An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4

Activity-based protein profiling (ABPP) is a versatile
strategy
for identifying and characterizing functional protein sites and compounds
for therapeutic development. However, the vast majority of ABPP methods
for covalent drug discovery target highly nucleophilic amino acids
such as cysteine or lysine. Here, we report a methionine-directed
ABPP platform using Redox-Activated Chemical 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
three new ligandable methionine sites. We then synthesized a methionine-targeting
covalent ligand library bearing a diverse array of heterocyclic, heteroatom,
and stereochemically rich substituents. ABPP screening of this focused
library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric
M169 site. This compound inhibited kinase activity in a dose-dependent
manner on purified protein and in breast cancer cells. Further investigation
of 1oxF11 found prominent cation-π and H-bonding interactions
stabilizing the binding of this fragment at the M169 site. Quantitative
mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast
cancer cell lysates. Further biochemical analyses revealed cross-talk
between M169 oxidation and T172 phosphorylation, where M169 oxidation
prevented phosphorylation of the activating T172 site on CDK4 and
blocked cell cycle progression. By identifying a new mechanism for
allosteric methionine redox regulation on CDK4 and developing a unique
modality for its therapeutic intervention, this work showcases a generalizable
platform that provides a starting point for engaging in broader chemoproteomics
and protein ligand discovery efforts to find and target previously
undruggable methionine sites.