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

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.

Figure 1. Molecular modeling reveals potential sites of adduct formation at KOR

(A) The C-2 position of salvinorin A is in close proximity to Y313^7.36 and C315^7.38 in the wt KOR. Ribbons (white) indicate the position of the backbone, and a Connolly channel surface (green) describes the regions of steric accessibility within the activated receptor. An enlarged region of accessibility in the intracellular portion of the helical bundle is indicative of an activated GPCR. (B) The RB-64 thiocyanate group is in close proximity to C315^7.38. (C) The mechanisms for covalent-labeling of cysteine involve nucleophilic substitution at C-22 or the adjacent S atom. The molecular weight change for the modified peptides is 431 (i) or 463 (ii) depending on the site of substitution.

Figure 2. A comparison of the binding modes of Salvinorin A and the +431 and +463 adducts

The color of the backbone ribbons and displayed carbon atoms identify the receptor-ligand complex. A) wt KOR: Salvinorin A (white); +431 adduct (green); +463 adduct (magenta). B) F314^7.37C-C315^7.38S KOR double mutant: +431 adduct (cyan); +463 adduct (orange). C) all receptor-ligand complexes displayed in the same frame of reference. See text for details.

Figure 3. RB-64 induces wash-resistant inhibition of KOR binding at C315

(A) Dose-response curve for RB-64 for wash-resistant inhibition of binding at 4 °C (2 h) yielding an EC₅₀ of 1.2 µM. (B) Time-course study at 4 °C with a maximally-effective dose of RB-64 (10 µM) yielding t_1/2 = 0.4 h. (C) Cells expressing KOR were exposed for 10 µM/3 h to RB-64, salvinorin A, naloxone and vehicle (DMSO only). After incubation, the cell membranes were extensively washed (at least three times); the residual binding was determined by [³H]diprenorphine saturation binding. Data presented are B_max values for 2–4 independent experiments. (D) B_max values of various mutants after being labeled with RB-64, salvinorin A and naloxone. B_max values were expressed as a percentage of vehicle control. Each bar represents the mean ± SEM for two to four independent experiments. An asterisk (*) indicates that RB-64 labeling was significantly different (p < 0.01) from the reference (vehicle) by ANOVA. A hash mark (#) indicates that RB-64 labeling was significantly different (p < 0.05) than that of naloxone by ANOVA.

Figure 4. Fragmentation pathways for salvinorin A, RB-48 and RB-64

(A) Deduced structures of the major identified fragments. (B) MS/MS spectra for salvinorin A, RB-48 and RB-64 (from top to bottom), whose precursor ions are 433, 467 and 490 m/z respectively.

Figure 5. Identification of adduct at C315 via model KOR peptides

The MS spectra of unlabeled (A) and RB-64-labeled (B) peptide Ac-YFCIALGY-Na, MS/MS spectrum with 1475.5 m/z is shown in (C), which contains several major fragments from the RB-64 modified peptide.

Figure 6. RB-64 is a potent psychotomimetic agent in vivo

(A) Startle responses to the 120-db stimulus for animals administered different doses of salvinorin A or RB-64. A hash mark (#) indicates p < 0.05 in comparisons of startle responses to 0.5 mg/kg salvinorin A or 0.01 mg/kg RB-64. (B) Percent PPI to the 4-, 8-, 12-, and 16-db prepulses for animals given various doses of salvinorin A. (C) Percent PPI to the 4-, 8-, 12-, and 16-db prepulses for animals given various doses of RB-64. N = 10–16 mice/treatment. (D) Percent PPI to the 4-, 8-, 12-, and 16-db prepulses for animals administered 2 mg/kg salvinorin A or 10mg/kg Nor-BNI followed by 2 mg/kg salvinorin A. N = 6 mice/treatment. (E) Percent PPI to the 4-, 8-, 12-, and 16-db prepulses for animals administered 0.1 mg/kg RB-64 or 10mg/kg Nor-BNI followed by 0.1 mg/kg RB-64. N = 6 mice/treatment. An asterisk (*) indicates p < 0.05 compared to vehicle controls; a plus sign (+) indicates p < 0.05 in comparisons of prepulse-dependent PPI at the 4-, 8-, 12-, and 16-dB prepulses within a single treatment.