Shotgun Kinetic Target-Guided Synthesis Approach Enables the Discovery of Small-Molecule Inhibitors against Pathogenic Free-Living Amoeba Glucokinases

[Image: see text] Pathogenic free-living amoebae (pFLA) can cause life-threatening central nervous system (CNS) infections and warrant the investigation of new chemical agents to combat the rise of infection from these pathogens. Naegleria fowleri glucokinase (NfGlck), a key metabolic enzyme involved in generating glucose-6-phosphate, was previously identified as a potential target due to its limited sequence similarity with human Glck (HsGlck). Herein, we used our previously demonstrated multifragment kinetic target-guided synthesis (KTGS) screening strategy to identify inhibitors against pFLA glucokinases. Unlike the majority of previous KTGS reports, our current study implements a “shotgun” approach, where fragments were not biased by predetermined binding potentials. The study resulted in the identification of 12 inhibitors against 3 pFLA glucokinase enzymes—NfGlck, Balamuthia mandrillaris Glck (BmGlck), and Acanthamoeba castellanii Glck (AcGlck). This work demonstrates the utility of KTGS to identify small-molecule binders for biological targets where resolved X-ray crystal structures are not readily accessible.