Mono-quinoxaline-induced DNA structural alteration leads to ZBP1/RIP3/MLKL-driven necroptosis in cancer cells

Evading the cellular Apoptosis mechanism by modulating multiple pathways poses a sturdy barrier to effective chemotherapy. Cancer cell adeptly resists the Apoptosis signaling pathway by regulating anti and pro-apoptotic proteins to escape cell death. Nevertheless, bypassing the apoptotic pathway through Necroptosis, an alternative programmed cell death process, maybe a potential therapeutic modality for apoptosis-resistant cells. However, synthetic mono-quinoxaline-based intercalator-induced cellular Necroptosis as an anti-cancer perspective remains under-explored. To address this concern, we undertook the design and synthesis of quinoxaline-based small molecules (3a-3l). Our approach involved enhancing the π-surface of the mandatory benzyl moiety to augment its ability to induce DNA structural alteration via intercalation, thereby promoting cytotoxicity across various Cancer cell lines (HCT116, HT-29, and HeLa). Notably, the potent compound 3a demonstrated the capacity to induce DNA damage in Cancer cells, leading to the induction of ZBP1-mediated Necroptosis in the RIP3-expressed cell line (HT-29), where Z-VAD effectively blocked apoptosis-mediated cell death. Interestingly, we observed that 3a induced RIP3-driven Necroptosis in combination with DNA hypomethylating agents, even in the RIP3-silenced cell lines (HeLa and HCT116). Overall, our synthesized compound 3a emerged as a promising candidate against various cancers, particularly in apoptosis-compromised cells, through the induction of Necroptosis.

Apoptosis; Cytotoxicity; Necroptosis; RIP3/MLKL pathway; intercalation,5-aza-2′-deoxycytidine.