Fragment mapping-guided virtual screening identifies a small-molecule modulator of NPM1 oligomerization.

Nucleophosmin 1 (NPM1) is a multifunctional nucleolar protein whose oligomeric assembly is essential for its cellular functions and is implicated in human malignancies, including acute myeloid leukemia. Targeting protein-protein interaction interfaces such as the NPM1 oligomerization surface remains challenging due to their shallow and extended structural features. Here, we combined knowledge-based in silico fragment mapping with structure-based virtual screening to identify small molecules targeting the NPM1 oligomerization interface. Fragment mapping revealed recurrent aromatic and hydrogen-bonding features within surface grooves, which were used to construct three-dimensional pharmacophore models. A multistep workflow integrating pharmacophore filtering and molecular docking enabled prioritization of candidate compounds. Experimental validation using a split Renilla luciferase complementation assay identified STK947495 as a compound that reproducibly reduced NPM1-dependent luminescence in a concentration-dependent manner. Biochemical analysis further showed an increase in monomeric species under non-boiled SDS-PAGE conditions, consistent with disruption of oligomeric assembly. Although the observed activity was modest, these results provide experimental evidence that the NPM1 oligomerization interface is amenable to small-molecule modulation. More broadly, this study demonstrates that fragment mapping-guided virtual screening is a practical strategy for identifying ligands targeting protein-protein interaction interfaces.