Babesia duncani Pyruvate Kinase Inhibitor Screening and Identification of Key Active Amino Acid Residues
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Published:2024-06-04
Issue:6
Volume:12
Page:1141
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ISSN:2076-2607
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Container-title:Microorganisms
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language:en
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Short-container-title:Microorganisms
Author:
Li Fangjie1, Zhao Pengfei1, Wang Sen1, Luo Wanxin1, Xia Yingjun1, Li Dongfang1ORCID, He Lan123, Zhao Junlong123
Affiliation:
1. State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China 2. Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China 3. Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture of the People’s Republic of China, Wuhan 430070, China
Abstract
Babesia duncani (B. duncani), 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, B. duncani 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 B. duncani. 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 B. duncani, providing insights for babesiosis prevention and drug development.
Funder
National Natural Science Foundation of China National Key Research and Development Program of China Fundamental Research Funds for the Central Universities in China
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