Acetylation is the most dynamic protein translational modification often associated with increased DNA accessibility and transcription. These acetylated histones recruit transcription and remodeling factors, and their deregulation could result in aberrant expression of survival and growth-promoting genes. Recognition of acetylated lysine is principally mediated by bromodomains (BRDs). Recent studies have shown that BRD9 is preferentially used by cancers that harbor SMARCB1 abnormalities such as malignant rhabdoid tumors and sarcomas. BRD9 is an essential component of the SWI/SNF chromatin remodeling complex, and a critical target required in acute myeloid leukemia. As the biological function of BRD9 in tumorigenesis becomes clear, bromodomain of BRD9 has become a new hot target for effective tumor treatment method.
BRD9 has a different architecture than other bromodomains. Due to larger hydrophobic cavity of BRD9, it can recognize longer propionyl and butyryl marks on lysine. Thus, N-butyryl-lysine (BuK) can selectively bind to BRD9. Our group is specialized in the amber suppression-based noncanonical amino acid (ncAA) mutagenesis technique. We propose to extend this technique using phage-displayed ncAA-containing peptide libraries for the identification of high-affinity and highly selective BRD9 inhibitors.
Phage display is a technique for rapid screening of potential ligands. It is facilitated through the creation of a genetic fusion between a randomized peptide sequence and pIII, a phage coat protein. This direct link between genotype and phenotype allows for peptide screening. We utilized Phage-assisted, Active site Directed Ligand Evolution approach to target BRD9. To identify the binders, we choose 7mer phagemid library which generates 1.5x1010 randomized possible peptides displayed on PIII of bacteriophages. The peptides screened were tested for binding using Bio-Layer Interferometry and inhibition by Alpha Screen assay. Based on SARs second-generation focused selection was done to screen for more potent peptides. Studies resulted in identification of BRD9 binders with increased specificity and affinity. The estimated IC50 for peptide was 0.74 μM and Kd was determined to be 0.53 μM. Second generation selection peptide inhibits protein with IC50 0.54 μM and Kd value 0.104 μM. Selected peptides successfully bind and inhibit BRD9, and we aim to further optimize its cellular target engagement and on-target effects.
