An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4
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This paper presents a new methionine-directed activity-based protein profiling (ABPP) platform to identify and target functional methionine sites on proteins for covalent ligand discovery.
Using Redox-Activated Chemical Tagging (ReACT) probes that target methionine residues, the authors identified three hyperreactive, ligandable methionine sites (M169, M264, M275) on the cancer-related protein cyclin-dependent kinase 4 (CDK4). They designed and synthesized a focused library of 179 oxaziridine fragments to screen against CDK4. Fragment 1oxF11 was identified as binding to the M169 site selectively over other CDK isoforms.
Biochemical assays showed 1oxF11 inhibits CDK4 activity in vitro and in cells. Cell viability assays also showed 1oxF11 decreases viability of certain cancer cell lines. Further analyses revealed an interesting crosstalk mechanism where oxidation of M169 prevents phosphorylation of the adjacent T172 site needed to activate CDK4. This identifies a potential new redox-dependent vulnerability in CDK4 regulation.
In summary, this paper introduces a versatile methionine-targeting ABPP approach to identify new functional sites on proteins and develop selective covalent ligands, showcasing its application to discover a new allosteric regulatory mechanism in CDK4. The platform provides a framework to expand covalent ligand discovery efforts beyond commonly targeted residues like cysteine and lysine.
https://pubs.acs.org/doi/full/10.1021/jacs.2c04039
