RESUMO
Salvinorin A (1), from the sage Salvia divinorum, is a potent and selective kappa opioid receptor (KOR) agonist. We screened other salvinorins and derivatives for binding affinity and functional activity at opioid receptors. Our results suggest that the methyl ester and furan ring are required for activity but that the lactone and ketone functionalities are not. Other salvinorins showed negligible binding affinity at the KOR. None of the compounds bound to mu or delta opioid receptors.
Assuntos
Diterpenos/síntese química , Receptores Opioides kappa/agonistas , Animais , Diterpenos/química , Diterpenos/farmacologia , Diterpenos Clerodânicos , Ensaio Radioligante , Ratos , Relação Estrutura-AtividadeRESUMO
5-HT(2A) serotonin receptors represent the principal molecular targets for LSD-like hallucinogens and atypical antipsychotic drugs. It has been proposed that a dysregulation of 5-HT(2A) receptor-mediated signaling may contribute to the pathogenesis of schizophrenia and related diseases. A major mechanism for the attenuation of GPCR signaling following agonist activation typically involves the phosphorylation of serine and/or threonine residues by various kinases. Ser/Thr phosphorylation leads to the binding of accessory proteins and the uncoupling of the G proteins, thereby preventing further signaling. The molecular mechanisms by which 5-HT(2A) receptors are desensitized are unknown, and to date, no residues essential for agonist-mediated desensitization have been identified. Thus, we mutated, individually or in groups, all of the 37 serines and threonines in the cytoplasmic domains of the 5-HT(2A) receptor and assessed the effects of these mutations on agonist-mediated desensitization. We discovered that mutation of two residues, S421 in the C-terminal tail and S188 in the second intracellular loop, to alanine resulted in a significant block of agonist-induced desensitization. Intriguingly, a single-nucleotide polymorphism, of unreported frequency, at the S421 locus has been reported (S421F); the S421F mutation, like the S421A mutation, significantly attenuated agonist-mediated desensitization. Taken together, these findings indicate that the process of agonist-mediated desensitization of 5-HT(2A) receptors requires the presence of two nonconserved serine residues located in distinct intracellular loops.
Assuntos
Receptores de Serotonina/metabolismo , Serina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Dados de Sequência Molecular , Ésteres de Forbol/química , Ésteres de Forbol/farmacologia , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Quipazina/farmacologia , Ratos , Receptor 5-HT2A de Serotonina , Receptores de Serotonina/química , Receptores de Serotonina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/genética , Serotonina/farmacologia , Treonina/genética , Treonina/metabolismo , TransfecçãoRESUMO
Ephedrine is a long-studied stimulant available both as a prescription and over-the-counter medication, as well as an ingredient in widely marketed herbal preparations, and is also used as a precursor for the illicit synthesis of methamphetamine. Ephedrine is related to phenylpropanolamine, a decongestant removed from the market place due to concerns that its use increased the risk of hemorrhagic stroke. Standard pharmacology texts emphasize that ephedrine is both a direct and indirect adrenergic agonist, activating adrenergic receptors both by direct agonist activity as well as by releasing norepinephrine via a carrier-mediated exchange mechanism. Chemically, ephedrine possesses two chiral centers. In the present study, we characterized the stereoisomers of ephedrine and the closely related compounds pseudoephedrine, norephedrine, pseudonorephedrine (cathine), methcathinone, and cathinone at biogenic amine transporters and a large battery of cloned human receptors (e.g., "receptorome"). The most potent actions of ephedrine-type compounds were as substrates of the norepinephrine transporter (EC50 values of about 50 nM) followed by substrate activity at the dopamine transporter. Screening the receptorome demonstrated weak affinity at alpha2-adrenergic and 5-hydroxytryptamine7 receptors (Ki values 1-10 microM) and no significant activity at beta-adrenergic or alpha1-adrenergic receptors. Viewed collectively, these data indicate that the pharmacological effects of ephedrine-like phenylpropanolamines are likely mediated by norepinephrine release, and although sharing mechanistic similarities with, they differ in important respects from those of the phenylpropanonamines methcathinone and cathinone and the phenyisopropylamines methamphetamine and amphetamine.
Assuntos
Aminas Biogênicas/metabolismo , Efedrina/farmacologia , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso , Simportadores/metabolismo , Adrenérgicos/farmacologia , Alcaloides/química , Alcaloides/farmacologia , Animais , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Efedrina/química , Humanos , Masculino , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Simportadores/efeitos dos fármacosRESUMO
The serotonin (5-hydroxytryptamine) 2A receptor (5-HT2A) is an important G protein-coupled receptor (GPCR) that mediates the effects of hallucinogens and is the target of a number of commonly prescribed medications including atypical antipsychotics, antidepressants, and anxiolytics. The 5-HT2A receptor possesses a canonical Type I PDZ-binding domain (X-Ser/Thr-X-Phi) at the carboxyl terminus and has been predicted, but never demonstrated, to interact with PDZ domain-containing proteins. We discovered that PSD-95, a prototypic PDZ domain-containing protein, directly associates with the 5-HT2A receptor and regulates 5-HT2A receptor-mediated signaling and trafficking in HEK-293 cells. Co-immunoprecipitation studies revealed that the native 5-HT2A receptor, but not a mutant lacking the PDZ-binding domain, interacted directly with PSD-95. The association with PSD-95 enhanced 5-HT2A receptor-mediated signal transduction, a novel action of PSD-95 on GPCRs. The augmentation of 5-HT2A receptor signaling by PSD-95 was not accompanied by alteration in the kinetics of 5-HT2A receptor desensitization but was associated with the inhibition of agonist-induced 5-HT2A receptor internalization. Additional studies demonstrated that 5-HT2A receptor and PSD-95 were co-localized in clusters on the cell surface of HEK-293 cells. Taken together, the present work elucidates novel roles for PSD-95 in regulating the functional activity and intracellular trafficking of 5-HT2A receptors and possibly other GPCRs.