Targeting G Protein‐Coupled Receptors by Capture Compound Mass Spectrometry: A Case Study with Sertindole

Unbiased chemoproteomic profiling of small‐molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein‐coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates. We report a capture compound (CC)‐based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure–activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell‐based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most‐active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100 fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug–protein interactions. Target acquired: Unbiased chemoproteomic profiling of small‐molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes, including GPCRs, have to be tractable. Our new living‐cell capture compound (CC) mass spectrometry workflow broadens the chemoproteomic arsenal to access GPCRs based on specific small‐molecule–protein interactions.