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We have previously identified 2\u2010phenoxy\u20101,4\u2010naphthoquinone as an inhibitor of both Trypanosoma brucei<\/jats:italic> and human GAPDH<\/jats:styled-content>. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2\u2010phenoxy\u20101,4\u2010naphthoquinone binds the GAPDH<\/jats:styled-content> catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2\u2010phenoxy\u20101,4\u2010naphthoquinone and its analogs with four GAPDH<\/jats:styled-content> homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non\u2010covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2\u2010phenoxy\u20101,4\u2010naphthoquinone displays selectivity for GAPDH<\/jats:styled-content>s over two other cysteine\u2010dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.<\/jats:p>","DOI":"10.1111\/cbdd.12941","type":"journal-article","created":{"date-parts":[[2017,1,13]],"date-time":"2017-01-13T01:56:22Z","timestamp":1484272582000},"page":"225-235","source":"Crossref","is-referenced-by-count":20,"title":["Molecular basis for covalent inhibition of glyceraldehyde\u20103\u2010phosphate dehydrogenase by a 2\u2010phenoxy\u20101,4\u2010naphthoquinone small molecule"],"prefix":"10.1111","volume":"90","author":[{"given":"Stefano","family":"Bruno","sequence":"first","affiliation":[{"name":"Department of Pharmacy University of Parma Parma Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elisa","family":"Uliassi","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology Alma Mater Studiorum \u2010 University of Bologna Bologna Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mirko","family":"Zaffagnini","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology Alma Mater Studiorum \u2010 University of Bologna Bologna Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Federica","family":"Prati","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology Alma Mater Studiorum \u2010 University of Bologna Bologna Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christian","family":"Bergamini","sequence":"additional","affiliation":[{"name":"Department of Pharmacy and Biotechnology Alma Mater Studiorum \u2010 University of Bologna Bologna Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Riccardo","family":"Amorati","sequence":"additional","affiliation":[{"name":"Department of Chemistry \u201cG. 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