Understanding the mode-of-action of TRPC4/5 ion channel inhibitors TRP channels are non-selective cation channels that are sensitive to a wide variety of chemical and physical stimuli, such as temperature, physical force, toxic metal ions and dietary chemicals. For example, capsaicin and noxious heat activate TRPV1 channels, while menthol and noxious cold activate TRPM8 channels. TRP channels all consist of tetramers of TRP proteins. Our research focuses on the closely related TRPC4 and TRPC5 channels, including heteromeric TRPC1/C4 and TRPC1/C5 channels, which are emerging as potential therapeutic targets for the treatment of anxiety disorders,1,2 renal cancer3 and cardiovascular inflammation.4,5 However, target validation studies and drug discovery of these channels is impeded by the lack of potent and channel-specific inhibitors. Yet the lack of understanding of the mode-of-action of small molecule inhibitors of TRPC4/5 currently prevents rational design of such inhibitors. In this project, we use a combination of chemical, biochemical and biophysical approaches to identify the mode-of-action of small molecule TRPC4/5 inhibitors. We initially determined structure-activity relationships and selectivity of TRPC4/5 inhibitors against a panel of ion channels – by calcium imaging and (automated) electrophysiology – which allowed us to select two promising series of TRPC4/5 inhibitors for more detailed studies. First, we identified natural and synthetic flavonols as potent and selective TRPC4/5 inhibitors.6 The most potent flavonols inhibited TRPC4/5 in excised membrane patches, and did not perturb the integrity of model membranes, which suggests they inhibit the TRPC4/5 channels through a direct mechanism such as a well-defined binding site. Second, we used structure-activity relations of a newly identified picomolar TRPC4/5 inhibitor, which shows selectivity for heteromeric TRPC1/C4 and TRPC1/C5 channels, to develop chemical probes incorporating diazirine photocrosslinkers and alkyne handles. These probes retained activity against TRPC4/5 channels and are currently being used in chemical proteomics experiments to profile cellular (off-)targets and identify an inhibitor binding site of TRPC4/5 channels. Key references: (1) A. Riccio et al., Cell, 2009, 137, 761–772. (2) L. Yang et al., PLoS One, 2015, 1–15. (3) Y. Akbulut et al., Angew. Chem., Int. Ed., 2015, 54, 3787–3791. (4) A. Alzoubi et al., Am. J. Pathol., 2013, 183, 1779–1788. (5) M. Francis et al., Am. J. Pathol., 2016, 186, 1701–1709. (6) J. Naylor et al,. Br. J. Pharmacol., 2016, 173, 562–574. .