{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T22:27:41Z","timestamp":1791325661228,"version":"4.3.1"},"reference-count":44,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T00:00:00Z","timestamp":1747958400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Science and Technology Development Fund","award":["0043\/2023\/AFJ"],"award-info":[{"award-number":["0043\/2023\/AFJ"]}]},{"name":"Science and Technology Development Fund","award":["RP\/FCA-12\/2023"],"award-info":[{"award-number":["RP\/FCA-12\/2023"]}]},{"name":"Science and Technology Development Fund","award":["32270446"],"award-info":[{"award-number":["32270446"]}]},{"name":"Macao Polytechnic University","award":["0043\/2023\/AFJ"],"award-info":[{"award-number":["0043\/2023\/AFJ"]}]},{"name":"Macao Polytechnic University","award":["RP\/FCA-12\/2023"],"award-info":[{"award-number":["RP\/FCA-12\/2023"]}]},{"name":"Macao Polytechnic University","award":["32270446"],"award-info":[{"award-number":["32270446"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["0043\/2023\/AFJ"],"award-info":[{"award-number":["0043\/2023\/AFJ"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["RP\/FCA-12\/2023"],"award-info":[{"award-number":["RP\/FCA-12\/2023"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32270446"],"award-info":[{"award-number":["32270446"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background: Human African trypanosomiasis (HAT), caused by Trypanosoma brucei, is a neglected tropical disease with limited treatments, highlighting the pressing need for new drugs. Cell division cycle-2-related kinase 12 (CRK12), a pivotal protein involved in the cell cycle regulation of T. brucei, has emerged as a promising therapeutic target for HAT, yet effective CRK12 inhibitors remain lacking. Methods: An integrated strategy combining computational modeling, virtual screening, molecular dynamics (MD) simulations, and experimental validation was adopted to discover potential inhibitors against CRK12. By using the predicted and refined 3D structure of CRK12 from AlphaFold2 and MD simulation, over 1.5 million compounds were screened based on multiple-scale molecular docking, and 26 compounds were selected for evaluation of biological activity based on anti-T. brucei bioassays. Dose\u2013response curves were generated for the most potent inhibitors, and the interaction mechanism between the top four compounds and CRK12 was explored by MD simulations and MM\/GBSA binding free energy analysis. Results: Of the 26 compounds, six compounds demonstrated sub-micromolar to low-micromolar IC50 values (0.85\u20133.50 \u00b5M). The top four hits, F733-0072, F733-0407, L368-0556, and L439-0038, exhibited IC50 values of 1.11, 1.97, 0.85, and 1.66 \u00b5M, respectively. Binding free energy and energy decomposition analyses identified ILE335, VAL343, PHE430, ALA433, and LEU482 as hotspot residues for compound binding. Hydrogen bonding analysis demonstrated that these compounds can form stable hydrogen bonds with the hinge residue ALA433, ensuring their stable binding within the active site. Conclusions: This study establishes a robust and cost-effective pipeline for CRK12 inhibitor discovery, identifying several novel inhibitors demonstrating promising anti-HAT activity. The newly discovered scaffolds exhibit structural diversity distinct from known CRK12 inhibitors, providing valuable lead compounds for anti-trypanosomal drug development.<\/jats:p>","DOI":"10.3390\/ph18060778","type":"journal-article","created":{"date-parts":[[2025,5,23]],"date-time":"2025-05-23T06:16:03Z","timestamp":1747980963000},"page":"778","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Discovery of Novel CRK12 Inhibitors for the Treatment of Human African Trypanosomiasis: An Integrated Computational and Experimental Approach"],"prefix":"10.3390","volume":"18","author":[{"given":"Qin","family":"Li","sequence":"first","affiliation":[{"name":"Faculty of Applied Sciences, Macao Polytechnic University, Macao SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayi","family":"Luo","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenggong","family":"Fu","sequence":"additional","affiliation":[{"name":"Faculty of Applied Sciences, Macao Polytechnic University, Macao SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenqingqing","family":"Kang","sequence":"additional","affiliation":[{"name":"Faculty of Applied Sciences, Macao Polytechnic University, Macao SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lingling","family":"Wang","sequence":"additional","affiliation":[{"name":"Faculty of Applied Sciences, Macao Polytechnic University, Macao SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2687-741X","authenticated-orcid":false,"given":"Henry","family":"Tong","sequence":"additional","affiliation":[{"name":"Faculty of Applied Sciences, Macao Polytechnic University, Macao SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaorong","family":"Lun","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4422-4968","authenticated-orcid":false,"given":"Qianqian","family":"Zhang","sequence":"additional","affiliation":[{"name":"Faculty of Applied Sciences, Macao Polytechnic University, Macao SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4709-1507","authenticated-orcid":false,"given":"Dehua","family":"Lai","sequence":"additional","affiliation":[{"name":"MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9284-3667","authenticated-orcid":false,"given":"Huanxiang","family":"Liu","sequence":"additional","affiliation":[{"name":"Faculty of Applied Sciences, Macao Polytechnic University, Macao SAR, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,5,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Sutherland, C.S., Yukich, J., Goeree, R., and Tediosi, F. 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