CoMFA analyses of C-2 position Salvinorin A analogs at the kappa-opioid receptor provides insights into epimer selectivity

The highly potent and kappa-opioid receptor (KOR)-selective hallucinogen salvinorin A and selected analogs have been analyzed using the 3D quantitative structure-affinity relationship technique Comparative Molecular Field Analysis (CoMFA) in an effort to derive a statistically significant and predictive model of salvinorin affinity at the KOR and to provide additional statistical support for the validity of previously proposed structure-based interaction models. Two CoMFA models of salvinorin A analogs substituted at the C-2 position are presented. Separate models were developed based on the radioligand used in the kappa-opioid binding assay, [3H]diprenorphine or [125I]6β-iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan ([125I]IOXY). For each dataset, three methods of alignment were employed: a receptor-docked alignment derived from the structure-based docking algorithm GOLD, another from the ligand-based alignment algorithm FlexS, and a rigid realignment of the poses from the receptor-docked alignment. The receptor-docked alignment produced statistically superior results compared to either the FlexS alignment or the realignment in both datasets. The [125I]IOXY set (Model 1) and [3H]diprenorphine set (Model 2) gave q2 values of 0.592 and 0.620, respectively, using the receptor-docked alignment, and both models produced similar CoMFA contour maps that reflected the stereoelectronic features of the receptor model from which they were derived. Each model gave significantly predictive CoMFA statistics (Model 1 PSET r2 = 0.833; Model 2 PSET r2 = 0.813). Based on the CoMFA contour maps, a binding mode was proposed for amine-containing salvinorin A analogs that provides a rationale for the observation that the β-epimers (R-configuration) of protonated amines at the C-2 position have a higher affinity than the corresponding β-epimers (S-configuration)

Salvinorin A: Fragment Synthesis and Modeling Studies

Salvinorin A is a non-nitrogenous, selective kappa opioid receptor agonist with potent hallucinogenic properties. Because Salvinorin A has no basic nitrogen, it does not readily adhere to the “message-address” concept of selectivity for the opioid receptors. Therefore, a better understanding of how salvinorin A and its analogs interact with the kappa opioid receptor may shed some light on how salvinorin A obtains its potency and selectivity. The structure-affinity relationships (SAFIR) of salvinorin A and its analogs along with a discussion of the selectivity of the opioid receptors, is presented. A fragment of salvinorin A, methyl-3-acetoxy-4-oxocyclohexanecarboxylate, was synthesized to determine if the B, C and D rings are or are not necessary for binding to the opioid receptors. The fragment was found not to bind to the kappa, delta or mu receptor which reinforces the importance of the B, C and D rings in the binding of salvinorin A to the kappa opioid receptor. Homology models of the kappa, delta and mu opioid receptors were constructed based on inactive bovine rhodopsin, light-activated bovine rhodopsin and the human beta-2 adrenergic receptors. The program MODELLER was also used to construct the kappa opioid receptor. Two comparative molecular field analysis (CoMFA) studies are then presented which compared three different types of alignment methods. The alignment methods employed included a receptor-docked alignment in which the salvinorin A analogs were docked into a model of the kappa opioid receptor using the program GOLD. The docked poses for this alignment were chosen based on their similarity to our postulated model of salvinorin A in the kappa opioid receptor. In our model the furan oxygen forms hydrogen bonds with Q115(2.60) and Y320(7.43), the methoxy oxygen of the C-4 position ester group may form a hydrogen bond with Y312(7.35) and the methyl group of the C-2 position acetoxy moiety forms a hydrophobic interaction with Y313(7.36). These interactions are consistent with mutagenesis studies. The other alignment methods employed were a FlexS alignment and a realignment of the receptor-docked poses using the Fit Atoms function within SYBYL. Only the receptor-docked alignment method resulted in robust and predictive CoMFA models which indicates that the analogs may bind to the kappa opioid receptor in a similar but non-identical way. In addition, information from the CoMFA models based on the receptor-docked alignment led to a postulated binding mode for a set of amine analogs of salvinorin A which were not part of the original data set. Docking studies have the positively charged C-2 position amine group interacting with E209(XL2.49) while the furan oxygen and C-4 position ester group interacts with the same residues as in our model of salvinorin A in the kappa opioid receptor. The studies presented here not only support our postulated model of salvinorin A binding to the kappa opioid receptor but may also explain the trend of the beta epimers of the amine analogs to have a higher affinity than the corresponding alpha epimers. Site-directed mutagenesis studies could provide data to support or refute the postulated models of the amines docked in the kappa opioid receptor presented here