BPS2026 – Dynamic analysis of ion channel gating using BRET: Unveiling temporal signatures for drug discovery

Ion channels are crucial drug targets for various therapeutic applications, but their high-throughput screening (HTS) remains challenging and expensive. To address this, we developed innovative bioluminescence resonance energy transfer (BRET) probes to monitor ligand-gated and polymodally activated ion channels in real-time within live cells. These tools enable the dynamic assessment of ion channel conformational changes, protein-protein interactions, trafficking, and subnanometric Ca 2+ and K + concentrations near the channel pore. Using BRET probes targeting TRPV1 conformational changes and coupling to calmodulin, we screened the Prestwick library of 1280 FDA/EMA-approved compounds. This multiparametric analysis revealed the potential of BRET assays in drug discovery campaigns. Building on these results, we recently designed BRET probes to measure selective Ca 2+ and/or K + flux dynamics in the TRPV1 and P2X4/7 nanoenvironment. These new assays identified transient, compound-specific ion flux patterns, revealing four distinct temporal signatures. By evaluating compound-induced responses immediately post-injection, we uncovered key temporal profiles amenable to mathematical modeling. These signatures may offer predictive value for identifying modulators of ligand-gated ion channels. Our findings highlight the importance of temporal kinetics in ion channel drug screening, presenting a refined strategy to enhance precision, reduce costs, and accelerate the development of novel therapeutics.