Analgesic Buxus alkaloids with Enhanced Selectivity for the Low-Voltage-Gated Calcium Channel Cav 3.2 over Cav 3.1 through a New Binding Mode.

Low-voltage-gated calcium channels (LVGCCs; Cav 3.1-3.3) represent promising drug targets for epilepsy, pain, and essential tremor. At present, modulators with heightened selectivity for a subtype of LVGCCs are still highly desired. In this study we explored three classes of Buxus alkaloids and identified 9(10/19)abeo-artanes Buxusemine H and Buxusemine L (BXSL) as an unprecedented type of Cav 3.2 inhibitors. Particularly, BXSL exhibited Cav 3.2 inhibition comparable to Z944, a non-subtype-selective LVGCCs inhibitor under clinical trial. While lacking specificity for Cav 3.3, BXSL showed a 30-fold selectivity of Cav 3.2 over Cav 3.1. As compared to several well-known inhibitors, the experimental and computational studies suggested BXSL exhibits a distinct binding mode to Cav 3.2, notably through the essential interaction with serine-1543 in domain III. Furthermore, BXSL showed minimal impact on various recombinant and native nociceptive ion channels, while significantly reducing the excitability of isolated mouse dorsal root ganglion neurons. Animal studies in wild-type and Cav 3.2 knock-out mice revealed that BXSL (5 mg/kg), by inhibiting Cav 3.2, exhibits an analgesic effect equivalent to Z944 (10 mg/kg) or mibefradil (10 mg/kg). Moreover, we proposed a structural rationale for the high selectivity of 9(10/19)abeo-artane-type alkaloids towards Cav 3.2 over Cav 3.1. This study introduces a novel analgesic agent and valuable molecular insight for structure-based innovative Cav 3.2 drug development.