Exploiting NMR Ensemble Heterogeneity Enables Small Molecule Discovery against Dynamic Protein–Protein Interfaces

High Resolution Image Download MS PowerPoint Slide Protein–protein interactions governed by conformationally heterogeneous domains remain difficult to drug because ligand-competent states are often absent from single static structures. Here, we present AtlasNMR, a statistical framework that transforms multimodel NMR ensembles into screening-ready conformational hypotheses for small molecule discovery. Using the neuronal nitric oxide synthase (nNOS) PDZ domain that engages the adaptor protein CAPON (NOS1AP) as a model system, AtlasNMR identified two representative conformational states capturing the dominant and minor populations of the NMR ensemble. Ensemble-based virtual screening, followed by consensus ranking, yielded MC-3, a small molecule modulator that disrupts the NOS1-NOS1AP interaction in live cells and directly engages the nNOS PDZ domain. MC-3 produced convergent neuroprotective effects in disease-relevant neuronal models by reducing amyloid-β-induced cytotoxicity, suppressing NMDA-driven nitrosative stress, and attenuating pathological tau phosphorylation, while exhibiting a balanced early lead-like ADME and safety profile. Together, this work establishes a generalizable strategy for exploiting NMR ensemble heterogeneity to enable small molecule discovery against dynamic protein–protein interfaces.