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Consequently, the development of drugs with novel modes of action (MOAs) is urgently required. In this study, we discovered and characterized two potent inhibitors, Pi-1-58 and Pi-2-26, targeting the prokaryotic ubiquitin-like protein (Pup) ligase PafA of Mtb. Pi-1-58 was identified through computer-aided drug screening. The binding mode of Pi-1-58 and PafA was investigated through molecular docking, followed by experimental validations. Based on the core structure of Pi-1-58, we developed a more potent inhibitor, Pi-2-26, through structure-guided drug design. Both Pi-1-58 and Pi-2-26 exhibited selective and specific inhibition of PafA according to biochemical and cell-based assays. Importantly, the inhibitors demonstrated significant inhibition on Mtb survival in the presence of nitric oxide, mimicking the in vivo nitrogen limited environment that Mtb encountered in macrophage. Our findings provide a comprehensive understanding of the structural and functional aspects of these PafA inhibitors and establish a solid foundation for the development of novel therapeutics against tuberculosis.<\/jats:p>","DOI":"10.3390\/ijms252313189","type":"journal-article","created":{"date-parts":[[2024,12,9]],"date-time":"2024-12-09T04:01:37Z","timestamp":1733716897000},"page":"13189","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Structure-Based Screening and Optimization of PafA Inhibitors with Potent Anti-Tuberculosis Activity"],"prefix":"10.3390","volume":"25","author":[{"given":"Hewei","family":"Jiang","sequence":"first","affiliation":[{"name":"Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China"},{"name":"Lingang Laboratory, Shanghai 200031, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin","family":"Xie","sequence":"additional","affiliation":[{"name":"Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Wang","sequence":"additional","affiliation":[{"name":"Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China"},{"name":"Lingang Laboratory, Shanghai 200031, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Chen","sequence":"additional","affiliation":[{"name":"Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunxiao","family":"Zheng","sequence":"additional","affiliation":[{"name":"Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuening","family":"Wang","sequence":"additional","affiliation":[{"name":"Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shujuan","family":"Guo","sequence":"additional","affiliation":[{"name":"Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tao","family":"Wang","sequence":"additional","affiliation":[{"name":"Institute of Systems Biology, Shenzhen Bay Laboratory, Peking University Shenzhen Graduate School, Shenzhen 518055, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Bi","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4999-3598","authenticated-orcid":false,"given":"Xuelian","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8482-1185","authenticated-orcid":false,"given":"Jianfeng","family":"Pei","sequence":"additional","affiliation":[{"name":"Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9210-1823","authenticated-orcid":false,"given":"Shengce","family":"Tao","sequence":"additional","affiliation":[{"name":"Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"100603","DOI":"10.1016\/j.eclinm.2020.100603","article-title":"The potential impact of the COVID-19 pandemic on the tuberculosis epidemic a modelling analysis","volume":"28","author":"Cilloni","year":"2020","journal-title":"eClinicalMedicine"},{"key":"ref_2","unstructured":"WHO (2023, December 17). 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