The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop the first small-molecule NUDT15 inhibitors to elucidate its biological functions, and potentially for improving NUDT15-dependent chemotherapeutics. Lead compound TH1760, demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We further employed thiopurine potentiation as a proxy functional read-out and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity is via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.

Highlights d NUDT15 hydrolyzes the active metabolites of antiherpes ganciclovir d Depletion of NUDT15 via RNAi potentiates the anti-HCMV efficacy of ganciclovir d Nanomolar NUDT15 inhibitor TH8321 demonstrates submicromolar cellular activity d Application of TH8321 potentiates the anti-HCMV efficacy of ganciclovir

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