Mechanistic insights into DCPIB inhibition of VRAC: Electrostatic control and binding plasticity.

Abstract

The volume-regulated anion channel (VRAC), formed by LRRC8 heteromers, regulates cell volume and is a promising therapeutic target. DCPIB is the most widely used VRAC inhibitor, yet its binding mechanism remains poorly defined. Although structural studies suggested a role of residue R103 in LRRC8A in DCPIB binding, its functional relevance has been unclear. Here, we combined electrophysiology in Xenopus oocytes and LRRC8-/- HEK293T cells with MD simulations and rational design of DCPIB analogs. Mutations of R103 abolished ATP-dependent block of VRAC but did not affect inhibition by DCPIB or the related inhibitor carbenoxolone (CBX), indicating distinct binding mechanisms. Simulations revealed two DCPIB-binding modes: one involving R103 and another engaging K51 and hydrophobic residues. In R103F mutants, DCPIB retained binding via π-stacking and alternative electrostatic contacts, explaining its persistent inhibition. Attempts to disrupt these interactions by mutating K51 led to nonfunctional channels. To further probe the role of electrostatic groups in the binding of DCPIB to VRAC, we synthesized derivatives varying charge and hydrophobicity. Functional assays and steered MD showed that analogs retaining the carboxylate group maintain high-affinity binding, whereas esterified forms exhibit rapid unbinding and reduced potency. Biphenyl substitutions increased binding energy but imposed steric penalties, suggesting a trade-off between affinity and accessibility. Our findings reveal an adaptable binding mechanism for DCPIB, contrasting with described lipid-displacement mechanisms for other VRAC inhibitors, and establish electrostatic complementarity as the primary determinant of high-affinity block. These insights provide a framework for rational design of next-generation VRAC inhibitors with improved specificity.