Structural and Biochemical Basis for Intracellular Kinase Inhibition by Src-specific Peptidic Macrocycles

Abstract

Protein kinases are attractive therapeutic targets because their dysregulation underlies many diseases, including cancer. The high conservation of the kinase domain and the evolution of drug resistance, however, pose major challenges to the development of specific kinase inhibitors. We recently discovered selective Src kinase inhibitors from a DNA-templated macrocycle library. Here, we reveal the structural basis for how these inhibitors retain activity against a disease-relevant, drug-resistant kinase mutant, while maintaining Src specificity. We find that these macrocycles display a degree of modularity: two of their three variable groups interact with sites on the kinase that confer selectivity, while the third group interacts with the universally conserved catalytic lysine and thereby retains the ability to inhibit the “gatekeeper” kinase mutant. We also show that these macrocycles inhibit migration of MDA-MB-231 breast-tumor cells. Our findings establish intracellular kinase inhibition by peptidic macrocycles, and inform the development of potent and specific kinase inhibitors.

Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-L-phenylalanine

“…The two structures reported here represent the first reported X-ray crystal structures with pNO 2 F incorporated into a protein structure using the amber codon-suppression technology. Previous structures containing the pNO 2 F residue contained pNO 2 F on a synthesized short peptide fragment (4-8 residues; Rose et al, 1996;Lizak et al, 2011;Aleem et al, 2016;Napió rkowska et al, 2017) or a semi-synthetic protein in which the pNO 2 F was synthesized in a peptide that was later ligated to an expressed protein fragment (Baril et al, 2017). The Asp133pNO 2 F sfGFP and Asn149pNO 2 F sfGFP crystal structures illustrate clear density for the pNO 2 F residue at the specified position, indicating that pNO 2 F can be sitespecifically incorporated to produce a stable pNO 2 F group.…”

Second-generation DNA-templated macrocycle libraries for the discovery of bioactive small molecules

“…Despite its modest size compared with subsequent industrial DNA-encoded libraries 9, 11, 42 , this initial library was of sufficient quality and diversity to serve as a source of potent and selective inhibitors of proteins including kinases and insulin-degrading enzyme (IDE) protease, ultimately leading to biological discoveries and the validation in vivo of new targets for therapeutic intervention. 43–46 …”

Selective targeting of the inactive state of hematopoietic cell kinase (Hck) with a stable curcumin derivative

“…The MDA-MB-231 cells rely on Src signalling for cell migration and metastasis ( Figure 4 b). ( 44 , 45 ) We observed that 4 inhibits the autophosphorylation of Src in the cell ( Figure 4 d) and significantly impaired the wound healing rates (IC 10 and IC 50 dose) ( Figure S8b-c ). The migration process involves the TNF-α induced activation of Src signalling cascade and translocation of ERK1/2 to the nucleus.…”