A Novel Approach for the Development of Selective Cdk4 Inhibitors:  Library Design Based on Locations of Cdk4 Specific Amino Acid Residues

Abstract

Identification of a selective inhibitor for a particular protein kinase without inhibition of other kinases is critical for use as a biological tool or drug. However, this is very difficult because there are hundreds of homologous kinases and their kinase domains including the ATP binding pocket have a common folding pattern. To address this issue, we applied the following structure-based approach for designing selective Cdk4 inhibitors: (1) identification of specifically altered amino acid residues around the ATP binding pocket in Cdk4 by comparison of 390 representative kinases, (2) prediction of appropriate positions to introduce substituents in lead compounds based on the locations of the altered amino acid residues and the binding modes of lead compounds, and (3) library design to interact with the altered amino acid residues supported by de novo design programs. Accordingly, Asp99, Thr102, and Gln98 of Cdk4, which are located in the p16 binding region, were selected as first target residues for specific interactions with Cdk4. Subsequently, the 5-position of the pyrazole ring in the pyrazol-3-ylurea class of lead compound (2a) was predicted to be a suitable position to introduce substituents. We then designed a chemical library of pyrazol-3-ylurea substituted with alkylaminomethyl groups based on the output structures of de novo design programs. Thus we identified a highly selective and potent Cdk4 inhibitor, 15b, substituted with a 5-chloroindan-2-ylaminomethyl group. Compound 15b showed higher selectivity on Cdk4 over those on not only Cdk1/2 (780-fold/190-fold) but also many other kinases (>430-fold) that have been tested thus far. The structural basis for Cdk4 selective inhibition by 15b was analyzed by combining molecular modeling and the X-ray analysis of the Cdk4 mimic Cdk2-inhibitor complex. The results suggest that the hydrogen bond with the carboxyl group of Asp99 and hydrophobic van der Waals contact with the side chains of Thr102 and Gln98 are important. Compound 15b was found to cause cell cycle arrest of the Rb(+) cancer cell line in the G(1) phase, indicating that it is a good biological tool.

Synthetic Studies toward Aryl-(4-aryl-4H-[1,2,4]triazole-3-yl)-amine from 1,3-Diarylthiourea as Urea Mimetics

“…For example, while some of these urea-based inhibitors bind to an allosteric site causing a significant rearrangement of the target protein structure 9,10 others bind to the active site such as the ATP pocket with the urea function participating in a bi-dentate hydrogen bond. 11 Bioisosteric replacements of the amide and thioamide functions included heterocyclic systems such as the [1,2,4]-and [1,3,4]oxadiazoles and [1,2,4]triazoles. 12 Such replacements led to [1,2,4]triazoles which were active antimycobacterials 13 and potent, selective 5-HT 1D receptor agonists.…”

Discovery of nitroaryl urea derivatives with antiproliferative properties

“…TE671 cancer cell line was the most sensitive against CDDP. Calculated GI 50 ± SD values for TE671 and RK33 cells were 56.66 ± 1.2 mM and 83.33 ± 1.1 mM respectively demonstrating greater potency of some newly synthesized compounds on tested cell lines. Healthy human cells showed the greatest resistance against CDDP in MTT assay, the calculated GI 50 value was 146.66 ± 1.1 mM.…”

“…The GI 50 values were calculated using nonlinear regression analysis on GraphPad Prism 5.04 platform. The selectivity index (SI) was calculated as GI 50 for normal cell line/GI 50 for cancer cell line ratio. The data were collected from three independent experiments, each in 8 wells (n ¼ 24).…”

“…Aryl substituted urea derivatives have been discovered as potent inhibitors of various kinases [44][45][46][47] and sorafenib is a wellknown example of a drug used for treatment of some cancers 48 . We decided to pursue CDK inhibition because of the similarity of our compounds and those described in the literature taking urea moiety into account 21,49,50 . Moreover a link between inhibition of CDKs and antiproliferative effect has been well documented [51][52][53][54] .…”

Fragment-Based Discovery of 7-Azabenzimidazoles as Potent, Highly Selective, and Orally Active CDK4/6 Inhibitors

“…Hydrogen bonding between the ligands and the side chain of Lys43 and the backbone of Asp163 also seems to contribute to a potent and selective CDK4/6 profile. 21,23 Examination of the structure in Figure 1 suggested that optimal growth vectors to target Lys43 would be provided by replacing the pyrrole with a pyridine ring. The pyridine analogue B (Scheme 1, 57% inhibition at 30 μM, LE = ca.…”