Dynamics of inhibitor binding and regulation of protein tyrosine kinases

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PROJECT SUMMARY. Protein kinases are a large family of ubiquitous signaling enzymes in human cells. Their dysregulation often underlies diseases such as cancer, making them excellent therapeutic targets, when drug specificity can be achieved. However, the high structural and sequence conservation of the protein kinase catalytic domains has complicated the development of specific inhibitors. The few clinically-successful kinase inhibitors achieve specificity in part by binding only to distinct kinase conformations. While the analysis of thousands of X-ray crystal structures of protein kinases has shown that a single kinase domain can access different active and inactive conformations, little is known about how kinases interconvert between the conformations. The rationale of this proposal is that a quantitative understanding of the stability of these conformations and the dynamics of their interconversion are key to understanding kinase activity, regulation and ligand binding in health and disease states. The objective of this project is to describe the kinetic and equilibrium parameters for the conformational interconversions within the kinase domains of tyrosine kinases Src, Abl, Brk and the promiscuous drug-binding tyrosine kinase DDR1. This proposal is part of a continuum of research centered around four questions that concern the role of conformational dynamics of protein kinases in kinase regulation (Q1), allosteric modulation (Q2), ligand binding kinetics (Q3) and drug specificity/kinase promiscuity (Q4): Q1: What are the thermodynamics and kinetics of conformational exchange in tyrosine kinases? Q2: How are allosteric signals communicated through protein domains and how can binding sites for allosteric regulators be predicted? Q3: What are the molecular determinants of ligand-binding kinetics? Q4: Why do some kinases bind inhibitors promiscuously and how can specific inhibitors with cellular potency be developed? The PI and his team will study these questions through a combination of structural methods (X-ray and NMR), ligand binding kinetics, protein engineering, chemical biology and computational methods. A network of productive collaborations supports this project. The impact of this project is to provide clinicians with the mechanism of resistance mutations, cell biologists with parameters to understand kinase signaling and medicinal chemists with parameters to modulate ligand binding kinetics. The long-term goal is to lay the foundation for the design of safe and effective, sufficiently specific, inhibitors of disease-associated protein kinases.