Identification of novel functionally selective κ-opioid receptor scaffolds.

The κ-opioid receptor (KOR)-dynorphin system has been implicated in the control of affect, cognition, and motivation, and is thought to be dysregulated in mood and psychotic disorders, as well as in various phases of opioid dependence. KOR agonists exhibit analgesic effects, although the adverse effects produced by some KOR agonists, including sedation, dysphoria, and hallucinations, have limited their clinical use. Interestingly, KOR-mediated dysphoria, assessed in rodents as aversion, has recently been attributed to the activation of the p38 mitogen-activated protein kinase pathway following arrestin recruitment to the activated KOR. Therefore, KOR-selective G protein-biased agonists, which do not recruit arrestin, have been proposed to be more effective analgesics, without the adverse effects triggered by the arrestin pathway. As an initial step toward identifying novel biased KOR agonists, we applied a multifaceted screening strategy utilizing both in silico and parallel screening approaches. We identified several KOR-selective ligand scaffolds with a range of signaling bias in vitro. The arylacetamide-based scaffold includes both G protein- and ß-arrestin-biased ligands, while the endogenous peptides and the diterpene scaffolds are G protein biased. Interestingly, we found scaffold screening to be more successful than library screening in identifying biased ligands. Many of the identified functionally selective ligands are potent selective KOR agonists that are reported to be active in the central nervous system. They therefore represent excellent candidates for in vivo studies aiming at determining the behavioral effects mediated by specific KOR-mediated signaling cascades.

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, 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.

Short synthesis of a novel class of salvinorin A analogs with hemiacetalic structure.

Novel semisynthetic analogs of salvinorin A, a full agonist having extraordinary affinity as well as selectivity for the κ-opioid receptor (KOR), were obtained in good yields. The derivatives are remarkable for their unusual and unique hemiacetal structure in the salvinorin series of compounds. The formation of the hemiacetal occurs with epimerization at C-12, thus preserving the original configuration of salvinorin A. The dimethyl ester derivative of the hemiacetal was found to have an affinity for both KOR and MOR (µ-opioid receptor).

Convenient synthesis and in vitro pharmacological activity of 2-thioanalogs of salvinorins A and B.

To study drug-receptor interactions, new thio-derivatives of salvinorin A, an extremely potent natural kappa-opioid receptor (KOR) agonist, were synthesized. Obtained compounds were examined for receptor binding affinity. Analogs with the same configuration at carbon atom C-2 as in natural salvinorin A showed higher affinity to KOR than their corresponding epimers.