TRPV2 is the least studied member of the vanilloid TRP subfamily despite its emerging relevance in cancer metastasis, pain, and inflammation. Although several small-molecule TRPV2 modulators have been reported, including the natural products piperlongumine (PL) and cannabidiol, all lack selectivity, complicating the interpretation of phenotypic readouts and functional insights into the role of the channel in health and disease. Here, we report a series of PL-based derivatives rationally designed to maintain TRPV2 antagonism while eliminating covalent off-target activity associated with the electrophilic groups present in PL. Using electrophysiological and calcium fluorescence imaging assays in HEK293T cells and DRG nociceptors, we identified HKC54 as the most potent TRPV2 antagonist to date (IC50 = 0.4 μM), displaying ∼50-fold selectivity over TRPV1 and ∼70-fold selectivity over TRPA1. Cellular thermal shift assays demonstrated direct TRPV2 engagement, and molecular dynamics and docking studies suggest a near-identical binding mode of the derivatives to PL. To assess proteome-wide selectivity, we pursued an unbiased chemoproteomic strategy and developed photoaffinity probes derived from PL and noncovalent derivative HKC22. Whereas the PL-based probe labeled many established covalent and noncovalent PL targets (e.g., GSTP1, GSTO1, STAT3, and KEAP1), no off-targets were detected for HKC22, suggesting high selectivity for TRPV2. Finally, PL derivatives inhibited cancer cell migration in vitro and suppressed metastasis in vivo, underscoring the therapeutic potential of selective TRPV2 antagonists.
Kiely-Collins et al. (Wed,) studied this question.