Tuberculosis (TB) is still a threat to humans worldwide. The rise of drug-resistant TB strains has escalated the need for developing effective anti-TB agents. Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is essential for thymidylate biosynthesis to maintain the DNA integrity. In Mycobacterium tuberculosis, dUTPase provides the sole source for thymidylate biosynthesis, which also has the specific five-residue loop and the binding pockets absent in human dUTPase. Therefore, dUTPase has been regarded as a promising anti-TB drug target. Herein, we used a luminescence-based dUTPase assay to search for the inhibitors target M. tuberculosis dUTPase (Mt-dUTPase) and identified compound F0414 as a potent Mt-dUTPase inhibitor with an IC₅₀ of 0.80 ± 0.09 μM. F0414 exhibited anti-TB activity with low cytotoxicity. Molecular docking model and site-directed mutation experiments revealed that P79 was the key residue in the interaction of Mt-dUTPase and F0414. Moreover, F0414 was shown to have stronger binding with Mt-dUTPase than with Mt-P79A-dUTPase by surface plasmon resonance (SPR) detection. Interestingly, F0414 exhibited insensitivity and weak directly binding on human dUTPase compared with that on Mt-dUTPase. All the results highlight that F0414 is the first compound reported to have anti-TB activity by inhibiting Mt-dUTPase, which indicates the potential application in anti-TB therapy.

结核病 (TB) 仍然是全世界人类的威胁。耐药性结核病菌株的兴起加剧了开发有效抗结核病药物的需求。脱氧尿苷 5'-三磷酸核苷酸水解酶 (dUTPase) 对于胸苷酸生物合成以维持 DNA 完整性至关重要。在结核分枝杆菌, dUTPase 提供胸苷酸生物合成的唯一来源，它还具有特定的五残基环和人 dUTPase 中不存在的结合口袋。因此，dUTPase 被认为是一种很有前景的抗结核药物靶点。在此，我们使用基于发光的 dUTPase 分析来寻找抑制剂目标结核分枝杆菌dUTPase (Mt-dUTPase) 并将化合物 F0414 鉴定为有效的 Mt-dUTPase 抑制剂，IC ₅₀为 0.80 ± 0.09 μM。F0414 具有抗结核活性和低细胞毒性。分子对接模型和定点突变实验表明，P79 是 Mt-dUTPase 和 F0414 相互作用的关键残基。此外，通过表面等离子共振 (SPR) 检测显示 F0414 与 Mt-dUTPase 的结合比与 Mt-P79A-dUTPase 的结合更强。有趣的是，与 Mt-dUTPase 相比，F0414 对人 dUTPase 表现出不敏感和弱直接结合。所有结果都强调 F0414 是第一个报道的通过抑制 Mt-dUTPase 具有抗结核活性的化合物，这表明其在抗结核治疗中的潜在应用。