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 small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve 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 also employed thiopurine potentiation as a proxy functional readout 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 takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.

NUDIX 水解酶 NUDT15 最初与消毒氧化核苷酸有关，但后来被证明可以水解活性硫嘌呤代谢物 6-thio-(d)GTP，从而决定了这种白血病和炎症性疾病标准治疗的临床反应. 尽管如此，它的生理作用仍然难以捉摸。在这里，我们试图开发小分子 NUDT15 抑制剂以阐明其生物学功能并可能改善 NUDT15 依赖性化疗。先导化合物 TH1760 通过直接和特异性结合到 NUDT15 催化袋中并在低微摩尔范围内与细胞 NUDT15 结合，表现出低纳摩尔生化效力。我们还使用硫嘌呤增强作为代理功能读数，并证明 TH1760 通过增强核酸中 6-硫代-(d)GTP 的积累使细胞对 6-硫鸟嘌呤敏感。一种经过生化验证的无活性结构类似物 TH7285 证实，硫嘌呤毒性增加是通过直接抑制 NUDT15 发生的。总之，TH1760 代表了第一个用于询问 NUDT15 生物学和潜在治疗途径的化学探针。