Potent Ca_V3.2 channel inhibitors exert analgesic effects in acute and chronic pain models

Pain is the most common presenting physical symptom and a primary reason for seeking medical care, which chronically affects people's mental health and social life. Ca_V3.2 channel plays an essential role in the peripheral processing maintenance of pain states. This study was designed to identify novel drug candidates targeting the Ca_V3.2 channel. Whole-cell patch-clamp, cellular thermal shift assay, FlexStation, in vivo and in vitro Ca_V3.2 knock-down, site-directed mutagenesis, and double-mutant cycle analysis were employed to explore the pain-related receptors and ligand-receptor direct interaction. We found that toddaculin efficiently inhibits the Ca_V3.2 channel and significantly reduced the excitability of dorsal root ganglion neurons and pain behaviors. The Carbonyl group of coumarins directly interacts with the pore domain of Ca_V3.2 via van der Waals (VDW) force. Docking with binding pockets further led us to identify glycycoumarin, which exhibited more potent inhibition on the Ca_V3.2 channel and better analgesic activity than the parent compound. Toddaculin and its analog showed beneficial therapeutic effects in pain models. Toddaculin binding pocket on Ca_V3.2 might be a promising docking site for the design of drugs.