LIBX-A401: A Novel Selective Inhibitor of Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) and Its Binding Mode

Abstract

Lipid metabolism is essential for cellular homeostasis, and its disruption is linked to various diseases. Acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), a pivotal enzyme in lipid metabolism, has emerged as a therapeutic target for ferroptosis-related conditions and cancer. Antidiabetic drug rosiglitazone is the reference ACSL4 inhibitor. However, its potent activity on peroxisome proliferator-activated receptor gamma (PPARγ), a key regulator of lipid metabolism, represents a significant limitation. Here, we report the discovery and characterization of LIBX-A401 (compound 9), a potent ACSL4 inhibitor derived from rosiglitazone but devoid of PPARγ activity. We showed that its binding to ACSL4 is ATP-dependent. Using Hydrogen-Deuterium Exchange Mass Spectrometry, we demonstrate that ATP binding stabilizes the ACSL4 C-terminus, with LIBX-A401 further stabilizing this domain and altering the fatty acid gate-region. Photoaffinity labeling with a diazirine probe identified A329 within the fatty acid binding site, while molecular dynamics simulations and mutagenesis highlighted Q302 as critical for LIBX-A401 binding. LIBX-A401 exhibits significant anti-ferroptotic properties in cells, supported by demonstrated target engagement. These findings position LIBX-A401 as a valuable tool for studying ACSL4 in ferroptosis-related diseases and cancer, while its elucidated binding mode paves the way to the rational design of improved ACSL4 inhibitors with therapeutic potential.