharmacology , Binding Sites , Biological Availability , Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Cells, Cultured , Humans , Models, Molecular , Protein Isoforms/agonists , Protein Structure, Tertiary , Rats , Solubility , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacology , Transcription, Genetic
16.
Am J Pharmacogenomics ; 4(2): 119-28, 2004.
Article in English | MEDLINE | ID: mdl-15059034

ABSTRACT

BACKGROUND AND OBJECTIVES: A number of recent studies surveying single nucleotide polymorphisms within the exonic regions of human genes have revealed a significant number of such variants, including many non-synonymous variants. This highlights the need to directly identify, within individual clinically well-defined patients, those variants that alter protein function as well as structure. We report on the development of a novel phenotypic screening process that combines high-throughput molecular cloning techniques with functional expression utilizing the cell-based assay R-SAT. METHODS: We applied the phenotypic screening process to an analysis of the m1 muscarinic acetylcholine receptor (CHRM1) gene in a cohort of 74 individuals, including 48 diagnosed with neurodegenerative disease, primarily Alzheimer disease, who have been stratified according to their clinical response to the acetylcholinesterase inhibitor donepezil. Phenotypic screening of the CHRM1 gene involved PCR-based amplification from genomic DNA and heterologous expression in mammalian cells. RESULTS: Phenotypic screening yielded functional responses to the agonist carbachol displaying a mean potency (-pEC(50)+/- standard deviation) of 5.8 +/- 0.2, which did not differ from that observed with expression of the wild-type receptor gene (6.0 +/- 0.3). No altered levels of constitutive receptor activity were observed. Dideoxy sequencing did not reveal any non-synonymous variants in the coding exon of this gene within this clinical cohort, while detecting three synonymous variants. CONCLUSION: The results confirm that the m1 receptor gene (CHRM1) is not highly polymorphic in the human population, suggesting that genetic variation within the coding exon of this gene is not a contributing factor to the clinical variability observed during treatment of dementia with cholinergic enhancement therapies.


Subject(s)
Dementia/drug therapy , Dementia/genetics , Drug Delivery Systems , Neurodegenerative Diseases/drug therapy , Neurodegenerative Diseases/genetics , Receptor, Muscarinic M1/drug effects , Receptor, Muscarinic M1/genetics , Aged , Cholinesterase Inhibitors/therapeutic use , Cohort Studies , DNA/analysis , DNA/genetics , Donepezil , Female , Genetic Testing , Humans , Indans/therapeutic use , Male , Nootropic Agents/therapeutic use , Phenotype , Piperidines/therapeutic use , Polymorphism, Genetic/genetics , Receptors, Muscarinic/genetics , Reverse Transcriptase Polymerase Chain Reaction
17.
J Pharmacol Exp Ther ; 310(3): 943-51, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15102927

ABSTRACT

The primary purpose of the present series of experiments was to characterize the in vitro and in vivo pharmacology profile of 2-(4-methoxy-phenyl)-N-(4-methyl-benzyl)-N-(1-methyl-piperidin-4-yl)-acetamide hydrochloride (AC-90179), a selective serotonin (5-HT2A) receptor inverse agonist, in comparison with the antipsychotics haloperidol and clozapine. The secondary purpose was to characterize the pharmacokinetic profile of AC-90179. Like all atypical antipsychotics, AC-90179 shows high potency as an inverse agonist and competitive antagonist at 5HT2A receptors. In addition, AC-90179 exhibits antagonism at 5HT2C receptors. In contrast, AC-90179 does not have significant potency for D2 and H1 receptors that have been implicated in the dose-limiting side effects of other antipsychotic drugs. The ability of AC-90179 to block 5-HT2A receptor signaling in vivo was demonstrated by its blockade of the rate-decreasing effects of the 5-HT2A agonist, (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride, under a fixed ratio schedule of reinforcement. Similar to clozapine and haloperidol, AC-90179 attenuated phencyclidine-induced hyperactivity. Although haloperidol impaired acquisition of a simple autoshaped response and induced cataleptic-like effects at behaviorally efficacious doses, AC-90179 and clozapine did not. Furthermore, unlike haloperidol and clozapine, AC-90179 did not decrease spontaneous locomotor behavior at efficacious doses. Limited oral bioavailability of AC-90179 likely reflects rapid metabolism rather than poor absorption. Taken together, a compound with a similar pharmacological profile as AC-90179 and with increased oral bioavailability may have potential for the treatment of psychosis.


Subject(s)
Benzamides/pharmacology , Piperidines/pharmacology , Serotonin 5-HT2 Receptor Antagonists , Serotonin Antagonists/pharmacology , 3T3 Cells , Animals , Benzamides/adverse effects , Benzamides/blood , Biological Availability , Brain/metabolism , Caco-2 Cells , Catalepsy/chemically induced , Cell Membrane Permeability/drug effects , Female , Humans , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver/drug effects , Motor Activity/drug effects , Nose/drug effects , Piperidines/adverse effects , Piperidines/blood , Radioligand Assay , Rats , Rats, Wistar , Serotonin Antagonists/adverse effects , Serotonin Antagonists/blood
18.
Biochem Pharmacol ; 67(3): 479-90, 2004 Feb 01.
Article in English | MEDLINE | ID: mdl-15037200

ABSTRACT

The 5-HT1A receptor is a critical mediator of serotonergic (5-HT) function. We have identified 13 potential single nucleotide polymorphisms resulting in amino acid changes throughout the human 5-HT1A receptor. The pharmacological profiles of these 13 polymorphic variants were then characterized using a high-throughput assay based on ligand-dependent transformation of NIH/3T3 cells. The majority of the polymorphic variants displayed wild-type pharmacological profiles in response to a panel of well-established agonists at the 5-HT1A receptor. However, the A50V polymorphic variant, which had an alanine to valine substitution in transmembrane 1, exhibited a loss of detectable response to 5-HT. Interestingly, all other agonists tested, including buspirone, lisuride, and (+)8-OH-DPAT, exhibited efficacies similar to that of the wild-type receptor. The competitive antagonist, methiothepin, also displayed a 19-fold decrease in potency at the A50V variant receptor. However, both 5-HT and methiothepin were able to compete for [3H]WAY-100635 binding to the A50V variant with affinities similar to the wild-type receptor. Moreover, the Bmax of [3H]WAY-100635 binding was 14-fold lower for the A50V variant than for the wild-type receptor. Thus, the A50V receptor variant exhibited ligand-specific functional alterations in addition to lower expression levels. These data suggest a previously unappreciated role for transmembrane 1 in mediating 5-HT response at the 5-HT1A receptor. Furthermore, individuals that potentially harbor the A50V polymorphism might display aberrant affective behaviors and altered responses to drugs targeting the 5-HT1A receptor.


Subject(s)
Polymorphism, Genetic , Receptor, Serotonin, 5-HT1A/metabolism , 3T3 Cells , Animals , COS Cells , Cells, Cultured , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Mice , Mutagenesis, Site-Directed , Radioligand Assay , Receptor, Serotonin, 5-HT1A/drug effects , Receptor, Serotonin, 5-HT1A/genetics , Serotonin/metabolism , Serotonin Antagonists/pharmacology , Sulfur Radioisotopes
19.
Biochem Pharmacol ; 67(7): 1279-84, 2004 Apr 01.
Article in English | MEDLINE | ID: mdl-15013843

ABSTRACT

Many naturally occurring peptides exhibit a high degree of promiscuity across G-protein coupled receptor subtypes. The degree to which this phenomenon occurs, and its physiological significance is not well characterized. In addition, many 'orphan' peptides exist for which there are no known receptors. Therefore, to identify novel interactions between biologically active peptides and G-protein coupled receptors, a library of nearly 200 peptides was screened against the human calcitonin (hCTr), human Parathyroid Hormone (PTH1R), human Corticotropin Releasing Factor (CRF1), and the human Glucagon-like peptide (GLP1) receptors using a cell-based functional assay (Receptor Selection and Amplification Technology). Functional profiling revealed that the 'orphan peptide' PHM-27 selectively activated the hCTr; no activity was observed at the PTH1, CRF1, or GLP1 receptors. PHM-27 was a potent agonist at the hCTr, with similar efficacy as human calcitonin, and a potency of 11 nM. These results were confirmed in cyclic AMP assays. Responses to calcitonin and PHM-27 could be suppressed by the antagonist salmon calcitonin (8-32). In competition binding studies, salmon calcitonin (8-32), calcitonin, and PHM-27 were each able to inhibit (125)I-calcitonin from cell membranes containing transiently expressed hCTr. These results indicate that the orphan peptide PHM-27 is a potent agonist at the hCTr.


Subject(s)
Peptide PHI/pharmacology , Receptors, Calcitonin/agonists , 3T3 Cells , Amino Acid Sequence , Animals , Binding, Competitive , Cells, Cultured , Cyclic AMP/metabolism , Humans , Mice , Molecular Sequence Data , Receptors, Calcitonin/metabolism
20.
Mol Pharmacol ; 65(3): 770-7, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14978256

ABSTRACT

The angiotensin II type 1 (AT1) receptor is the primary effector for angiotensin II (Ang II), a key peptide regulator of blood pressure and fluid homeostasis. AT1 receptors are involved in the pathogenesis of several cardiovascular diseases, including hypertension, cardiac hypertrophy, and congestive heart failure, which are characterized by significant interindividual variation in disease risk, progression, and response to pharmacotherapy. Such variation could arise from genomic polymorphisms in the AT1 receptor. To pursue this notion, we have pharmacologically characterized seven known and putative nonsynonymous AT1 receptor variants. Functional analysis using the cell-based assay receptor selection and amplification technology (R-SAT) revealed that three variants (AT1-G45R, AT1-F204S, and AT1-C289W) displayed altered responses to Ang II and other AT1 receptor agonists and antagonists. Agonist responses to Ang II were absent for AT1-G45R and significantly reduced in potency for AT1-C289W (11-fold) and AT1-F204S (57-fold) compared with the wild-type (WT) receptor. AT1-F204S also displayed reduced relative efficacy (57%). Quantitatively similar results were obtained in two additional functional assays, phosphatidyl inositol hydrolysis and extracellular signal-regulated kinase activation. Radioligand binding studies revealed that AT1-G45R failed to bind Ang II, whereas cell surface staining clearly showed that it trafficked to the cell surface. AT1-C289W and AT1-F204S displayed reduced binding affinities of 3- and 5-fold and reduced cell surface expression of 43 and 60% of that observed for the WT receptor, respectively. These data demonstrate that polymorphic variation in the human AT1 receptor induces loss of functional phenotypes, which may constitute the molecular basis of variability of AT1 receptor-mediated physiological responses.


Subject(s)
Angiotensin II/metabolism , Polymorphism, Genetic , Receptor, Angiotensin, Type 1/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Genetic Variation , Humans , Molecular Sequence Data , Protein Conformation , Receptor, Angiotensin, Type 1/genetics
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