The Design And Use Of Covalently Bound Agonists To Probe Kappa-Opioid Receptors

Salvinorin A, the most potent naturally occuring hallucinogen, has gained great attention since the Kappa‐Opioid Receptor (KOR) was identified as its principal molecular target (Roth et al, PNAS 2002). Previously, extensive efforts were made to understand how salvinorin A binds to and activates KOR. Our goal here was to design a series of ligands capable of covalently binding to KOR to further explore the ligand‐receptor interactions at atomic level. From substituted cysteine accessibility methods (SCAM) we knew that C315(7.38) is water accessible and highly reactive to methanethiosulfonate (MTS) reagents, therefore C315(7.38) could provide an anchoring point for proximity‐accelerated chemical coupling reaction. We designed a series of covalent binding ligands of variable size and electrophilic properties. Thus far, two compounds–RB‐48 and RB‐64–were the most promising candidates after extensive molecular pharmacology examinations. They showed high affinity, high efficacy and irreversible binding properties. We are currently using these ligands along with mass spectrometry to identify the anchoring residue(s). This study will facilitate our goals to elucidate the structure and function of KOR, a family A GPCR, which is involved in analgesia, pain, mood and reward. To our knowledge, this is the first design and application of site‐directed chemical coupling ligands to probe KOR.