Chemical Tools for the Selective Control of Proteins: Protein Kinases and Acetyl Transferases

Abstract

Post-translational modifications (PTMs) of proteins are now known to increase the complexity of biological signaling networks. The ability to selectively control the activity of individual proteins and enzymes involved in PTMs enables studies of biological systems and in designing therapeutic agents. The phosphorylation of serine, threonine, and tyrosine catalyzed by protein kinases play an important role in signaling and when deregulated are involved in different types of cancer amongst other diseases. Therefore, selective regulation of kinase activity by inhibition could provide a means to understand signaling and also aid in developing therapies. The design of specific kinase inhibitors is challenging because most inhibitors target the common ATP-binding site, leading to several off-target hits with adverse effects. With this in mind, a fragment-based bivalent strategy was developed to potentially increase affinity and selectivity. Our approach seeks to target the ATP-binding site with a small molecule while simultaneously targeting any available site on the kinase with a cyclic peptide that potentially increases selectivity and affinity. The bivalent approach has produced effective and selective inhibitors for cAMP-dependent protein kinase (PKA). Herein, we extended this approach to Aurora kinase (Aurora A), RSK1 and MSK1. Using our optimized selection conditions, we have successfully selected several cyclic peptide ligands to later generate bivalent ligands for these kinases. This strategy may prove a robust method to discover new allosteric sites on kinases as well as other proteins, furthermore it also has the potential to provide insight towards the design of new ATP targeted approaches. A second area of research focuses on PTMs involved in the acetylation of lysine residues by enzymes dubbed histone acetyl transferases (HATs) or lysine acetyl transferases (KATs). Recent studies have shown that a large fraction of the proteome may be modifies by KATs but it is challenging to develop uniquely selective inhibitors as the KATs like Kinases have similar active sites. Towards the goal of developing a method to control a single KAT in the presence of other KATs, we designed ligand-gated split-proteins based on a sequence dissimilarity based approach. Potential fragmentation sites were identified by insertion of a 25-residue loop insertion. Successful loop insertion mutants provided guidance for the dissection of KATs into fragments tethered to FKBP and FRB that cannot spontaneously assemble, but that do upon the addition of a ligand, rapamycin to generate catalytically active. The method was successfully applied to design the very first split-KAT, GCN5,which only showed activity in the presence of an added small molecule. The method was shown to be potentially general by application to a second KAT, PCAF. The above studies provide potentially new approaches for selectively targeting and studying PTMs catalyzed by kinases and lysine acetyl transferase.