A Chemical Proteomic Probe for the Mitochondrial Pyruvate Carrier Complex

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• Target engagement assays are vital for understanding drug mechanisms of action.
• Integral membrane transporters, like the mitochondrial pyruvate carrier (MPC), are difficult targets for cellular engagement studies.
• Covalent modification of proteins offers a strategy for target identification and engagement profiling.

Purpose of the Study:

• To develop a chemical probe for assessing cellular engagement of the mitochondrial pyruvate carrier (MPC).
• To investigate the mechanism of action of the MPC inhibitor UK-5099.
• To demonstrate the utility of click chemistry for studying drug-target interactions in complex biological systems.

Main Methods:

• Chemical proteomic discovery of alpha-chloroacetamide (αCA) compounds targeting MPC2.
• Synthesis of an alkyne-modified αCA probe (YY4-yne).
• Application of YY4-yne in click chemistry-enabled western blotting and mass spectrometry-based proteomics.
• Cellular studies using YY4-yne to assess UK-5099 engagement with MPC2.

Main Results:

• Discovery of αCA compounds that covalently modify cysteine-54 (C54) of the MPC2 subunit.
• YY4-yne successfully served as a cellular engagement probe for MPC2.
• UK-5099 demonstrated remarkable selectivity for engaging MPC2 in human cells.
• Evidence supporting a reversible covalent binding model for UK-5099 inhibition of MPC.

Conclusions:

• The YY4-yne probe enables quantitative assessment of MPC2 engagement by small molecules in cells.
• UK-5099 inhibits the MPC complex through selective, reversible covalent binding to MPC2 C54.
• This work provides a novel chemical proteomic approach for studying membrane transporter-small molecule interactions.