In silico and in vitro analysis of the genotoxic and mutagenic potential of STING modulators

The STING pathway plays a crucial role in dsDNA homeostasis and has been implicated in several diseases, including cancer and autoimmune disorders. Consequently, efforts have been made to develop modulators (agonists and antagonists) of the STING pathway. However, it is imperative to clinically evaluate the mutagenic and genotoxic potential effects of these novel molecules. For this purpose, an exhaustive list of STING modulators was subjected to knowledge- and expert-based in silico mutagenicity evaluations. A few antagonists were predicted to be positive for mutagenicity by Derek, and a few agonists were predicted to be positive (moderate reliability) by Vega. Based on the varying results of the computational approaches (Derek and Vega) for prediction of mutagenicity, two of each STING agonists and antagonists were evaluated using the bacterial reverse mutation test (Ames test; with and without metabolic activation) at concentrations of 1.935-250 microg/well. Additionally, the compounds (5, 10, and 20 microM) were subjected to single-cell gel electrophoresis (comet) assay to evaluate DNA damage in HepG2 cells. In silico analysis predicted that multiple STING modulators are mutagenic. In addition, the Ames test results demonstrated that only C-178, a STING antagonist, is mutagenic, while the other antagonist (SN-001) and both agonists (CMA and SR-717) are not mutagenic at the concentrations evaluated. C-178 is mutagenic, even without metabolic activation. Although not significantly different, there was a dose-dependent increase in comet tail length with C-178, thereby confirming genotoxicity, even in mammalian cells. The other STING modulators were inactive as per the comet assay. In summary, although mutagenic potential is not chemical class-specific, our findings highlight the carcinogenic potential of STING modulators, such as the covalent binder C-178. Caution must be exercised when developing novel STING modulators to avoid toxicological liabilities. Additionally, knowledge- and expert-based in silico evaluation of mutagenicity could help quickly identify toxicological potential.