Water-soluble 4-(dimethylaminomethyl)heliomycin exerts greater antitumor effects than parental heliomycin by targeting the tNOX-SIRT1 axis and apoptosis in oral cancer cells

The antibiotic heliomycin (resistomycin), which is generated fromStreptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays and molecular docking simulations to identify and validate the intracellular targets of heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH), in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. Consistent with ourin silicostudies, our cellular thermal shift assays (CETSA) revealed that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase called tNOX or ENOX2. The direct binding of 4-dmH to tNOX inhibited the activity of tNOX and enhanced its ubiquitin-proteasomal protein degradation in both SAS and HSC-3 cells. Moreover, the inhibition of tNOX by 4-dmH decreased the oxidation of NADH to NAD+which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, ourin vitroandin vivofindings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells.