Fragment Screening and Druggability Assessment for the CBP/p300 KIX Domain through Protein-Observed 19F NMR Spectroscopy

19F NMR spectroscopy of labeled proteins is a sensitive method for characterizing structure, conformational dynamics, higher‐order assembly, and ligand binding. Fluorination of aromatic side chains has been suggested as a labeling strategy for small‐molecule ligand discovery for protein–protein interaction interfaces. Using a model transcription factor binding domain of the CREB binding protein (CBP)/p300, KIX, we report the first full small‐molecule screen using protein‐observed 19F NMR spectroscopy. Screening of 508 compounds and validation by 1H–15N HSQC NMR spectroscopy led to the identification of a minimal pharmacaphore for the MLL‐KIX interaction site. Hit rate analysis for the CREB‐KIX and MLL‐KIX sites provided a metric to assess the ligandability or “druggability” of each interface informing future medicinal chemistry efforts. The structural information from the simplified spectra and data collection speed, affords a new screening tool for analysis of protein interfaces and discovery of small molecules. A full small‐molecule screen has been achieved using protein‐observed 19F NMR (PrOF NMR) spectroscopy of a model transcription factor binding domain of the CREB binding protein (CBP)/p300, KIX. This study demonstrates the applicability of PrOF NMR as a tool for library screening, ligand discovery, and druggability assessment.