Angew. Chem. Int. Ed.. 2019
doi:10.1002/anie.201914391
Target engagement assays are crucial for establishing the mechanism‐of‐action of small molecules in living systems. Integral membrane transporters, due to their specialized biophysical properties and activity assays, can present a challenging protein class for assessing cellular engagement by small molecules. Here, we describe the chemical proteomic discovery of alpha‐chloroacetamide (aCA) compounds that covalently modify cysteine‐54 (C54) of the MPC2 subunit of the mitochondrial pyruvate carrier (MPC) complex. We leverage this finding to create an alkyne‐modified aCA, YY4‐yne, that serves as a versatile cellular target engagement probe for MPC2 in click chemistry‐enabled western blotting or global mass spectrometry‐based proteomic experiments. Using YY4‐yne, we demonstrate that UK‐5099, an alpha‐cyanocinnamate inhibitor of the MPC complex, first discovered more than 30 years ago, but still with a poorly defined mechanism‐of‐action, engages MPC2 with remarkable selectivity in human cells. These findings support a model where UK‐5099 inhibits the MPC complex by binding to C54 of MPC2 in a covalent reversible manner that can be quantified in cells using the YY4‐yne probe.
