A high-resolution respirometer (HRR)-based screen identifies AYNE as a pharmacological inhibitor of VLCAD. (A) An HRR screen identified AYNE as an inhibitor of C16-supported ETF respiration. AML2 cells were incubated with a screen compound (10 µM) or a solvent vehicle for 1 hour, and C16-supported ETF respiration was assessed. AYNE (compound 151) is highlighted by a red arrow. Structure of AYNE is to the right. (B) Schematic diagram highlighting the pull-down of VLCAD in AYNE-treated AML2 cells. In brief, following a 3-hour treatment, AYNE-treated AML2 cells were lysed and exposed to an anti-VLCAD antibody coupled to magnetic beads. Magnetic separation produces 2 fractions from the lysate: the flow-through fraction (containing all lysate components except VLCAD) and the VLCAD-enriched fraction (containing only VLCAD). Both fractions underwent immunoblotting to confirm VLCAD pull-down or MS analysis to quantify AYNE. Created using BioRender. (C) Immunoblot showing isolation of VLCAD from the flow-through fractions (lanes 1 and 2) into VLCAD-enriched fractions (lanes 3 and 4) by magnetic co-IP. (D) Identification of AYNE in VLCAD-enriched fractions: chromatograms showing elution of a commercial AYNE standard (top panel) and a VLCAD-enriched fraction (bottom panel). The red arrows indicate AYNE elutes at 5.6 minutes. (E) MS quantification of AYNE in VLCAD-enriched fractions from AYNE-treated AML2 cells. Data in (E) are mean ± standard deviation; n.d., not detected.