Identification of a First-In-Class Small Molecule CAPON Binder Using Affinity Selection-Mass Spectrometry Screening.

NOS1AP (CAPON) is an adaptor protein of neuronal nitric oxide synthase (nNOS) correlated with Alzheimer's disease progression, making it an attractive yet unexplored therapeutic target. To assess its chemical tractability, we employed affinity selection-mass spectrometry (AS-MS) to screen approximately 10,000 small molecules for CAPON binding, identifying 52 initial hits. These compounds were further evaluated for true binding interactions and potential autofluorescence or quenching effects using the Dianthus platform. Five compounds were selected for quantitative affinity determination by microscale thermophoresis (MST). Among these, compound MA32 exhibited a dissociation constant (Kd) of 74 μM. MA32 thus represents the first AS-MS-identified small-molecule binder of CAPON. This study establishes a workflow for small-molecule discovery against novel, previously uncharacterized targets.