Center for Structural Biology, National Cancer Institute, Frederick, MD 21702, USA.
Center for Structural Biology, National Cancer Institute, Frederick, MD 21702, USA; Current Address: Thoracic and Gastrointestinal Malignancies Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Bioorg Med Chem Lett. 2024 Nov 15;113:129977. doi: 10.1016/j.bmcl.2024.129977. Epub 2024 Sep 25.
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but is absent in mammals. Yet, it is not the target of any existing antibiotics. Hence, this enzyme is an attractive target for developing novel antimicrobial agents. A wealth of structural and mechanistic information has provided solid basis for structure-based design of HPPK inhibitors. Our bisubstrate inhibitors were initially created by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups (HPA, n = 2, 3, or 4), among which HPA exhibited the highest binding affinity (K = 0.47 ± 0.04 μM). Further development was carried out based on high-resolution structures of HPPK in complex with HPA. Replacing the phosphate bridge with a piperidine linked thioether eliminated multiple negative charges of the bridge. Substituting the pterin moiety with 7,7-dimethyl-7,8-dihydropterin improved the binding affinity. Arming the piperidine ring with a carboxyl group and oxidizing the thioether further enhanced the potency, resulting in a druglike inhibitor of HPPK (K = 0.047 ± 0.007 μM). None of these inhibitors, however, exhibits bacterial cell permeability. It is most likely due to the lack of active folate transporters in bacteria. Replacing the pterin moiety with a 7-deazagaunine moiety, we have obtained a novel bisubstrate inhibitor (HP-101) showing observable cell permeability toward a Gram-positive bacterium. Here, we report the in vitro activity of HP-101 and its structure in complex with HPPK, providing a framework for structure-based further development.
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 ATP 与 6-羟甲基-7,8-二氢蝶呤（HP）之间的焦磷酸基转移。HPPK 对于微生物是必需的，但在哺乳动物中不存在。然而，它不是任何现有抗生素的靶标。因此，该酶是开发新型抗菌药物的有吸引力的靶标。丰富的结构和机制信息为基于结构的 HPPK 抑制剂设计提供了坚实的基础。我们的双底物抑制剂最初是通过将 6-羟甲基蝶呤通过 2、3 或 4 个磷酸基团与腺苷连接（HPA，n=2、3 或 4）而创建的，其中 HPA 表现出最高的结合亲和力（K=0.47±0.04μM）。进一步的开发是基于 HPPK 与 HPA 复合物的高分辨率结构进行的。用连接哌啶的硫醚取代磷酸桥消除了桥的多个负电荷。用 7,7-二甲基-7,8-二氢蝶呤取代蝶呤部分提高了结合亲和力。在哌啶环上装备一个羧基并氧化硫醚进一步增强了效力，得到了 HPPK 的类药性抑制剂（K=0.047±0.007μM）。然而，这些抑制剂都没有表现出细菌细胞通透性。这很可能是由于细菌中缺乏活性叶酸转运蛋白。用 7-脱氮鸟嘌呤取代蝶呤部分，我们获得了一种新型双底物抑制剂（HP-101），对革兰氏阳性菌表现出可观察到的细胞通透性。在此，我们报告了 HP-101 的体外活性及其与 HPPK 复合物的结构，为基于结构的进一步开发提供了框架。
