Li Fangjie, Zhao Pengfei, Wang Sen, Luo Wanxin, Xia Yingjun, Li Dongfang, He Lan, Zhao Junlong
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China.
Microorganisms. 2024 Jun 4;12(6):1141. doi: 10.3390/microorganisms12061141.
(), a protozoan parasite prevalent in North America, is a significant threat for human health. Given the regulatory role of pyruvate kinase I (PyK I) in glycolytic metabolism flux and ATP generation, PyK I has been considered the target for drug intervention for a long time. In this study, PyK I (BdPyK I) was successfully cloned, expressed, and purified. Polyclonal antibodies were confirmed to recognize the native BdPyK I protein (56 kDa) using Western blotting. AlphaFold software predicted the three-dimensional structure of BdPyK I, and molecular docking with small molecules was conducted to identify potential binding sites of inhibitor on BdPyK I. Moreover, inhibitory effects of six inhibitors (tannic acid, apigenin, shikonin, PKM2 inhibitor, rosiglitazone, and pioglitazone) on BdPyK I were examined under the optimal enzymatic conditions of 3 mM PEP and 3 mM ADP, and significant activity reduction was found. Enzyme kinetics and growth inhibition assays further confirmed the reliability of these inhibitors, with PKM2 inhibitor, tannic acid, and apigenin exhibiting the highest selectivity index as specific inhibitors for . Subsequently, key amino acid residues were mutated in both BdPyK I and Homo sapiens pyruvate kinase I (HPyK I), and two differential amino acid residues (isoleucine and phenylalanine) were identified between HPyK I and BdPyK I through PyK activity detection experiments. These findings lay foundation for understanding the role of PyK I in the growth and development of , providing insights for babesiosis prevention and drug development.
（）是一种在北美流行的原生动物寄生虫，对人类健康构成重大威胁。鉴于丙酮酸激酶I（PyK I）在糖酵解代谢通量和ATP生成中的调节作用，长期以来PyK I一直被视为药物干预的靶点。在本研究中，成功克隆、表达并纯化了PyK I（BdPyK I）。使用蛋白质免疫印迹法证实多克隆抗体可识别天然BdPyK I蛋白（56 kDa）。AlphaFold软件预测了BdPyK I的三维结构，并进行了与小分子的分子对接以确定抑制剂在BdPyK I上的潜在结合位点。此外，在3 mM磷酸烯醇式丙酮酸（PEP）和3 mM二磷酸腺苷（ADP）的最佳酶促条件下，检测了六种抑制剂（单宁酸、芹菜素、紫草素、丙酮酸激酶M2（PKM2）抑制剂、罗格列酮和吡格列酮）对BdPyK I的抑制作用，发现其活性显著降低。酶动力学和生长抑制试验进一步证实了这些抑制剂的可靠性，其中PKM2抑制剂、单宁酸和芹菜素作为针对（此处原文缺失相关内容）的特异性抑制剂表现出最高的选择性指数。随后，对BdPyK I和人类丙酮酸激酶I（HPyK I）中的关键氨基酸残基进行了突变，并通过PyK活性检测实验确定了HPyK I和BdPyK I之间的两个差异氨基酸残基（异亮氨酸和苯丙氨酸）。这些发现为理解PyK I在（此处原文缺失相关内容）生长发育中的作用奠定了基础，为巴贝斯虫病的预防和药物开发提供了见解。
