Etomoxir repurposed as a promiscuous fatty acid mimetic chemoproteomic probe

Abstract

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.

Keywords: Biochemistry; Chemical compound; Enzymology; Molecular biology; Molecular interaction.

Graphical abstract

Figure 1

Click-enabled etomoxir inhibits fatty acid oxidation but promiscuously binds an array of proteins (A) Structures of etomoxir, etomoxir-azide, and etomoxir-alkyne. (B) Etomoxir (Eto) and Click etomoxir inhibit fatty acid oxidation in HEK293T cells stably expressing TMP-dependent Cpt1a. (C and D) Acylcarnitine and fatty acid analysis or D. Untargeted lipidomic analysis of primary hepatocytes from wild-type or Cpt1a liver-specific KO mice treated with vehicle, 100μM etomoxir or etomoxir-alkyne in the presence of 60μM palmitate and 120μM oleate. (E) In gel fluorescence of primary mouse hepatocytes labeled with 100μM Click etomoxir for 24hrs. (F) Western analysis of dose-dependent labeling in primary mouse hepatocytes treated with 100μM etomoxir-alkyne for 24hrs and labeled with TAMRA-azide and probed for TAMRA. (G) Etomoxir competes for binding of Etomoxir-alkyne targets. Western blot for TAMRA in primary hepatocytes prelabeled with Etomoxir from 200 to 0 μM competes for binding of Etomoxir-alkyne 0.1μM for 2 h. (H) Primary hepatocytes showing the localization of Click-etomoxir in situ. Scale bar: 10μm. Data are expressed as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. WT DMSO vs. treatments; ###p < 0.001. Cpt1aKO DMSO vs. Etomoxir.

Figure 2

Click-enabled etomoxir labels proteins in vivo (A) 3 mg/kg etomoxir or etomoxir-alkyne was injected intraperitoneally over three days. The on-target Cpt1b can be visualized in heart and skeletal muscle via Western blot. (B) 3 mg/kg etomoxir-alkyne was injected IP over three days into control Cpt1b floxed mice or skeletal muscle specific KO of Cpt1b. (C) 3 mg/kg etomoxir-alkyne was injected IP over three days into control Cpt1a floxed mice or liver specific KO of Cpt1a. (D) Localization of etomoxir-alkyne (red) in the liver of wild-type mice injected with etomoxir or etomoxir-alkyne intraperitoneally. Scale bar: 10μm.

Figure 3

Proteomic identification of etomoxir binding proteins (A) Workflow of proteomics identification including proteins identified in primary hepatocytes. (B) Follow up immunoprecipitation-Western blots for Eto-Alkyne bound mitochondrial proteins. (C) Follow up immunoprecipitation-Western blots for Eto-Alkyne bound peroxisomal proteins.

Figure 4

Peroxisomes are major targets of etomoxir in hepatocytes (A) Co-localization of etomoxir-alkyne with peroxisomal or mitochondrial markers show peroxisomal puncta (green) and mitochondria (YFP) with TAMRA (red). Scale bar: 10μm. (B) Identification of etomoxir binding proteins in control, Cpt1a KO and Pex5 KO primary hepatocytes. (C) Proteomic identification of etomoxir binding proteins enriched, unchanged, or reduced in Pex5 KO primary hepatocytes by label free quantitative proteomics.