Anil Purohit

Constitutive Activity of G-Protein Coupled Receptors: Emphasis on Serotonin Receptors

Several lines of evidence indicate that G-protein coupled receptors (GPCR) may exist in a state that allows a tonic level of stimulation in vivo (constitutive activity). Several native forms of GPCR, when expressed in recombinant cell lines, display significant signal transduction stimulation in the absence of activating ligand. Many GPCR, including three serotonin receptors, display robust constitutive activation upon the mutation of a single amino acid, indicating mutations producing inappropriate constitutive activation may be etiological factors in diseases. If constitutive activity of GPCR is as common a phenomenon as some researchers suspect, this would suggest significant alterations in the classical model of ligand-receptor interactions. One of the most significant implications of constitutive activity for pharmacologists and medicinal chemists, is the possibility of developing drugs that lower the level of constitutive activity. Such compounds have been termed inverse agonists . These drugs, in theory, would have different physiological effects, and therefore possibly different therapeutic potential, than classical competitive receptor antagonists ( neutral antagonists ). Theoretical issues concerning constitutive activity in the GPCR family and some of the evidence supporting the existence of constitutive activity in the GPCR family is reviewed. Studies are presented demonstrating the procedures for producing and characterizing constitutive activated forms of serotonin receptors, including the demonstration of inverse agonist activity of drugs on these receptors.

Arylguanidine and arylbiguanide binding at 5-HT3 serotonin receptors: a QSAR study.

For a series of monosubstituted arylguanidines, 5-HT3 receptor affinity was found generally related to the electron withdrawing nature of the substituent at the aryl 3-position and the lipophilicity of the 4-position substituent. A broader examination of 35 arylguanidines and arylbiguanides revealed that affinity could be described by molecular polarizability, a Chi index term (8chiP), and the sum of all (-Cl) E-State values (SsCl) in the molecule.

γ-Carbolines: binding at 5-HT5A serotonin receptors

Abstract Screening of various agents resulted in the identification of 5-methyl-1,2,3,4-tetrahydro-γ-carboline ( 1 ; K i =5,300 nM) as a compound with modest affinity for mouse 5-HT 5A receptors. Structure–affinity studies were conducted resulting in 5-methyl-2-[3-(4-fluorophenoxy)propyl]-1,2,3,4-tetrahydro-γ-carboline ( 17 ; K i =13 nM). Although 17 also binds at 5-HT 2 receptors, it serves as a novel lead for the further development of 5-HT 5A ligands.

Stable expression of constitutively activated mutant h5HT6 and h5HT7 serotonin receptors: inverse agonist activity of antipsychotic drugs

RationaleIn order to determine the possible relationship between antipsychotic drug properties and inverse agonist activity at h5HT6 and h5HT7 receptors, constitutively activated forms of these receptors were created by site-specific mutagenesis. Typical and atypical antipsychotic drugs were assayed for their potencies as inverse agonists at these mutated receptors.ObjectivesStable cell lines expressing constitutively activated forms of the h5HT6 and h5HT7 receptors were created. Typical and atypical antipsychotic drugs demonstrating high to moderate affinities for the h5HT6 and h5HT7 receptors were assayed for their potencies in reversing the agonist-independent activity (inverse agonist activity).ResultsThe E322R h5HT6 mutant and the S267K h5HT7 mutant displayed sufficiently robust agonist-independent activity when expressed in stable cell lines to allow the detailed, concentration-dependent, investigation of the inverse agonist activity of typical and atypical antipsychotic drugs. All the drugs tested displayed inverse agonist activity at both the activated h5HT6 and h5HT7 receptors. Native forms of these receptors did not display any constitutive activity. Interestingly, atypical antipsychotic drugs displayed potent inverse agonist activity, relative to typical antipsychotic drugs, at the h5HT7 receptor. LSD displayed neutral antagonist properties at the mutant h5HT7 receptor.ConclusionsSite-specific mutations in the third intracellular loop of the Gs-coupled h5HT6 and h5HT7 receptors produce constitutive activation. Antipsychotic drugs display inverse agonist activity at the activated receptors. The inverse agonist mechanism-of-action of the atypical antipsychotic drugs at the h5HT7 receptors may be different from the typical antipsychotic drugs as these drugs displayed far higher potencies as inverse agonists at the h5HT7 receptor.

Conformationally-restricted analogues and partition coefficients of the 5-HT3 serotonin receptor ligands meta-chlorophenylbiguanide (mCPBG) and meta-chlorophenylguanidine (mCPG).

The present investigation examined two features of arylbiguanide and arylguanidine 5-HT(3) ligands: conformation and partition coefficients. Several conformationally-constrained analogues of mCPBG (2) and mCPG (11; K(i)=32 nM) were prepared and of these only 2-amino-5-chloro-3,4-dihydroquinazoline (14; K(i)=34 nM) retained high affinity. The partition coefficient of compound 11 (LogP(app)=-0.64) was less than that of its corresponding arylbiguanide 2 (LogP(app)=-0.38). The quinazoline structure may represent a pharmacologically-active conformation of these agents, and the arylbiguanides were found more lipid soluble than their arylguanidine counterparts at physiological pH.

Constitutive Activity of Brain Serotonin Receptors: Inverse Agonist Activity of Antipsychotic Drugs

Several lines of evidence indicate that G-protein coupled receptors (GPCR) may exist in a state that allows a tonic level of stimulation in vivo (constitutive activity). Several native forms of GPCR, when expressed in recombinant cell lines, display significant signal transduction stimulation in the absence of activating ligand. Many GPCR, including four serotonin receptors, display robust constitutive activation upon the mutation of a single amino acid, indicating mutations producing inappropriate constitutive activation may be etiological factors in diseases. If constitutive activity of GPCR is as common a phenomenon as some researchers suspect, this would suggest significant alterations in the classical model of ligand-receptor interactions. One of the most significant implications of constitutive activity for pharmacologists and medicinal chemists, is the possibility of developing drugs that lower the level of constitutive activity. Such compounds have been termed “inverse agonists”. These drugs, in theory, would have different physiological effects, and therefore possibly different therapeutic potential, than classical competitive receptor antagonists (“neutral antagonists”). In this review, theoretical issues concerning constitutive activity in the GPCR family and evidence supporting the existence of constitutively active GPCR are discussed. Data demonstrating the activation of human 5-HT 2A, 5-HT2C, 5-HT6, and 5-HT7 receptors by single amino acid substitutions are presented. These studies demonstrate the procedures for producing and characterizing constitutively active forms of serotonin receptors, including the demonstration of inverse agonist activity of drugs on these receptors.

Formaldehyde-fixed semen is suitable and safer for leukocyte detection and DNA amplification**Supported by the Faulkner Institute For Reproductive Medicine, Boston, Massachusetts and in part by grant CA53899-03, National Institutes of Health, Bethesda, Maryland.

This pilot study indicates the use of formalin-fixation may offer a significant advance in the cytologic and pathological evaluation of semen. We are reporting this novel method to stimulate further assessment of this safer approach to semen analysis. The immunohistochemistry and PCR methods described here may also prove useful in improving the safety of studies of pathogen-infected cells in other laboratory settings.