Development of a Lysine‐Reactive Targeted Covalent Inhibitor for the P300/CBP‐Associated Factor Bromodomain Through Structure‐Based Design

Epigenetics is defined by changes in heritable phenotypes that do not involve a change in DNA sequence. P300/CBP‐associated factor (PCAF) is an important epigenetic regulatory protein that can alter chromatin through a histone acetyltransferase domain, while also serving as an epigenetic reader through a C‐terminal bromodomain. PCAF promotes the transcription of the HIV‐1 genome and is implicated in the development of glioblastoma. The currently reported PCAF inhibitors are non‐covalent and require high concentration to maintain target occupancy. Here, we explore a new approach using covalent inhibition. Starting with a lead scaffold (BZ1), test‐molecules were rationally designed for selectively targeting PCAF by installing lysine‐reactive groups onto the lead scaffold to enable covalent bond formation with the nonconserved lysine residue in the PCAF bromodomain. The inhibition, selectivity, and kinetic properties ( k inact / K I ) of these molecules were evaluated using intact protein mass spectrometry, while biophysical and cellular data were employed to verify the covalent mechanism and in‐cell target engagement. After optimization, we developed the first PCAF covalent inhibitor, 10 , which labeled PCAF covalently in vitro and engages PCAF in cells. The covalent inhibitor, 10 , represents a useful starting point for future inhibitor optimization and heterobifunctional molecule development.