Chemical Tools for Profiling the Allosteric Regulation and Interactomes of Inhibitor-Bound Protein Kinases

Abstract

This dissertation describes the development of a chemoproteomic method for profiling the interaction network of inhibitor-bound kinase complexes and chemical probes for understanding how ATP-competitive inhibitors can allosterically modulate tyrosine kinases that contain a Src-like regulatory architecture. Small molecule inhibitors often only block a subset of the cellular functions of their protein targets. In many cases, how inhibiting only a portion of a multifunctional protein’s functions affects the state of the cell is not well understood. Therefore, tools that allow the systematic characterization of the cellular interactions that inhibitor-bound proteins make would be of great utility, especially for multifunctional proteins. In the second chapter of this thesis, I describe a chemoproteomic strategy for interrogating the cellular localization and interactomes of inhibitor-bound kinases. By developing a set of orthogonal inhibitors that contain a trans-cyclooctene (TCO) click handle, we are able to enrich and characterize the proteins complexed to a drug-sensitized variant of the multidomain kinase Src. We show that Src’s cellular interactions are highly influenced by the intermolecular accessibility of its regulatory domains, which can be allosterically modulated through its ATP-binding site. Furthermore, we find that the signaling status of the cell also has a large effect on Src’s interactome. Finally, we demonstrate that our TCO-conjugated probes can be used as a part of a proximity ligation assay to study Src’s localization and interactions in situ. Together, our chemoproteomic strategy represents a comprehensive method for studying the localization and interactomes of inhibitor-bound kinases and, potentially, other druggable protein targets. Small molecule kinase inhibitors that stabilize distinct ATP-binding site conformations can differentially modulate the global conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP-binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects are general to other tyrosine kinases. In the third chapter of this thesis, I describe the development of chemical probes that allow us to deconvolute which features in the ATP-binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src’s regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix C. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src’s helix C in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Taken together, our study highlights that an ATP-competitive inhibitor’s interactions with helix C can have a major influence on the global conformation of some tyrosine kinases in vitro and in cells.