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

Salvinorin A, the most potent naturally occurring hallucinogen, targets the kappa-opioid receptor (KOR). Researchers designed and synthesized novel irreversible salvinorin A-derived ligands, RB-64 and RB-48, as active state probes of KOR. Based on molecular modeling, they targeted cysteine residue C315(7.38) for covalent binding. Both compounds were extraordinarily potent and selective KOR agonists in vitro and in vivo. RB-64 showed wash-resistant inhibition of binding requiring a free cysteine near the binding pocket. Mass spectrometry confirmed C315(7.38) as the anchoring residue and suggested a biochemical mechanism for covalent binding. These findings provide direct evidence of a free cysteine in the agonist-bound KOR state and insights into salvinorin A's binding and activation mechanism.

Study at a glance

Abstract

Salvinorin A, the most potent naturally occurring hallucinogen, has attracted an increasing amount of attention since the kappa-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.