A Chemical Proteomic Probe for the Mitochondrial Pyruvate Carrier Complex

Abstract

Target engagement assays are crucial for establishing the mechanism‐of‐action of small molecules in living systems. Integral membrane transporters can present a challenging protein class for assessing cellular engagement by small molecules. The chemical proteomic discovery of alpha‐chloroacetamide (αCA) compounds that covalently modify cysteine‐54 (C54) of the MPC2 subunit of the mitochondrial pyruvate carrier (MPC) is presented. This finding is used to create an alkyne‐modified αCA, YY4‐yne, that serves as a cellular engagement probe for MPC2 in click chemistry‐enabled western blotting or global mass spectrometry‐based proteomic experiments. Studies with YY4‐yne revealed that UK‐5099, an alpha‐cyanocinnamate inhibitor of the MPC complex, 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.