Weak Modulation of Human Glycolysis Isomerases by Metabolites: A Screening Study

Modulation of enzyme activity by metabolites represents the most efficient and rapid way of controlling metabolism. Exploring novel enzyme-metabolite interactions could provide insights into metabolic regulation as well as facilitate the discovery of new enzyme modulators with potential utility in therapeutic development. The interactions between enzymes and metabolites can range from strong (K d ≤ nM) to weak (K d = µM-mM), where the latter are generally overlooked, partly because of the difficulties in studying these subtle interactions. In this study, metabolite screening was performed for two human isomerases in the glycolytic pathway: glucose-6-phosphate isomerase (GPI) and triosephosphate isomerase (TPI), whose ligandability is still poorly characterized and requires further investigation. With a combination of activity and binding assays, the screening revealed multiple novel weak inhibitors for the two targets, causing partial attenuation of their activity, with K d and K i in the mM range. Ligand binding sites for several inhibitors were solved by X-ray crystallography; four GPI inhibitors were found in the substrate binding site, and two TPI inhibitors in a non-substrate binding site within the active site region. Together, our findings underscore the prevalence of weak interactions in modulating enzyme activities. The observed target-ligand interactions might provide new structural insights for the design of human GPI and TPI inhibitors and to explore the potential of the two enzymes as therapeutic targets for glycolysis inhibition.