NMR strategies to probe allosteric effects of inhibitor binding on AML-associated Src-family kinases

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

Investigators

Abstract

The Src tyrosine kinase family has been validated as a target for drug development against many forms of cancer including myeloid leukemias. A major clinical challenge with kinase-targeted drug therapy has been the emergence of drug-resistant mutants that escape ATP-binding site inhibitors. To overcome this obstacle, the Smithgall lab (co-I on this proposal), focuses on the development of complementary pairs of ATP-site and allosteric inhibitors. This approach aims to enhance target selectivity and eliminate the potential for acquired resistance. Protein NMR is an ideal method for characterizing conformational changes induced by both ATP- site and allosteric inhibitor interaction in solution. Particularly, when an allosteric inhibitor binding site is occluded in a crystal by the packing effect or cannot be co-crystallized, NMR serves a powerful and orthogonal alternative method to crystallography. The goal of the proposed research, therefore, is to design a tailored NMR strategy for investigating inhibitor-induced conformational changes in the Src family kinase Hck, which has been implicated in the etiology of acute myeloid leukemia (AML). In Src family kinase, tryptophan residues are located at several important sites. There are ~10 tryptophan residues in Src family kinase. They are mostly conserved and positioned at key domain interaction sites. As shown for Hck, the indole amide signals are observed without severe signal overlaps. We hypothesize that this unique characteristic will make Trp indole resonances in Hck highly sensitive probes for investigating allosteric modulator effects on remote regions of the protein (Aim 1). We also hypothesize that site-specific 19F detection will be a useful tool to observe resonance changes in key Src-family kinase sites in response to ligand binding (Aim 2). We test these hypotheses by performing NMR experiments. Based on results in both aims, we will formulate a comprehensive NMR strategy for evaluating and categorizing allosteric conformational changes in Hck upon small molecule interaction. Successful completion of this study will significantly advance the development of both ATP-site and allosteric inhibitors targeting the AML-associated target kinase, Hck.