Design, synthesis and biological evaluation of triazolothiadiazole derivatives as FSP1 inhibitors for sensitizing cancer cells to ferroptosis

Abstract

Ferroptosis suppressor protein 1 (FSP1) is a recently identified ferroptosis suppressor that functions independently of the glutathione peroxidase reductase 4 (GPX4)-mediated pathway. Mechanistically, FSP1 mitigates ferroptosis by catalyzing the reduction of ubiquinone to ubiquinol and vitamin K (VK) to hydroquinone, thereby reducing lethal lipid peroxidation through the neutralization of free radicals. In this study, we designed and synthesized 32 compounds to systematically explore their structure-activity relationship (SAR) with the aim of identifying potent and novel FSP1 inhibitors. Among these, compound 39, a triazolothiadiazole derivative, exhibited the most significant inhibitory activity against FSP1, with an IC₅₀ value of 35 nM. In vitro cellular assays demonstrated that compound 39 markedly enhanced RSL3-induced lipid peroxide (LPO) accumulation and sensitized cancer cells from diverse tissue origins to RSL3-induced ferroptosis. Furthermore, by exploiting the FSP1-mediated reduction of VK, compound 39 effectively augmented ferroptosis in HT1080 cells pretreated with RSL3 and VK through its potent inhibition of FSP1 activity. To the best of our knowledge, this study represents the first pharmacochemical investigation dedicated to the systematic design and synthesis of FSP1 inhibitors. Collectively, our findings underscore the profound impact of compound 39 on tumor ferroptosis, providing a promising foundation for the development of FSP1 inhibitors as potential therapeutic agents in cancer treatment.