Molecular basis for covalent inhibition of glyceraldehyde‐3‐phosphate dehydrogenase by a 2‐phenoxy‐1,4‐naphthoquinone small molecule

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.