Etomoxir repurposed as a promiscuous fatty acid mimetic chemoproteomic probe
Joseph Choi,
Danielle M. Smith,
Ye Jin Lee,
Danfeng Cai,
Mohammad J. Hossain,
Tamara J. O’Connor,
Pragney Deme,
Norman J. Haughey,
Susanna Scafidi,
Ryan C. Riddle,
Michael J. Wolfgang
Affiliations
Joseph Choi
Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
Danielle M. Smith
Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
Ye Jin Lee
Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
Danfeng Cai
Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
Mohammad J. Hossain
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
Tamara J. O’Connor
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
Pragney Deme
Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
Norman J. Haughey
Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
Susanna Scafidi
Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
Ryan C. Riddle
Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, USA; Research and Development Service, Baltimore VA Medical Center, Baltimore, MD, USA
Michael J. Wolfgang
Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA; Corresponding author
Summary: Etomoxir has been used for decades as a popular small molecule inhibitor of carnitine palmitoyltransferase I, Cpt1, to block mitochondrial fatty acid β-oxidation. To test the specificity of etomoxir, we generated click chemistry–enabled reagents to label etomoxir binding proteins in situ. Etomoxir bound to Cpt1, but also bound to a large array of diverse proteins that metabolize and transport fatty acids in the cytoplasm, peroxisome, and mitochondria. Many of the most abundant proteins identified in primary hepatocytes were peroxisomal proteins. The loss of Pex5, required for the import of peroxisomal matrix proteins, eliminated many of these etomoxir-labeled proteins. By utilizing the promiscuous, covalent, and fatty acid mimetic properties of etomoxir, etomoxir targets of fatty acid ω-oxidation were revealed following the loss of Pex5. These data demonstrate that etomoxir is not specific for Cpt1 and is not appropriate as a tool to distinguish the biological effects of fatty acid oxidation.
