Structure and Characterization of a Covalent Inhibitor of Src Kinase.

Deepak Gurbani,Asim K Bera,Nathaniel J Henning,Kenneth D Westover,Guangyan Du,Nathanael S Gray,Tinghu Zhang,Suman Rao

doi:10.3389/fmolb.2020.00081

Abstract

Unregulated Src activity promotes malignant processes in cancer, but no Src-directed targeted therapies are used clinically, possibly because early Src inhibitors produce off-target effects leading to toxicity. Improved selective Src inhibitors may enable Src-directed therapies. Previously, we reported an irreversible Src inhibitor, DGY-06-116, based on the hybridization of dasatinib and a promiscuous covalent kinase probe SM1-71. Here, we report biochemical and biophysical characterization of this compound. An x-ray co-crystal structure of DGY-06-116: Src shows a covalent interaction with the kinase p-loop and occupancy of the back hydrophobic kinase pocket, explaining its high potency, and selectivity. However, a reversible analog also shows similar potency. Kinetic analysis shows a slow inactivation rate compared to other clinically approved covalent kinase inhibitors, consistent with a need for p-loop movement prior to covalent bond formation. Overall, these results suggest that a strong reversible interaction is required to allow sufficient time for the covalent reaction to occur. Further optimization of the covalent linker may improve the kinetics of covalent bond formation.

Highlights

SRC was among the first oncogenes to be discovered (Stehelin et al, 1976) and encodes a nonreceptor protein tyrosine kinase that regulates many cancer-related cellular processes including mitogenesis, angiogenesis, adhesion, invasion, migration, and survival (Sen and Johnson, 2011)
We subsequently showed that SM-1-71 could be optimized for Src inhibition by hybridization with dasatinib (Figures 1A–D, Figure S1; Du et al, 2020)
We evaluated the relative potency of inhibitors on Src enzymatic activity using a mobility shift assay (MSA), which measures phosphorylation of a peptide substrate of Src

Summary

Introduction

SRC was among the first oncogenes to be discovered (Stehelin et al, 1976) and encodes a nonreceptor protein tyrosine kinase that regulates many cancer-related cellular processes including mitogenesis, angiogenesis, adhesion, invasion, migration, and survival (Sen and Johnson, 2011). Src activity drives malignant phenotypes in hematologic and solid cancers including breast, prostate, lung, colorectal, and pancreatic cancer (Araujo and Logothetis, 2010; de Felice et al, 2016; Appel et al, 2017). Genetic ablation of Src in animal models reverses cancer phenotypes without systemic toxicity (Trevino et al, 2006; Ammer et al, 2009; Marcotte et al, 2012), suggesting that Src inhibition may be effective in treating certain cancers (Araujo and Logothetis, 2009; Zhang et al, 2009; Chen et al, 2014; Anderson et al, 2017; Appel et al, 2017). Selective Src inhibition has not been demonstrated as a driver of efficacy for any of the clinically used multi-targeted Src drugs.

Methods

Results

Conclusion