6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but absent in mammals; therefore, it is an attractive target for developing novel antimicrobial agents. Previously, based on our studies of the structure and mechanism of HPPK, we created first-generation bisubstrate inhibitors by linking 6-hydroxymethylpterin to adenosine through phosphate groups, and developed second-generation inhibitors by replacing the phosphate bridge with a linkage that contains a piperidine moiety. Here, we report third-generation inhibitors designed based on the piperidine-containing inhibitor, mimicking the transition state. We synthesized two such inhibitors, characterized their protein-binding and enzyme inhibition properties, and determined their crystal structures in complex with HPPK, advancing the development of such bisubstrate analog inhibitors.

6-羟甲基-7,8-二氢蝶呤焦磷酸激酶 (HPPK) 是叶酸生物合成途径中的关键酶。它催化焦​​磷酸从 ATP 转移到 6-羟甲基-7,8-二氢蝶呤 (HP)。HPPK 对微生物至关重要，但在哺乳动物中不存在；因此，它是开发新型抗菌剂的一个有吸引力的目标。此前，我们基于对HPPK结构和机制的研究，通过磷酸基团将6-羟甲基蝶呤与腺苷连接，创建了第一代双底物抑制剂，并通过用含有哌啶的连接取代磷酸桥，开发了第二代抑制剂部分。在这里，我们报告了基于含哌啶抑制剂设计的第三代抑制剂，模仿过渡态。我们合成了两种此类抑制剂，表征了它们的蛋白质结合和酶抑制特性，并确定了它们与 HPPK 复合物的晶体结构，从而推进了此类双底物类似物抑制剂的开发。