Novel Kinase Allostery FRET Assays for Mechanistic Studies and Drug Design

University Of Minnesota, Minneapolis MN

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Abstract

PROJECT SUMMARY/ABSTRACT. Signal transduction by protein kinases controls many aspects of cell development and proliferation. Deregulation of kinases has been linked to many cancers, and kinase inhibitors are an important class of chemotherapeutic drugs. However, most currently available inhibitors are poorly selective (because they target the conserved ATP binding site), and clinical resistance within one year is nearly universal. There is thus an increasing demand for new and more specific kinase inhibitors. The focus of regulation for most Ser/Thr kinases is on the conformational transition between active and autoinhibited states. Each kinase has a unique set of allosteric mediators of this transition, including small molecule ligands, post- translational modifications of the kinase, and protein-protein interactions. Active and autoinhibited states are distinguished by the conformations of a few conserved structural features, including the kinase activation loop. In allosteric activation, according to x-ray crystallography, activation loop residues move several nanometers, unblocking the substrate peptide binding site and positioning a conserved Asp residue for catalysis. Yet, very little is known about how these conformational changes occur in solution and their role in kinase activity because there are currently no real-time assays for the structural state and dynamics of the kinase. In this proposal, we describe the use of a novel assay determining the conformation of the activation loop of human Aurora A kinase (AurA), a key regulator of mitosis which has been implicated in cancer pathogenesis. I use site-directed mutagenesis to incorporate two fluorescent probes into a single AurA molecule, and I then measure the distance between them using the nanometer-scale measurement technique of Förster resonance energy transfer (FRET). I will use this assay, in conjunction with activity assays, point mutation of critical residues, and state-of-the-art time-resolved FRET and kinetics techniques, to define the conformational ensembles and structural elements governing AurA activation (Aim 1). I will also use this assay to characterize and screen AurA allosteric inhibitor drugs using FRET (Aim 2). I will elucidate the binding modes of existing inhibitors and will improve upon currently available high-throughput drug screening methods for allosteric inhibitors which bind specifically outside the kinase active site. We anticipate that our FRET assay will lead to major advances in drug discovery and the understanding of allosteric regulation. Under this award, I will train for three years as a postdoctoral scholar at the University of Minnesota with Dr. Nicholas Levinson, an expert in kinase structural biology, and Dr. David Thomas, an expert in fluorescence spectroscopy and drug discovery methods. Under their dual mentorship, I will develop skills in writing, presenting, and collaboration, as well as laboratory techniques in biophysics, structural biology, and drug development. This training will prepare me to be an independent researcher studying mechanisms of protein allostery and its applications for drug design.