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TRPC1/4/5 channels are non-specific cation channels implicated in a wide variety of diseases, and TRPC1/4/5 inhibitors have recently entered the first clinical trials. However, fundamental and translational studies require a better understanding of TRPC1/4/5 channel regulation by endogenous and exogenous factors. Although several potent and selective TRPC1/4/5 modulators have been reported, the paucity of mechanistic insights into their modes-of-action remains a barrier to the development of new chemical probes and drug candidates. The xanthine class of modulators includes the most potent and selective TRPC1/4/5 inhibitors described to date, as well as TRPC5 activators. Our previous studies suggest that xanthines interact with a, so far, elusive pocket of TRPC1/4/5 channels that is essential to channel gating. Targeting this pocket may be a promising strategy for TRPC1/4/5 drug discovery. Here we report the first structure of a small molecule-bound TRPC1/4/5 channel – human TRPC5 in complex with the xanthine Pico145 – to 3.0 Å. We found that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. Our findings explain the mode-of-action of xanthine-based TRPC1/4/5 modulators, and suggest a structural basis for TRPC1/4/5 modulation by endogenous factors such as (phospho)lipids and Zn2+ ions. These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.

Several types of drug-resistant epileptic encephalopathies of infancy have been associated with mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel subunit KNa1.1. These mutations are commonly gain-of-function, increasing channel activity, therefore inhibition by drugs is proposed as a stratified approach to treat disorders. To date, quinidine therapy has been trialled with several patients, but mostly with unsuccessful outcomes, which has been linked to its low potency and lack of specificity. Here we describe the use of a cryo-electron microscopy-derived KNa1.1 structure and mutational analysis to identify the quinidine biding site and identified novel inhibitors that target this site using computational methods. We describe six compounds that inhibit KNa1.1 channels with low- and sub-micromolar potencies, likely through binding in the intracellular pore vestibule. In preliminary hERG inhibition and cytotoxicity assays, two compounds showed little effect. These compounds may provide starting points for the development of novel pharmacophores for KNa1.1 inhibition, with the view to treating KCNT1-associated epilepsy and, with their potencies higher than quinidine, could become key tool compounds to further study this channel. Furthermore, this study illustrates the potential for utilising cryo-electron microscopy in ion channel drug discovery.