Romain Marteau

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. However, its reference inhibitor, rosiglitazone, has off‐target activity on peroxisome proliferator‐activated receptor gamma (PPARγ), a key regulator of lipid homeostasis. Here, the discovery of LIBX‐A401, a potent ACSL4 inhibitor derived from rosiglitazone devoid of PPARγ activity, is reported. Its binding to ACSL4 is ATP‐dependent, stabilizing the C‐terminal domain and altering the fatty acid gate region, as shown by Hydrogen‐Deuterium Exchange Mass Spectrometry. Photoaffinity labeling identified A329 within the fatty acid binding site, while molecular dynamics and mutagenesis highlighted Q302 as critical for LIBX‐A401 binding. LIBX‐A401 exhibits anti‐ferroptotic properties in cells, supported by target engagement. These findings establish LIBX‐A401 as a valuable tool to study ACSL4 in ferroptosis and cancer, while its elucidated binding mode paves the way for the rational design of improved inhibitors.

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. However, its reference inhibitor, rosiglitazone, has off‐target activity on peroxisome proliferator‐activated receptor gamma (PPARγ), a key regulator of lipid homeostasis. Here, the discovery of LIBX‐A401, a potent ACSL4 inhibitor derived from rosiglitazone devoid of PPARγ activity, is reported. Its binding to ACSL4 is ATP‐dependent, stabilizing the C‐terminal domain and altering the fatty acid gate region, as shown by Hydrogen‐Deuterium Exchange Mass Spectrometry. Photoaffinity labeling identified A329 within the fatty acid binding site, while molecular dynamics and mutagenesis highlighted Q302 as critical for LIBX‐A401 binding. LIBX‐A401 exhibits anti‐ferroptotic properties in cells, supported by target engagement. These findings establish LIBX‐A401 as a valuable tool to study ACSL4 in ferroptosis and cancer, while its elucidated binding mode paves the way for the rational design of improved inhibitors.