Cofactor Analogues as Active Site Probes in Lysine Acetyltransferases

Lysine acetyltransferases (KATs, also termed histone acetyltransferases, HATs) catalyze the acetylation of substrate lysine residues by employing the cofactor acetyl-coenzyme A (AcCoA), thereby providing a dynamic control mechanism of protein function. Because of their major involvement in cell development and homeostasis, small-molecule modulators of KAT activity are urgently needed to assess their therapeutic potential and for probing their underlying biology. Recent advances in the field suggest that targeting the cofactor binding site represents a promising strategy for identifying potent and selective ligands. Here, we present the synthesis of two functional cofactor-based chemical probes and their usage as mechanistic tools in a broadly applicable assay platform. A fluorescence polarization (FP)-based binding assay was combined with biolayer interferometry competition analysis and a FP competition activity immunoassay to enable easy, reliable, and profound evaluation of ligands that target the KAT cofactor binding site.