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

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.