Structure-Based Design, Synthesis, and Biochemical and Pharmacological Characterization of Novel Salvinorin A Analogues as Active State Probes of the κ-Opioid Receptor

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• 作者：Feng Yan ; Ruslan V. Bikbulatov ; Viorel Mocanu ; Nedyalka Dicheva ; Carol E. Parker ; William C. Wetsel ; Philip D. Mosier ; Richard B. Westkaemper ; John A. Allen ; Jordan K. Zjawiony ; Bryan L. Roth
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：July 28, 2009
• 年：2009
• 卷：48
• 期：29
• 页码：6898-6908
• 全文大小：1089K
• 年卷期：v.48,no.29(July 28, 2009)
• ISSN：1520-4995

文摘

Salvinorin A, the most potent naturally occurring hallucinogen, has attracted an increasing amount of attention since the κ-opioid receptor (KOR) was identified as its principal molecular target by us [Roth, B. L., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11934−11939]. Here we report the design, synthesis, and biochemical characterization of novel, irreversible, salvinorin A-derived ligands suitable as active state probes of the KOR. On the basis of prior substituted cysteine accessibility and molecular modeling studies, C315^7.38 was chosen as a potential anchoring point for covalent labeling of salvinorin A-derived ligands. Automated docking of a series of potential covalently bound ligands suggested that either a haloacetate moiety or other similar electrophilic groups could irreversibly bind with C315^7.38. 22-Thiocyanatosalvinorin A (RB-64) and 22-chlorosalvinorin A (RB-48) were both found to be extraordinarily potent and selective KOR agonists in vitro and in vivo. As predicted on the basis of molecular modeling studies, RB-64 induced wash-resistant inhibition of binding with a strict requirement for a free cysteine in or near the binding pocket. Mass spectrometry (MS) studies utilizing synthetic KOR peptides and RB-64 supported the hypothesis that the anchoring residue was C315^7.38 and suggested one biochemical mechanism for covalent binding. These studies provide direct evidence of the presence of a free cysteine in the agonist-bound state of the KOR and provide novel insights into the mechanism by which salvinorin A binds to and activates the KOR.