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, 56 (10), 3768-82

Development of Highly Potent and Selective Diaminothiazole Inhibitors of Cyclin-Dependent Kinases

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Development of Highly Potent and Selective Diaminothiazole Inhibitors of Cyclin-Dependent Kinases

Ernst Schonbrunn et al. J Med Chem.

Abstract

Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009-0.0015 μM) from a single hit compound with weak inhibitory activity (IC50 = 15 μM), discovered by high-throughput screening. Structure-based design was performed using 35 cocrystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4, and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 μM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics.

Figures

Figure 1
Figure 1. Interaction of HTS hit compound 1 with CDK2
(a) The co-crystal structure of CDK2 with hit 1 revealed a typical Type I mechanism of action by binding through the hinge-region of the ATP site. The enzyme is shown in grey with the hinge region (residues 81–83) highlighted in orange, gatekeeper residue Phe80 in red, and DFG motif in cyan. The exploded view shows the hydrogen bonding interactions between the inhibitor (yellow) and enzyme residues including Asp145 of the DFG motif. Water molecules are shown as cyan spheres. The 2Fo-Fc electron density map at 1.85 Å resolution is shown as blue mesh, contoured at 1σ around the inhibitor. The Fo-Fc omit map is shown in the supplementary material (Figure S4). (b) Schematic representation of hydrogen bonding (dotted lines) and hydrophobic interactions (green). PDB: 3QQK.
Figure 2
Figure 2. SAR milestones in the optimization of hit compound 1
Figure 3
Figure 3. Influence of R2 pyridine substituents on binding interactions with CDK2
Crystal structures of CDK2 liganded with the pyridine analogues 3 (a), 6 (b), and 10 (c). Meta-pyridine (3) attracts the ε-amino group of Lys33, whereas para-pyridine (10) induces a large conformational change in the P-loop allowing a water molecule to interact with the ring nitrogen. The ortho-pyridine (6) adopts a conformation unfavorable for interaction with CDK2. Figure 3d shows the differences in binding interactions upon superposition of the crystal structures. The enzymes are shown in grey with the hinge region (residues 81–83) colored in orange, the gatekeeper Phe80 residue in red, and Asp145 of the DFG motif in cyan. The hydrogen bonding interactions between the inhibitors (yellow) and enzyme residues are shown as black dotted lines. The key water molecules are shown as cyan spheres, with the 2Fo-Fc electron density map shown as a blue mesh around the inhibitor, and a green mesh around the water both contoured at 1σ. PDB entries 3QTQ, 3S0O, and 3R8Z.
Figure 4
Figure 4. Interaction of sulfonamide analogues 42 and 51 with CDK2
Crystal structures of the most potent inhibitors, 42 (a) and 51 (b). Addition of a para-phenyl sulfonamide moiety at R1 substantially increased the inhibitory activity through establishment of a hydrogen bonding network with residues of the front specificity pocket (top exploded view). Addition of an ortho-nitrophenyl group at R1 of 51 retained the high in vitro activity and substantially increased cellular activity. The exploded view (bottom) shows the unusual proximity of the nitro group to neighboring hydrophobic residues. The 2Fo - Fc electron density is displayed as blue mesh, contoured at 1σ around the compound. Hydrogen bonding and potential π-bonding interactions are shown as black and orange dotted lines, respectively. Fo-Fc electron density maps from refinements omitting the inhibitors are shown in the Supporting Information (Figure S5). Schematic representations of the respective CDK2-inhibitor interactions are shown in the right panel. Black dotted lines indicate hydrogen bonding interactions, while van-der-Waals (hydrophobic) interactions are shown in green. PDB entries 3QU0 and 3QXP.
Figure 5
Figure 5. Sulfonamide inhibitors tolerate diverse R2 substituents
Crystal structures of CDK2 liganded with 52 (a), 53 (b), 54 (c), and 55 (d), other highly potent inhibitors of the para-sulfonamide series of Table 1e. The respective 2Fo-Fc density maps, contoured at 1σ, are shown as blue mesh. Black, green, and orange dotted lines indicate potential hydrogen bonding, hydrophobic, and π-interactions, respectively. PDB entries 3QTZ, 3QTU, 3QTX, and 3RPV.
Figure 6
Figure 6. Profiling of compound 51 against a panel of 339 kinases
(a) Quantitative distribution of kinases binned according to the inhibition by compound 51. (b) Distribution of inhibited kinases within the human kinome. The color code for inhibition is indicated. Residual enzymatic activity was determined in single dose duplicate at a compound concentration of 0.1 µM. The ATP concentration was 10 µM. Staurosporine served as a positive control. The experiment was performed by Reaction Biology Corp. using a P33-radiolabel assay. The data sets are shown in Table S7.
Figure 7
Figure 7. Mechanism of action of compound 51
(a) Asynchronously growing A-673 sarcoma cells were treated with increasing concentrations of compound 51 for 24 h and cell lysates were analyzed for phosphorylation of Rb protein at Ser807/811 by immunoblotting (left). Dinaciclib (0.05 µM) served as a positive inhibitor control and actin as a loading control. Onset of apoptosis was assessed by measuring the activity of caspase 3 using a bioluminescent assay upon 24 h treatment of cells with increasing concentrations of compound 51 (▲), dinaciclib (●) or doxorubicin (■) (right). Dinaciclib and doxorubicin served as positive controls. The EC50 values for caspase-3 activation were 0.41 µM for compound 51, 0.011 µM for dinaciclib and 0.39 µM for doxorubicin, which served as a positive control for the onset of apotosis. (b) Asynchronously growing H929 myeloma cells were treated for 24 h with compound 51 or dinaciclib and phosphorylation of Rb protein was analyzed by immunoblotting (left). Onset of apoptosis was assessed by measuring the level of cleaved, activated caspase 3 using flow cytometry (right). Shown are the percentages of cells with increased caspase-3 levels upon treatment with 1.5 µM compound 51 or 0.005 µM dinaciclib from three experiments.
Scheme 1
Scheme 1

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