Small Molecule Modulation of NOS1AP for Molecularly Targeted Glioblastoma Therapy.

NOS1AP (CAPON) is a signaling adaptor implicated in glioblastoma (GBM) proliferation, but its chemical tractability remains largely unexplored. Here, affinity selection-mass spectrometry screening of 10,000 drug-like compounds identified NGM1, a small molecule scaffold that directly binds NOS1AP. Binding was confirmed by microscale thermophoresis (MST), yielding a dissociation constant of 11.9 ± 7.0 μM. Focused structure-activity analysis and molecular modeling identified structural features associated with NOS1AP recognition and provided a framework for compound optimization. In living cells, NGM1 produced a concentration-dependent reduction in the NanoBRET signal generated by the NOS1AP-NOS1 reporter pair, consistent with perturbation of the complex. NGM1 reduced viability in U87 and U251 GBM cells, with IC50 values of 12.7 ± 0.91 and 17.3 ± 1.04 μM, respectively, while normal human astrocyte viability remained above 50% at 150 μM. Mechanistic studies in U87 cells demonstrated reduced DNA synthesis, G0/G1 cell-cycle accumulation, and induction of apoptosis, accompanied by increased p53 and CDKN1A and decreased CDK6. Collectively, these findings establish the initial chemical tractability of NOS1AP and identify NGM1 as a chemical scaffold for investigating NOS1AP-associated signaling in GBM. The findings also provide a foundation for optimizing NOS1AP-directed compounds.