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Comparative Study
, 13 (14), 2296-305

Structure-based Discovery of the First Allosteric Inhibitors of Cyclin-Dependent Kinase 2

Comparative Study

Structure-based Discovery of the First Allosteric Inhibitors of Cyclin-Dependent Kinase 2

Giulio Rastelli et al. Cell Cycle.


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: BEAR; Cyclin-dependent kinase 2; allosteric inhibitors; protein kinase; structure-based drug design; virtual screening.


Chart 1. Chemical structures of the allosteric inhibitors 1–7 and those resulting from the hit expansion of compound 4.
Figure 1. Concentration-dependent displacement of ANS from CDK2. Panel (A) reports the displacement activity of compounds 1–7, while (B) shows the activity of six (4b–4g) of the nine compounds derived from the hit expansion of compound 4. Compounds 4a, 4h, and 4i did not show appreciable displacement activity.
Figure 2. Inhibition of Rb phosphorylation by compound 4 and SU9516 in MDA-MB231 cells. Proteins were separated on SDS page, electroblotted to nitrocellulose, and hybridized with antibodies against phoshorylated Rb (T821, left panel and S780, right panel). Blots were subsequently hybridized with antibodies against total Rb and actin (used as loading control). C, control, untreated cells; SU, cells treated with SU9516 (10 μM); 4, cells treated with compound 4 (4 μM).
Figure 3. Predicted binding modes of compounds 1–7 in the allosteric pocket of CDK2, highlighting (left) the location of each compound in the overall fold, (right) the close up of the allosteric site with selected residues interacting with the ligands. For comparative purposes, the first box shows the binding mode of the 2 ANS molecules present in the crystal structure of the binary complex (PDB code 1PXF).

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