Quantitative analysis of inhibitor-induced assembly disruption in human UDP-GlcNAc 2-epimerase using mass photometry

UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE/MNK) is the rate-limiting enzyme in sialic acid biosynthesis and a promising therapeutic target. We applied interferometric scattering microscopy (iSCAM) to investigate GNE oligomerization and its modulation by three small-molecule inhibitors (C5, C13, C15). Substrate binding (UDP-GlcNAc) stabilized tetramer formation by increasing dimer-dimer affinity 120-fold. All inhibitors destabilized tetramers in a concentration-dependent manner, with IC ₅₀ values in the low micromolar range. Using a modified Cheng-Prusoff equation, IC ₅₀ values were converted into K _i values. Schild analysis was applied to estimate an apparent K _B,app value and assess cooperative inhibition effects. Molecular docking confirmed competitive binding for all inhibitors and helped rationalize observed potency trends. While iSCAM has previously been used to study protein assembly, our work demonstrates its applicability for the label-free, quantitative characterization of small-molecule inhibitors affecting protein oligomerization. These findings provide a foundation for further mechanistic studies and underscore the potential of iSCAM in drug-target interaction profiling.