Therapeutic exploration of novel reactive oxygen species-mediated anti-cancer mechanism by modulating electron...

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. bc₁ complex), which encompasses four redox centers (Fe₂S₂, heme b_L, heme b_H, and heme c₁) and two native binding sites (Q_o and Q_i 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 bc₁ complex, thereby controlling the conformation of a key phenylalanine 90 (Phe90) residue, which acts as an internal ET switch between heme b_L and heme b_H. Our proposed mechanism would potentially enhance or reduce ET between heme b_L and heme b_H, 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_o, Q_i, and NQ sites of the bc₁ complex, obtaining 272 patented ligands that bind preferentially at the NQ site compared to the Q_o and Q_i 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.