Human glycolysis isomerases are inhibited by weak metabolite modulators

Modulation of enzyme activity by metabolites represents the most efficient and rapid way of controlling metabolism. Investigating enzyme–metabolite interactions can deepen our understanding of metabolic control and aid in identifying enzyme modulators with potential therapeutic applications. These interactions vary in strength, with dissociation constants ( K d ) ranging from strong (n m ) to weak (μ m –m m ). However, weak interactions are often overlooked due to the challenges in studying them. Despite this, weak modulators can reveal unknown binding modes and serve as starting points for compound optimization. In this study, we aimed to identify metabolites that weakly modulate the activity of human glucose‐6‐phosphate isomerase (GPI) and triosephosphate isomerase (TPI), which are potential therapeutic targets in tumor glycolysis. Through a combination of activity and binding assays, the screening revealed multiple weak inhibitors for the two targets, causing partial attenuation of their activity, with K d and K i in the low m m range. X‐ray crystallography revealed six orthosteric ligands binding to the active sites – four inhibitors of GPI and two of TPI. Our findings underscore the role of weak interactions in enzyme regulation and may provide structural insights that could aid the design of inhibitors targeting human GPI and TPI in cancer intervention.