An allosteric cyclin E-CDK2 site mapped by paralog hopping with covalent probes

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Over half of human genes have paralogs, posing challenges for targeted drug development.
• Chemical proteomics identifies cysteine residues reactive to electrophiles, often specific to protein subsets.

Purpose of the Study:

• To investigate if covalent compound-cysteine interactions can reveal ligandable pockets in paralogs lacking specific cysteines.
• To develop novel assays for identifying inhibitors of cyclin-dependent kinase 2 (CDK2) complexes.

Main Methods:

• Engineered a CCNE1 paralog mutant (N112C) to mimic CCNE2's cysteine reactivity.
• Utilized activity-based protein profiling and NanoBRET assays for compound screening.
• Employed X-ray crystallography to elucidate inhibitor binding sites.

Main Results:

• The CCNE1-N112C mutant selectively reacted with tryptoline acrylamides.
• Developed assays identified reversible inhibitors for both mutant and wild-type CCNE1:CDK2 complexes.
• Discovered a cryptic allosteric pocket at the CCNE1:CDK2 interface binding these inhibitors.

Conclusions:

• Electrophile-cysteine interactions mapped by chemical proteomics can identify druggable pockets in protein paralogs.
• This strategy expands the concept of protein ligandability beyond direct covalent modification.
• Provides a framework for discovering inhibitors targeting protein families with paralogs.