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.

Keywords: :

• Cyclin-dependent kinase 2
• protein kinase
• allosteric inhibitors
• virtual screening
• structure-based drug design
• BEAR

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Financial support from the Nando Peretti Foundation (grant NPF 2013–25) is gratefully acknowledged. Computational resources were provided by the ISCRA (Italian Super Computing Resource Allocation) grant “BEAR” Class A # HP10A6E2CI. We wish to thank Fabiana Caporuscio for critical reading of the manuscript.