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

Gonzalez-Valero, Angel; Reeves, Audrey G.; Page, Annika C. S.; Moon, Patrick J.; Miller, Edward; Coulonval, Katia; Crossley, Steven W. M.; Xie, Xiao; He, Dan; Musacchio, Patricia Z.; Christian, Alec H.; McKenna, Jeffrey M.; Lewis, Richard A.; Fang, Eric; Dovala, Dustin; Lu, Yipin; McGregor, Lynn M.; Schirle, Markus; Tallarico, John A.; Roger, Pierre P.; Toste, F. Dean; Chang, Christopher J.

doi:10.1021/jacs.2c04039

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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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Abstract

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. Finally, 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.

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY activity-based protein profiling bioconjugation chemoproteomics covalent fragment library redox signaling

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Gonzalez-Valero, Angel, Reeves, Audrey G., Page, Annika C. S., Moon, Patrick J., Miller, Edward, Coulonval, Katia, Crossley, Steven W. M., Xie, Xiao, He, Dan, Musacchio, Patricia Z., Christian, Alec H., McKenna, Jeffrey M., Lewis, Richard A., Fang, Eric, Dovala, Dustin, Lu, Yipin, McGregor, Lynn M., Schirle, Markus, Tallarico, John A., Roger, Pierre P., Toste, F. Dean, Chang, Christopher J.. 2022-12-09. An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4. https://doi.org/10.1021/jacs.2c04039

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