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

Area of Science:

• Biochemistry
• Structural Biology
• Neuroscience

Background:

• Protein-protein interactions involving flexible domains are challenging to target with drugs.
• Static protein structures often miss crucial ligand-binding conformations.

Purpose of the Study:

• To develop a method for small molecule discovery against dynamic protein interfaces using NMR data.
• To identify modulators of the neuronal nitric oxide synthase (nNOS) PDZ domain interaction with CAPON.

Main Methods:

• AtlasNMR framework to generate conformational hypotheses from NMR ensembles.
• Ensemble-based virtual screening and consensus ranking.
• In vitro and cellular assays to validate small molecule efficacy.

Main Results:

• AtlasNMR identified two key conformational states of the nNOS PDZ domain.
• The identified small molecule MC-3 effectively disrupts the NOS1-NOS1AP interaction.
• MC-3 demonstrated neuroprotective effects in disease models, reducing cytotoxicity and nitrosative stress.

Conclusions:

• AtlasNMR provides a generalizable strategy for drug discovery against dynamic protein interfaces.
• MC-3 represents a promising lead compound for neurological disorders.
• Exploiting NMR ensemble heterogeneity is key for targeting challenging protein interactions.