title: Molecular basis for covalent inhibition of glyceraldehyde-3-phosphate dehydrogenase by a 2-phenoxy-1,4-naphthoquinone small molecule.
abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2-phenoxy-1,4-naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. 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-phenoxy-1,4-naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2-phenoxy-1,4-naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non-covalent 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-phenoxy-1,4-naphthoquinone displays selectivity for GAPDHs over two other cysteine-dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.
authors.affiliation: Department of Pharmacy, University of Parma, Parma, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Bruno
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Bologna, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Uliassi
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Bologna, Italy.
authors.forename: Mirko
authors.initials: M
authors.lastname: Zaffagnini
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Bologna, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Prati
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Bologna, Italy.
authors.forename: Christian
authors.initials: C
authors.lastname: Bergamini
authors.affiliation: Department of Chemistry "G. Ciamician", Alma Mater Studiorum - University of Bologna, Bologna, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Amorati
authors.affiliation: Department of Pharmacy, University of Parma, Parma, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Paredi
authors.affiliation: Department of Pharmacy, University of Parma, Parma, Italy.
authors.forename: Marilena
authors.initials: M
authors.lastname: Margiotta
authors.affiliation: Department of Pharmaceutical Sciences, University of Milan, Milan, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Conti
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Maria Paola
authors.initials: MP
authors.lastname: Costi
authors.affiliation: Swiss Tropical & Public Health Institute, Basel, Switzerland.|University of Basel, Basel, Switzerland.
authors.forename: Marcel
authors.initials: M
authors.lastname: Kaiser
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Bologna, Italy.|CompuNet, Istituto Italiano di Tecnologia, Genova, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Cavalli
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Bologna, Italy.
authors.forename: Romana
authors.initials: R
authors.lastname: Fato
authors.affiliation: Department of Pharmacy and Biotechnology, Alma Mater Studiorum - University of Bologna, Bologna, Italy.
authors.forename: Maria Laura
authors.identifier: 0000-0002-1289-5361
authors.initials: ML
authors.lastname: Bolognesi
mesh_terms: D000981:Antiprotozoal Agents / Q000737:chemistry* / Q000494:pharmacology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy; D009285:Naphthoquinones / Q000737:chemistry* / Q000494:pharmacology*; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology*; D014344:Trypanocidal Agents / Q000737:chemistry / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000201:enzymology*
chemical_list: D000981:Antiprotozoal Agents; D009285:Naphthoquinones; D014344:Trypanocidal Agents; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C035342:1,4-naphthoquinone
keywords: covalent inhibition; glyceraldehyde-3-phosphate dehydrogenase; naphthoquinones