Salvinorin A, the most potent naturally-occurring hallucinogen, has gained great attention since the κ-opioid receptor (KOR) was identified as its principal molecular target (1). However, the molecular mechanisms by which salvinorin A, a small-molecule agonist, binds to and activates KOR was unclear. To understand these mechanisms, three aims were proposed for my dissertation research; correspondingly, I will report our findings in three parts (Chapter 3, Chapter 4 and Chapter 5) in this dissertation.
The primary goal (Chapter 3) is to identify the binding site of salvinorin A in KOR. A combination of site-directed mutagenesis and molecular modeling was applied to determine the structural features of KOR essential for the binding of Salvinorin A (2). Meanwhile, a series of naturally-occurring and synthetic salvinorin A derivatives was designed and assayed to compare their binding and functional properties (3-6).
The subsequent goal (Chapter 4) is to investigate KOR's conformational change during the activation process. In this part of the dissertation research, over-expression of Gα16 and Gαi2 were used to increase the coupling ratio between KOR and the Gα subunits (7). The substituted cysteine accessibility method (SCAM), utilizing the specific reaction between the thiolate groups (-S-) and 2-aminoethylmethanethiosulfonate (MTSEA), was applied to detect the conformational changes of the receptor (7). Intriguingly, these G protein-dependent conformational changes significantly increased the binding affinity of salvinorin A.
In PART III (Chapter 5), our goal is to further verify ligand-receptor interactions by designing a series of ligands capable of covalently binding to KOR. From our earlier work using the SCAM approach, we demonstrated that C3157.38 was both water accessible and highly reactive to methanethiosulfonate (MTS) reagents (7). Thus far, two compounds RB-48 and RB-64 (both with pM potency and extraordinary selectivity for KOR) have emerged as being suitable for affinity-labeling KOR. Our preliminary mass spectrometry data was consistent with C3157.38 as the labeling site.
Collectively, this research project has revealed the molecular mechanisms by which a small-molecule agonist selectively binds to and activates a Class A GPCR.