{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,25]],"date-time":"2025-10-25T04:47:44Z","timestamp":1761367664220,"version":"build-2065373602"},"reference-count":137,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,10,23]],"date-time":"2025-10-23T00:00:00Z","timestamp":1761177600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003141","name":"Secretariat of Science, Humanities, Technology, and Innovation","doi-asserted-by":"publisher","award":["CF-2019-01-170715"],"award-info":[{"award-number":["CF-2019-01-170715"]}],"id":[{"id":"10.13039\/501100003141","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007228","name":"Autonomous University of Baja California","doi-asserted-by":"crossref","award":["CPI\/300\/735\/E","300\/3806","300\/3816","300\/3839","300\/4272"],"award-info":[{"award-number":["CPI\/300\/735\/E","300\/3806","300\/3816","300\/3839","300\/4272"]}],"id":[{"id":"10.13039\/501100007228","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>Background\/Objectives: Naegleria fowleri is a free-living protozoan that causes primary amoebic meningoencephalitis, a rapidly progressing central nervous system infection with high mortality rates and limited treatment options. Targeting virulence-associated proteins is essential for effective drug development. Fowlerpain-1 (FWP1), a papain-like cysteine protease (CP) implicated in extracellular matrix degradation and host\u2013cell cytotoxicity, has been investigated as a therapeutic target. This study aimed to evaluate the FWP1 pocket geometry and stefin binding using an integrated in silico structural biology approach. Methods: A computational pipeline was used, including AlphaFold2-Multimer modeling of FWP1\u2013stefin complexes, 20-ns molecular dynamics simulations under NPT conditions for conformational sampling, and molecular mechanics Poisson\u2013Boltzmann surface area free energy calculations. Three natural CP inhibitors (stefins) were investigated. Structural stability was assessed using root mean square deviations, and binding profiles were characterized using protein\u2013protein interaction analysis. Results: Stable FWP1\u2013stefin interaction interfaces were predicted, with human stefin A showing favorable binding free energy. Two conserved motifs (PG and QVVAG) were identified as critical mediators of active-site recognition. Druggability analysis revealed a concave pocket with both hydrophobic and polar characteristics, consistent with a high-affinity ligand-binding site. Conclusions: This computational study supports a structural hypothesis for selective FWP1 inhibition and identifies stefins as promising scaffolds for developing structure-guided protease-targeted therapeutics against N. fowleri.<\/jats:p>","DOI":"10.3390\/ph18111606","type":"journal-article","created":{"date-parts":[[2025,10,24]],"date-time":"2025-10-24T00:47:36Z","timestamp":1761266856000},"page":"1606","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Structure-Based Insights into Stefin-Mediated Targeting of Fowlerpain-1: Towards Novel Therapeutics for Naegleria fowleri Infections"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-1435-8068","authenticated-orcid":false,"given":"Pablo A.","family":"Madero-Ayala","sequence":"first","affiliation":[{"name":"Science and Engineering Graduate Program, Autonomous University of Baja California, Tijuana 22390, Mexico"},{"name":"Biotechnology and Biosciences Research Group, School of Chemical Sciences and Engineering, Autonomous University of Baja California, Tijuana 22390, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6102-6966","authenticated-orcid":false,"given":"Rosa E.","family":"Mares-Alejandre","sequence":"additional","affiliation":[{"name":"Biotechnology and Biosciences Research Group, School of Chemical Sciences and Engineering, Autonomous University of Baja California, Tijuana 22390, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8303-6305","authenticated-orcid":false,"given":"Patricia L. A.","family":"Mu\u00f1oz-Mu\u00f1oz","sequence":"additional","affiliation":[{"name":"Biotechnology and Biosciences Research Group, School of Chemical Sciences and Engineering, Autonomous University of Baja California, Tijuana 22390, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7392-283X","authenticated-orcid":false,"given":"Samuel G.","family":"Mel\u00e9ndez-L\u00f3pez","sequence":"additional","affiliation":[{"name":"Biotechnology and Biosciences Research Group, School of Chemical Sciences and Engineering, Autonomous University of Baja California, Tijuana 22390, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8950-2079","authenticated-orcid":false,"given":"Marco A.","family":"Ramos-Ibarra","sequence":"additional","affiliation":[{"name":"Biotechnology and Biosciences Research Group, School of Chemical Sciences and Engineering, Autonomous University of Baja California, Tijuana 22390, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"30433","DOI":"10.1074\/jbc.R800035200","article-title":"Proteases: Multifunctional Enzymes in Life and Disease","volume":"283","author":"Bond","year":"2008","journal-title":"J. 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