Dual inhibition of carbonic anhydrase IX and glutathione peroxidase 4 as a novel strategy for ferroptosis-induced tumor cell death.

In this study, we explored a dual-target strategy combining the inhibition of human carbonic anhydrase IX (hCA IX), a tumor-associated isoform, and glutathione peroxidase 4 (GPX4), a key regulator of ferroptosis. We demonstrated that the simultaneous inhibition of hCA IX and GPX4 disrupts redox and iron homeostasis, thereby enhancing cell death via ferroptosis. Three series of compounds were rationally designed and synthesized based on the ML162 scaffold using an integrated structural approach and their enzymatic inhibition was evaluated in vitro. Several dual-target compounds exhibited significant antitumor activity, with 18a-c, 22abab and 22abcb inducing dose-dependent cell death. In vivo, intratumoral administration of the lead active compound, 22abcb, significantly prevented the growth of CA IX-expressing human breast cancer xenografts, compared to inactive 22abbb. The effect on tumour growth was significantly reversed by the ferroptosis inhibitor, Fer-1, confirming ferroptosis as the underlying mechanism. These findings highlight the synergistic potential of dual-target inhibitors in disrupting tumor-specific metabolic pathways and position them as a promising therapeutic strategy for solid tumors.