Re: Concerns Regarding the Validation of ZYS-1 as a Bona Fide ADAR1 Inhibitor

Wang et al. in Nature Cancer report ZYS-1 as an ADAR1 inhibitor and present biochemical and cellular evidence to support ZYS-1s direct interaction with and inhibition of ADAR1, including enzymatic assays, surface plasmon resonance (SPR), drug affinity responsive target stability (DARTS), and cellular thermal shift assays (CETSA). We raise critical methodological concerns regarding the validation of ZYS-1 as a bona fide ADAR1 inhibitor. Our primary concern relates to the misuse of an adenosine deaminase (ADA) activity assay kit for ADAR1 enzymatic activity characterization. Using established biochemical assays with direct inosine quantification, we evaluated ZYS-1s inhibitory activity against ADAR1 using physiologically relevant RNA substrates. The compound showed no inhibitory activity at concentrations up to 1 mM. We demonstrate that the ADA assay kit used by Wang et al. does not reflect ADAR1-specific activity, and ZYS-1 is unable to inhibit ADAR1 in well-validated biochemical assays. We urge for further validation using ADAR1-specific assays to establish ZYS-1 as a genuine ADAR1 inhibitor.