Article révisé par les pairs
Résumé : Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional proteinsites and compounds for therapeutic development. Yet, the vast majority of ABPP methods for covalent drug discovery target highlynucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-ActivatedChemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification.Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified threenew ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array ofheterocyclic, heteroatom, and stereochemically-rich substituents. ABPP screening of this focused library identified 1oxF11 as acovalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner onpurified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactionsstabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4in breast cancer cell lysates. Further biochemical analyses revealed crosstalk between M169 oxidation and T172 phosphorylation,where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. Byidentifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeuticintervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomicsand protein ligand discovery efforts to find and target previously undruggable methionine sites.

Documents en relation

DI-fusion