Therapeutic exploration of novel reactive oxygen species-mediated anti-cancer mechanism by modulating electron transfer in respiratory complex III.

Reactive oxygen species (ROS) are by-products of cellular aerobic metabolism, playing a crucial role in the development of multiple diseases, such as cancer. ROS are generated by various enzymes, including respiratory complexes such as respiratory complex III (a.k.a. bc complex), which encompasses four redox centers (FeS, heme b, heme b and heme c) and two native binding sites (Q and Q sites). In the current study, we explored a novel anti-cancer mechanism by binding electron tunneling (ET)-modulating agents at the newly discovered Non-Q or NQ site in the bc complex, thereby controlling the conformation of a key phenylalanine 90 (Phe90) residue, which acts as an internal ET switch between heme b and heme b. Our proposed mechanism would potentially enhance or reduce ET between heme b and heme b, ultimately reducing or increasing the cellular ROS levels. To explore our proposed mechanism, we performed extensive virtual screening of 1,489,806 ligands from the ZINC20 database against the Q, Q, and NQ sites of the bc complex, obtaining 272 patented ligands that bind preferentially at the NQ site compared to the Q and Q sites. Biochemical assays for the top hit ligands, examining their ROS-regulatory and cytotoxic activities against breast cancer MCF7 cells, led to the discovery of potential compounds that upregulate and downregulate ROS. We found significant cytotoxic activity for the single treatment of a lead-hit ROS upregulator and even more synergistic cytotoxic activity for the sequential treatment of ROS upregulator and ROS downregulator ligands, which could be reserved for more resistant cancer cells.