RESUMO
The structural basis to salvinorin A recognition of the kappa-opioid receptor is evaluated using a combination of site-directed mutagenesis and molecular-modeling techniques. The results show that salvinorin A recognizes a collection of residues in transmembrane II and VII, including Q115, Y119, Y313, I316, and Y320. The mutation of one hydrophobic residue in particular, I316, was found to completely abolish salvinorin A binding. As expected, none of the residues in transmembrane III or VI commonly associated with opiate recognition (such as D138 or E297) appear to be required for ligand binding. On the basis of the results presented here and elsewhere, a binding site model is proposed that aligns salvinorin A vertically within a pocket spanning transmembrane II and VII, with the 2' substituent directed toward the extracellular domains. The model explains the role that hydrophobic contacts play in binding this lipophilic ligand and gives insight into the structural basis to the mu-opioid receptor selectivity of 2'-benzoyl salvinorin (herkinorin).
Assuntos
Diterpenos/química , Diterpenos/farmacologia , Receptores Opioides kappa/efeitos dos fármacos , Animais , Sítios de Ligação , Diterpenos Clerodânicos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Camundongos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase/métodos , Ratos , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides delta/genética , Receptores Opioides kappa/química , Receptores Opioides kappa/genética , Receptores Opioides mu/efeitos dos fármacos , Receptores Opioides mu/genética , Relação Estrutura-AtividadeRESUMO
A series of xanomeline analogs were synthesized and evaluated for binding at the M(1) muscarinic acetylcholine receptor (M(1) receptor). Specifically, compounds that substitute the O-hexyl chain of xanomeline with polar, ionizable, or conformationally restricted moieties were assessed for their ability to bind to the M(1) receptor in a wash-resistant manner (persistent binding). From our screen, several novel ligands that persistently bind to the M(1) receptor with greater affinity than xanomeline were discovered. Results indicate that persistent binding may arise not only from hydrophobic interactions but also from ionic interactions with a secondary M(1) receptor binding site. Herein, a qualitative model that accounts for both binding scenarios is proposed and applied to understand the structural basis to wash-resistant binding and long-acting effects of xanomeline-based compounds.
Assuntos
Piridinas/síntese química , Piridinas/farmacologia , Receptor Muscarínico M1/antagonistas & inibidores , Tiadiazóis/síntese química , Tiadiazóis/farmacologia , Animais , Células CHO , Ácidos Carboxílicos/química , Cricetinae , Cricetulus , Concentração de Íons de Hidrogênio , Estrutura Molecular , Piridinas/química , Receptor Muscarínico M1/metabolismo , Relação Estrutura-Atividade , Tiadiazóis/químicaRESUMO
The cloning of the opioid receptors and subsequent use of recombinant DNA technology have led to many new insights into ligand binding. Instead of focusing on the structural features that lead to increased affinity and selectivity, researchers are now able to focus on why these features are important. Site-directed mutagenesis and chimeric data have often been at the forefront in answering these questions. Herein, we survey pharmacophores of several opioid ligands in an effort to understand the structural requirements for ligand binding and selectivity. Models are presented and compared to illustrate key sites of recognition for both opiate and nonopiate ligands. The results indicate that different ligand classes may recognize different sites within the receptor, suggesting that multiple epitopes may exist for ligand binding and selectivity.
Assuntos
Modelos Moleculares , Receptores Opioides/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Ligantes , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Receptores Opioides/química , Receptores Opioides/genética , Relação Estrutura-AtividadeRESUMO
Salvinorin A is a potent kappa opioid receptor (KOP) agonist with unique structural and pharmacological properties. This non-nitrogenous ligand lacks nearly all the structural features commonly associated with opioid ligand binding and selectivity. This study explores the structural basis to salvinorin A binding and selectivity using a combination of chimeric and single-point mutant opioid receptors. The experiments were designed based on previous models of salvinorin A that locate the ligand within a pocket formed by transmembrane (TM) II, VI, and VII. More traditional sites of opioid recognition were also explored, including the highly conserved aspartate in TM III (D138) and the KOP selectivity site E297, to determine the role, if any, that these residues play in binding and selectivity. The results indicate that salvinorin A recognizes a cluster of residues in TM II and VII, including Q115, Y119, Y312, Y313, and Y320. Based on the position of these residues within the receptor, and prior study on salvinorin A, a model is proposed that aligns the ligand vertically, between TM II and VII. In this orientation, the ligand spans residues that are spaced one to two turns down the face of the helices within the receptor cavity. The ligand is also in close proximity to EL-2 which, based on chimeric data, is proposed to play an indirect role in salvinorin A binding and selectivity.
