Imatinib binding to human c-Src is coupled to inter-domain allostery and suggests a novel kinase inhibition strategy.

Yuko Tsutsui,Franklin A Hays,Daniel Deredge,Patrick L Wintrode

doi:10.1038/srep30832

Abstract

Imatinib (Gleevec), a non-receptor tyrosine kinase inhibitor (nRTKI), is one of the most successful anti-neoplastic drugs in clinical use. However, imatinib-resistant mutations are increasingly prevalent in patient tissues and driving development of novel imatinib analogs. We present a detailed study of the conformational dynamics, in the presence and absence of bound imatinib, for full-length human c-Src using hydrogen-deuterium exchange and mass spectrometry. Our results demonstrate that imatinib binding to the kinase domain effects dynamics of proline-rich or phosphorylated peptide ligand binding sites in distal c-Src SH3 and SH2 domains. These dynamic changes in functional regulatory sites, distal to the imatinib binding pocket, show similarities to structural transitions involved in kinase activation. These data also identify imatinib-sensitive, and imatinib-resistant, mutation sites. Thus, the current study identifies novel c-Src allosteric sites associated with imatinib binding and kinase activation and provide a framework for follow-on development of TKI binding modulators.

Highlights

Exposure of an activation-loop (A-loop) containing Tyr[416] auto-phosphorylation site, rotation of helix C, and re-organization of the interface between kinase domain N- and C-lobes that result in stabilization of a hydrophobic spine composed of Leu[325], Met[314] in helix C, DFG, and the HRD motif (Fig. 1c–e)
Backbone amide hydrogen atoms undergo deuterium exchange when they are solvent-exposed while amide hydrogen atoms engaged in hydrogen bonds or buried in hydrophobic environment are protected from the exchange[23]
By monitoring the number of deuterium atoms incorporated in each peptic fragment as a function of deuterium labeling time, structural fluctuations in native state ensemble are probed in a region-specific manner (Fig. 2a–e)

Summary

Introduction

Exposure of an activation-loop (A-loop) containing Tyr[416] auto-phosphorylation site, rotation of helix C, and re-organization of the interface between kinase domain N- and C-lobes that result in stabilization of a hydrophobic spine composed of Leu[325], Met[314] in helix C, DFG, and the HRD motif (Fig. 1c–e). These structural changes stabilize a DFG-out conformer in a native state ensemble, suggesting conformational selection of imatinib binding[7]. The emergence of imatinib resistant mutations highlights the importance of such studies for developing new classes of specific TKIs15–17 This may require identification of potential drug target sites distal to the highly conserved kinase domain ATP binding pocket. Analysis of clinically-identified imatinib resistant mutation sites, together with our HDX-MS results, provides intriguing insight into the relationship between allostery and evolution of drug resistant mutations

Methods

Results

Conclusion