Abstract

Human protein kinases play essential roles in cellular signaling pathways and - if deregulated - are linked to diverse diseases such as cancer and inflammation or metabolic diseases. Because of their key role in disease development or progression, kinases have developed into major drug targets resulting in the approval of 89 kinase inhibitors by the FDA thus far. Within the drug discovery process, affinity of the developed inhibitors is the parameter used most often to predict efficacy in humans. However, binding kinetics has recently emerged as an important but largely neglected factor of kinase inhibitor efficacy. There is increasing evidence of correlation between prolonged drug-target residence time and improved drug efficacy. Moreover, inhibitor selectivity in cellular contexts can be modulated by altered residence times. To contribute to the understanding of the effect of long residence times in a cellular context we determined a structural mechanism that allows kinetic selection between two closely related kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2). Using an inhibitor series designed to probe the molecular mechanism of kinetic binding, residence times measured in vitro and in cells were determined and showed a strong correlation. Crystal structures and mutagenesis identified hydrophobic interactions with L567, adjacent to the DFG-motif, as being crucial to kinetic selectivity of FAK over PYK2. This specific interaction was observed only when the DFG-motif was stabilized into a helical conformation upon ligand binding to FAK. The interplay between the protein structural mobility and ligand-induced effect was found to be the key regulator of kinetic inhibitor selectivity for FAK over PYK2