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

Lipid metabolism affects many cellular processes essential for homeostasis, and its disruption is linked to various diseases. A key enzyme in these processes, acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), is a promising target for treating conditions involving ferroptosis and certain cancers. Rosiglitazone (ROSI) is a known ACSL4 inhibitor but its potent activity on peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor strongly involved in lipid metabolism constitutes an important limitation. This study focuses on developing novel ACSL4 inhibitors derived from ROSI, which lack PPARγ activity. Binding of the most potent compound of this series (9) relies on the prior binding of ATP. Hydrogen-Deuterium Exchange Mass Spectrometry (HDx-MS) demonstrated that ATP binding stabilizes the ACSL4 C-terminus, an effect enhanced by compound 9, which also alters important peptide sequences, including the fatty acid gate-domain. Photoaffinity Labeling (PAL) with a diazirine-based probe identified residue A329 in the fatty acid pocket. Molecular dynamics simulations and site-directed mutagenesis highlighted Q302 as critical for compound 9 binding. Thus, compound 9 (LIBX-A401) is a promising tool for studying ACSL4 in ferroptosis-related diseases and cancer, and the elucidation of its binding mode paves the way to the rational design of optimized inhibitors