Probing an allosteric pocket of CDK2 with small-molecules

The availability of well characterized allosteric modulators is of crucial importance for investigating allosteric regulation of protein function. In a recently identified inactive conformation of CDK2 an open allosteric pocket has been detected and proposed as a site to accommodate allosteric inhibitors. Previous structure-based approaches allowed the identification of a hit compound expected to bind to this pocket. Here, we report the characterization of this compound by X-ray crystallography, which surprisingly provided a chemical structure different from the one previously reported. Therefore, the compound was synthesized and completely characterized. X-ray structures of the synthesized and purchased compounds were superimposable. A reaction mechanism was proposed to explain the formation of the structure indicated by X-ray. Moreover, a stereoselective synthesis was developed to evaluate the biological activity of the pure stereoisomers. Modeling studies were performed to unveil the details of the interaction with CDK2. Then, the activity of the obtained compounds was evaluated with different biological assays. Mutagenesis experiments confirmed binding to the allosteric pocket. Finally, the allosteric ligands were shown to inhibit the growth of A549 and SKOV3 cancer cell lines. Therefore, this paper presents a thorough chemical and biological characterization of the first small-molecule ligands to be used as probes to study the allosteric modulation of CDK2 activity.