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.
Yan
Feng
F
Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Bikbulatov
Ruslan V
RV
Mocanu
Viorel
V
Dicheva
Nedyalka
N
Parker
Carol E
CE
Wetsel
William C
WC
Mosier
Philip D
PD
Westkaemper
Richard B
RB
Allen
John A
JA
Zjawiony
Jordan K
JK
Roth
Bryan L
BL
D002460:Cell Line; D045785:Diterpenes, Clerodane / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015335:Molecular Probes; D015394:Molecular Structure; D016296:Mutagenesis; D017473:Receptors, Opioid, kappa / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics
C573301:22-thiocyanatosalvinorin A; D045785:Diterpenes, Clerodane; D015335:Molecular Probes; D017473:Receptors, Opioid, kappa; C090499:salvinorin A