Thermal proteome profiling identifies the membrane-bound purinergic receptor P2X4 as a target of the autophagy inhibitor indophagolin.

Signaling pathways are frequently activated through signal-receiving membrane proteins, and the discovery of small molecules targeting these receptors may yield insights into their biology. However, due to their intrinsic properties, membrane protein targets often cannot be identified by means of established approaches, in particular affinity-based proteomics, calling for the exploration of new methods. Here, we report the identification of indophagolin as representative member of an indoline-based class of autophagy inhibitors through a target-agnostic phenotypic assay. Thermal proteome profiling and subsequent biochemical validation identified the purinergic receptor P2X4 as a target of indophagolin, and subsequent investigations suggest that indophagolin targets further purinergic receptors. These results demonstrate that thermal proteome profiling may enable the de novo identification of membrane-bound receptors as cellular targets of bioactive small molecules.