Studies on selectivity determinants of protein kinase inhibitor binding

Abstract

Protein kinases are involved in many essential cellular processes, and are regulated in a dynamic manner by the movement of domains or motifs via interaction with various proteins and substrates. Protein kinase deregulation can lead to a variety of diseases, including cancer and diabetes. Protein kinase A (PKA) has been a prototype to study the entire family, including for studies in drug discovery research. However, the high sequence similarities in the kinase domain of protein kinases hinders the development of target-specific inhibitors, and the use of PKA alone is insufficient. Mutants of PKA can act as surrogate targets to aid the design of target specificity. In this work, we use a combination of several biophysical methods to investigate the properties of PKA, PKA based surrogate kinases (PKAB3 & PKA-Au6), the cancer target Aurora kinase, including especially their interactions with inhibitors. We characterized a series of 32 enantiomerically pure inhibitors with respect to interactions with protein kinase A (PKA) and its mutant PKAB3 as a PKB surrogate. The ligands bind to the hinge region, ribose pocket, while their substituted aromatic rings (phenyl, thienyl) bind to the glycine-rich loop at the ATP site. Biological assays show their high potency against both PKA and PKAB3, generally with preference to PKA. The crystal structures reveal a multi-facetted network of ligand–glycine-rich loop interactions, with efficient water replacement contributing to the binding strength. Site-directed mutagenesis and biophysical characterization of the glycine–rich loop mutants (T51G and G55A-PKA) show weakened affinities against tested inhibitors: 1i, 1p, 1c and H-89. We also show structural effects of the aromatic residue phenylalanine that is highly conserved among the AGC kinase group, corresponding to Phe327 in PKA, and its role in inhibitor binding. The inhibitor-Phe327 interactions reveal a complex mix of favorable and unfavorable contacts: Phe327 can block inhibitors from occupying the ATP-binding pocket, but may also be displaced to enable tight binding. The PKA-based Aurora kinase B (PKA-AU6) model, which shares this Phe327, provides examples of crystal structures (PDB ID: 5N23) showing effects on the binding affinities of the pan-Aurora inhibitor AT9283, and also the novel tricyclic dianilino-pyrimidine inhibitors (1B & 2A). The pan-kinome inhibitor staurosporine is known to be able to displace Phe327. We also observe how bisubstrate inhibitors can be used for studies of strong protein peptide-interactions. In summary, this work advances the understanding of how subtle aspects of flexibility, water structure, and chemical interactions determine inhibitor binding kinetics and energetics. Such detailed understanding is required for truly effective structure based drug design.