D-DARTS: an alternative method for NaV1.5 affinity molecules identification based on dual-drug affinity responsive target stability

Drug affinity responsive target stability (DARTS) is a powerful label-free technique for detecting target engagement by measuring the increased resistance of a protein to proteolytic degradation upon ligand binding. However, its application to multi-transmembrane channel proteins has been limited due to their intrinsic structural instability and general resistance to protease digestion. To address this challenge, we developed a novel strategy termed Dual-DARTS (D-DARTS), which employs controlled proteolysis in an SDS-containing denaturing buffer to evaluate a target protein's dual stability—namely, resistance to both chemical denaturation and enzymatic degradation. Using this approach, we successfully identified affinity ligands for the NaV1.5 channel, including both inhibitors and agonists. Notably, D-DARTS enabled the discovery of poneratoxin, a peptide derived from bullet ants, as a high-affinity binder to NaV1.5, a finding validated through electrophysiological assays. Preliminary molecular docking analyses suggested that poneratoxin binds to an epitope analogous to that of the alkaloid agonist BTXB. The general utility of the D-DARTS method was further corroborated by its successful application to the mitochondrial membrane protein VDAC1. By enabling target profiling based on binding affinity rather than functional activity, D-DARTS provides a complementary alternative to electrophysiology-based screening for NaV1.5 channel binders, with advantages including operational simplicity, cost-efficiency, and reduced reliance on specialized equipment. This strategy is anticipated to be applicable to target identification and active molecule screening for a wide range of channel proteins and other multi-transmembrane proteins.