title: Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2.
abstract: Allosteric targeting of protein kinases via displacement of the structural αC helix with type III allosteric inhibitors is currently gaining a foothold in drug discovery. Recently, the first crystal structure of CDK2 with an open allosteric pocket adjacent to the αC helix has been described, prospecting new opportunities to design more selective inhibitors, but the structure has not yet been exploited for the structure-based design of type III allosteric inhibitors. In this work we report the results of a virtual screening campaign that resulted in the discovery of the first-in-class type III allosteric ligands of CDK2. Using a combination of docking and post-docking analyses made with our tool BEAR, 7 allosteric ligands (hit rate of 20%) with micromolar affinity for CDK2 were identified, some of them inhibiting the growth of breast cancer cell lines in the micromolar range. Competition experiments performed in the presence of the ATP-competitive inhibitor staurosporine confirmed that the 7 ligands are truly allosteric, in agreement with their design. Of these, compound 2 bound CDK2 with an EC50 value of 3 μM and inhibited the proliferation of MDA-MB231 and ZR-75-1 breast cancer cells with IC50 values of approximately 20 μM, while compound 4 had an EC50 value of 71 μM and IC50 values around 4 μM. Remarkably, the most potent compound 4 was able to selectively inhibit CDK2-mediated Retinoblastoma phosphorylation, confirming that its mechanism of action is fully compatible with a selective inhibition of CDK2 phosphorylation in cells. Finally, hit expansion through analog search of the most potent inhibitor 4 revealed an additional ligand 4g with similar in vitro potency on breast cancer cells.
authors.affiliation: Life Sciences Department; University of Modena and Reggio Emilia; Modena, Italy.
authors.forename: Giulio
authors.initials: G
authors.lastname: Rastelli
authors.affiliation: Life Sciences Department; University of Modena and Reggio Emilia; Modena, Italy.
authors.forename: Andrew
authors.initials: A
authors.lastname: Anighoro
authors.affiliation: IRCCS - Istituto di Ricerche Farmacologiche "Mario Negri"; Milan, Italy.
authors.forename: Martina
authors.initials: M
authors.lastname: Chripkova
authors.affiliation: IRCCS - Istituto di Ricerche Farmacologiche "Mario Negri"; Milan, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Carrassa
authors.affiliation: IRCCS - Istituto di Ricerche Farmacologiche "Mario Negri"; Milan, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Broggini
mesh_terms: D000494:Allosteric Regulation; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D017076:Computer-Aided Design; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D062105:Molecular Docking Simulation; D058990:Molecular Targeted Therapy; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D016160:Retinoblastoma Protein / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship
chemical_list: D000970:Antineoplastic Agents; D008024:Ligands; D047428:Protein Kinase Inhibitors; D016160:Retinoblastoma Protein; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2
keywords: BEAR; Cyclin-dependent kinase 2; allosteric inhibitors; protein kinase; structure-based drug design; virtual screening