Structure and Characterization of a Covalent Inhibitor of Src Kinase

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.

Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries

“…We posited that this was due to the size of the electrophile (~300 Da), such that it occupied only a fraction of the active site. To test this hypothesis, we competed SM-71, a recently published covalent SRC inhibitor 51,52 , with CL126 and measured the BRET signal (Supplementary Fig. 7d).…”

Combining Network Pharmacology with Molecular Docking for Mechanistic Research on Thyroid Dysfunction Caused by Polybrominated Diphenyl Ethers and Their Metabolites

“…The hydroxylated metabolites of BDE-47 and BDE-99 (6-OH-BDE47, 5-OH-BDE47, 3-OH-BDE47, 5-OH-BDE99, and 5′-OH-BDE99) and the sulfate metabolites (6-BDE47 sulfate, 5-BDE47 sulfate, 3-BDE47 sulfate, 5-BDE99 sulfate, and 5′-BDE99 sulfate) were also selected. The top 5 kernel targets screened by network pharmacology, including PIK3R1 [ 27 ] (PDB code: 1H9O), MAPK1 [ 28 ] (PDB code: 1TVO), SRC [ 29 ] (PDB code: 6E6E), RXRA [ 30 ] (PDB code: 1MVC), and TP53 [ 31 ] (PDB code: 6GGA), were used as docking targets with PBDEs and metabolites to verify the accuracy of the prediction. Information on targets and ligands involved in molecular docking is shown (Supplementary Table S3 ).…”

Structural insights into redox-active cysteine residues of the Src family kinases

“…5 B). Fully exposed to solvent, Cys-277 has been the target of various covalent inhibitors of Src, YES, FGR[ 71 , 73 ], in addition to being the focus of the redox-regulation of Src[ 21 , 74 , 75 ]. In the later context, oxidation of Cys-277 to a sulfenic acid is uniquely positioned opposite the folded A-loop in the autoinhibited structure, and oxidation to a sulfenic acid promotes the opening of this loop in the redox-regulation of Src[ 21 ].…”

“…In a general sense, a nucleophilic cysteine, which is ideally suited to be targeted by a drug molecule containing an electrophilic warhead is intuitively likely to be redox-active. For example, both EGFR Cys-797 [ 76 , 102 ] and Src Cys-277 [ 71 , 103 , 104 ] are sites where redox-active cysteines have been targeted by covalent inhibitors. While a series of definitive proteome-level studies comparing the sensitivity of cysteine residues to oxidants and drugs containing cysteine-targeting electrophiles has not been reported, it remains valuable to consider the potential translational applications of identifying reactive cysteines on the basis of their surrounding protein structure.…”