Identification of Druggable Binding Sites and Small Molecules as Modulators of TMC1

The sensory hair cells of the inner ear use the mechano-electrical transducer (MET) channels to convert the mechanical stimuli from sound or acceleration into electrical signals, allowing us to perceive sound and maintain balance. The pore-forming subunit of the MET channel is formed by Transmembrane channel-like (TMC) 1 and 2 proteins, which are nonselective cationic channels that also allow the permeation of ototoxic aminoglycosides and cisplatin into hair cells. In search for otoprotective compounds, numerous molecules have been reported to block and modulate the properties of the MET channels. One of them, the styryl fluorescent probe FM1-43, and its analogue AM1-43, are commonly used to evaluate MET channel functionality. However, the mechanism of interaction of these modulators with the TMCs remains largely unknown. In this work, we implemented both computational and experimental approaches to identify novel TMC1 modulators using in silico 3D-pharmacophore approach and free energy binding calculations. Our 3D-pharmacophore contains the structural features necessary for ligands to bind and modulate the activity of TMC1. It consists of two aromatic groups, one acceptor group, and at least one protonatable amine. The pipeline we implemented successfully identified several novel TMC1 compound modulators, which reduced dye uptake in cultured cochlear explants, indicating MET modulation activity. Our molecular docking and MM-GBSA experiments allowed us to identify three potential drug binding sites within the TMC1 pore. Furthermore, our study also supports the ligand-binding relationship between the TMC and TMEM16 proteins, providing novel insights suggesting that these proteins share common 3D-pharmacophoric features for their inhibition.