Structure-Based Design of a Potent and Selective Covalent Inhibitor for SRC Kinase That Targets a P-Loop Cysteine

Abstract

SRC is a major regulator of many signaling pathways and contributes to cancer development. However, development of a selective SRC inhibitor has been challenging, and FDA-approved SRC inhibitors, dasatinib and bosutinib, are multitargeted kinase inhibitors. Here, we describe our efforts to develop a selective SRC covalent inhibitor by targeting cysteine 277 on the P-loop of SRC. Using a promiscuous covalent kinase inhibitor (CKI) SM1-71 as a starting point, we developed covalent inhibitor 15a, which discriminates SRC from other covalent targets of SM1-71 including TAK1 and FGFR1. As an irreversible covalent inhibitor, compound 15a exhibited sustained inhibition of SRC signaling both in vitro and in vivo. Moreover, 15a exhibited potent antiproliferative effects in nonsmall cell lung cancer cell lines harboring SRC activation, thus providing evidence that this approach may be promising for further drug development efforts.

Structure and Characterization of a Covalent Inhibitor of Src Kinase

“…Our Src-SM1-71 crystal structure (PDB: 6ATE) revealed the Src p-loop in a kinked conformation (Rao et al, 2019). 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). Here, we present formal biochemical, biophysical, and structural characterization of Src inhibition by DGY-06-116.…”

“…Altogether, our interpretation of these findings is that a strong reversible interaction is required to allow sufficient time for the ploop to sample a kinked conformation compatible with covalent bond formation. We speculate that that optimization of the interaction between the p-loop and compound may increase the inactivation rate, leading to further improvements in compound selectivity in biological systems, since covalent bond formation appears to be the major driver of selectivity (Du et al, 2020). One way to do this would be to increase the length of the linker to the covalent warhead so the p-loop does not have to kink.…”

“…DGY-06-116 was previously characterized for the ability to bind Src (Du et al, 2020). Here, 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.…”

“…While the crystal structure clearly indicates covalent binding to Src, the enzymatic assay could not distinguish between DGY-06-116 and NJH-01-111. Nevertheless, clear differences are seen in the selectivity of these compounds (Du et al, 2020). Previously, we used the MSA to measure the inactivation rate of other covalent inhibitors (Tan et al, 2017).…”

Identification of Novel Src Inhibitors: Pharmacophore-Based Virtual Screening, Molecular Docking and Molecular Dynamics Simulations

“…The search for small molecules with an inhibitory activity toward Src kinases constitutes a growing field of study. Several compounds have entered clinical trials, with two compounds ultimately approved by the FDA: dasatinib, approved in 2006, and bosutinib, approved in 2012 [ 16 ]. However, dasatinib is known to inhibit over 40 kinases, while bosutinib inhibits over 45 kinases, making it impossible to use these compounds as selective mechanistic probes for Src-dependent pharmacology [ 17 , 18 ].…”