Modulating the Global Conformations of c-Src Kinase

Abstract

Protein Kinases are key regulators of important cellular processes and have been a validated therapeutic target for cancer over the past two decades. Kinase signaling is a combination of its catalytic function, through post-translational phosphorylation, and non-catalytic functions, often through protein-protein interactions. The design of kinase inhibitors to inhibit kinase signaling has solely focused on inhibition of the catalytic process. However, evidence of inhibitors modulating non-catalytic functions, through stabilizing distinct kinase conformations, has uncovered a novel strategy for modulating of both arms of kinase signaling. Unfortunately, the lack of tools to elucidate kinase conformational changes has stalled the targeting of these non-catalytic processes. The work herein highlights methods, both ligand and mutation induced, to modulate the global conformation of c-Src kinase. Characterization of these conformational changes was made possible by the development of our novel ‘Selective Proteolysis’ methodology that takes advantage of c-Src’s sensitivity to the protease thermolysin. Using this methodology we have characterized a panel of clinical c-Src mutations and have uncovered W121R to be a potential activating c-Src mutation. To improve the targeting strategies of c-Src, we developed series of ATP competitive, conformation-tunable inhibitors, to inhibit the catalytic function of c-Src, while simultaneously modulating its non-catalytic functions. Furthermore, we utilized MixMD methodology to identify two novel allosteric hotspots of c-Src that can be utilized to identify allosteric modulators of c-Src. This work demonstrates that inhibitors of c-Src can be used to modulate both catalytic and non-catalytic functions simultaneous. Additionally, that clinical mutations may be activating through stabilization of district protein conformations. We hope that others utilize this work as a stepping-stone towards developing the next generation of kinase targeted therapies with improved signaling modulation.