Pfizer Unveils Novel Strategy to Map Protein-Ligand Binding Pockets with Implications for Cancer Drug Discovery

At the forefront of biological research, scientists continuously explore the mysteries of proteins in hopes of finding new targets and methods for disease treatment. A recent study published by Pfizer has brought a new breakthrough. The article, titled "An allosteric cyclin E-CDK2 site mapped by paralog hopping with covalent probes," reveals an innovative research method and its significant findings in the study of cancer-related proteins.

A method to explore protein ligand-binding pockets by using "paralog-hopping" strategy and electrophile-cysteine interactions discovered in chemical proteomics, applied to the study of cyclin E, providing potential targets for the development of novel therapeutic drugs against cancer.

Through gel-based ABPP experiments, researchers were surprised to find that multiple tryptophan acrylamides showed specific reactivity towards the CCNE1-N112C mutant, with WX-02-520 exhibiting the strongest stereoselectivity.

Further NanoBRET experiments once again verified the specificity of this binding and identified a small molecule binding pocket near N112, which was like finding a beacon in the dark, pointing the way for subsequent research.

Researchers synthesized two 2,6-diazaspiro[3.4]octane inhibitors named I-125A and I-198. Through gel ABPP and NanoBRET assays, they demonstrated the ability of these inhibitors to bind to CCNE1-N112C and wild-type CCNE1:CDK2 complexes, while also inhibiting the binding of the tryptophan acrylamide stereo probe, offering a new approach for modulating protein activity.

X-ray crystallography once again demonstrated its power by resolving the crystal structures of I-125A and I-198 in complex with CCNE1:CDK2. The results revealed that they bind to a hidden allosteric pocket at the CCNE1:CDK2 interface, adjacent to N112 and distinct from the ATP binding site, providing crucial structural insights into the allosteric regulation mechanism of proteins.

Through ADP-Glo kinase assays, it was found that I-125A and I-198 could inhibit the kinase activity of purified CCNE1-N112C:CDK2 and wild-type CCNE1:CDK2 complexes. However, tryptophan acrylamide WX-02-308 and WX-02-14 did not exhibit this inhibitory effect. More interestingly, immunoprecipitation-mass spectrometry experiments revealed that tryptophan acrylamide could selectively stabilize the interaction between the CCNE1-N112C:CDK2 complex and CKS1B and CKS2, while I-125A did not have this effect, indicating that different small molecules exert distinct functional impacts on protein complexes.