Discovery of Potent Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) Inhibitors with Antiferroptotic Properties

Ferroptosis, an iron-dependent regulated cell death, is implicated in several diseases, including cancer and neurodegeneration. While most ferroptosis inhibitors act as radical-trapping antioxidants, direct modulation of pro-ferroptotic enzymes remains underexplored. Acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), a key regulator of ferroptosis, has emerged as a promising therapeutic target. Here, we report a fragment-based screening that identified a benzofuran hit (compound <b>8</b>, IC₅₀ = 33 μM), leading to the discovery of two selective ACSL4 inhibitors: compound <b>15b</b> (LIBX-A402, IC₅₀ = 0.33 μM) and compound <b>21</b> (LIBX-A403, IC₅₀ = 0.049 μM). Compound <b>21</b> is the most potent ACSL4 inhibitor reported to date and shows no activity against ACSL3. Molecular modeling and mutagenesis support its binding in the ACSL4 fatty acid pocket. The strong antiferroptotic activity of both compounds in cells, together with confirmed target engagement for <b>21</b>, underscores the relevance of ACSL4 as a target for ferroptosis modulation.