Understanding Src Kinase Regulatory Mechanisms and Drug Resistance Using Deep Mutational Scanning and Chemical Biology

Abstract

Protein phosphorylation controls a wide variety of cellular processes such as growth, differentiation, proliferation, and apoptosis in eukaryotes. Kinases are signaling enzymes that dictate cellular phosphorylation state by phosphorylating specific protein substrates. Over 530 protein kinases are encoded by the human genome and roughly half of these enzymes have at least one accessory domain in addition to the catalytic domain. The Src Family Kinases (SFKs) are a well-studied family of multi-domain, non-receptor tyrosine kinases. SFKs participate in numerous signal transduction pathways, and their misregulation is implicated in a variety of diseases, including cancer. Therefore, SFKs are of general interest as a model for understanding multi-domain kinase regulation and as potential drug targets. In this thesis, I describe my efforts to use the SFKs as models for understanding inhibitor selectivity and drug resistance, which are major challenges in the field. I show that it is possible to develop ATP-competitive inhibitors that are highly selective for the SFK Lyn over other members of the SFKs. Obtaining such selectivity is significant because the ATP-binding sites of SFKs are almost identical and obtaining selectivity amongst such closely related kinases has proven to be particularly challenging. I show that it is possible to achieve selectivity for Lyn by developing inhibitors that target a region called the helix C. With a series of sequence swap experiments, I demonstrate that sensitivity to these Lyn-selective inhibitors is due to the identity of the linker residues that control the conformational flexibility of helix C rather than any specific ATP-binding site interactions. Our strategy may hold promise for selectivity targeting other protein kinases. I also describe efforts to better understand how kinases develop resistance to ATP-competitive inhibitors. To do this, we used saturation mutagenesis and a yeast screening assay to comprehensively identify sites of ATP-competitive inhibitor resistance in Src. Using this methodology, resistance to the drug dasatinib and to a panel of conformation-selective, ATP-competitive inhibitors was profiled. Our efforts led to identification of mutations that provide general resistance to ATP-competitive inhibitors and mutations that uniquely affect specific modes of ATP-competitive inhibition. Interestingly, some of the strongest resistance mutations identified do not directly affect inhibitor binding but, instead, appear to confer resistance through other mechanisms. I describe comprehensive biochemical and biophysical analyses to better understand how a region on the N-terminal lobe of Src’s catalytic domain, which was previously identified as participating in the regulation of Src through an unknown mechanism, confers broad resistance. As a major challenge in the field of kinase drug discovery is the emergence of drug resistance, our efforts may help better inform the development of chemotherapeutic treatments that are less prone to resistance.