Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the κ-opioid receptor

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

Research output: Contribution to journalArticle

48 Scopus citations

Abstract

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, C3157.38 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 C3157.38. 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 C3157.38 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.

Original languageEnglish (US)
Pages (from-to)6898-6908
Number of pages11
JournalBiochemistry
Volume48
Issue number29
DOIs
StatePublished - Jul 28 2009

ASJC Scopus subject areas

  • Biochemistry

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    Yan, F., Bikbulatov, R. V., Mocanu, V., Dicheva, N., Parker, C. E., Wetsel, W. C., Mosier, P. D., Westkaemper, R. B., Allen, J. A., Zjawiony, J. K., & Roth, B. L. (2009). Structure-based design, synthesis, and biochemical and pharmacological characterization of novel salvinorin A analogues as active state probes of the κ-opioid receptor. Biochemistry, 48(29), 6898-6908. https://doi.org/10.1021/bi900605n