Angewandte Chemie International Edition · 2019 · 18 citations · 30 references
Life SciencesBiochemistryMolecular BiologyDrug DiscoveryMitochondrial BiologyProteomicsChemical Probe DiscoveryEnzyme InhibitionLigand BindingMass Spectrometry-based ProteomicsMitochondrial BiochemistryMitochondrial MetabolismMitochondrial Membrane TransportersMitochondrial ProteomicsReversible Covalent Bond FormationReversible Covalent ChemistryReversible Covalent InhibitionReversible Covalent InhibitorsSelectivityWestern Blotting
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.
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