Abstract 1351: Computational design and synthesis of non-peptidic inhibitors targeting the cyclin binding groove of CDK2/Cyclin A
Authors: Premnath Padmavathy N. , Bolger Joshua K. , Liu Shu , Campbell McInnes 
Published: June 2012

Inhibition of CDK2/Cyclin A (CDK2/CA) at G1/S phase of cell cycle controls cell proliferation and induces selective apoptosis of cancer cells. Neutralization of E2F-1by CDK2/CA phosphorylation is required prior to S-phase exit or apoptosis is triggered. E2F-1 is deregulated in many types of cancer and inhibition of CDK2/CA leads to selective cell death in these tumors. In our present study we target the substrate recruitment site knows as cyclin binding groove (CBG) for inhibition of CDK2/CA. The CBG recognizes a consensus sequence present on cyclin interacting proteins and the endogenous tumor suppressors knows cyclin binding motif (CBM) which has been minimized to the pentapeptide, RRLIF. It has been shown that the drug likeliness of the peptide can be improved by ligation of small molecules to truncated sequences. This was achieved using a unique strategy for designing inhibitors of protein-protein interactions known as REPLACE, in which small molecule fragments were identified by in silico methods. These were then ligated onto a truncated peptide sequence in order to replace critical residues, including the N-terminal Arginine moiety important for cyclin binding. Small molecule fragments for in silico docking were selected based on molecule weight, drug like features and possession of a carboxylic acid group for the formation of the peptide link. Docking was carried out using LigandFit and the crystal structure of 1-phenyl-1H-[1,2,4]triazole N-terminally capped peptides (Ncaps) in the CBG provided insight into molecular design. The 3,5 dichloro1-phenyl-1H-[1,2,4]triazole derivative linked to Arg-Leu-Asn-pF-Phe was previously shown to be a potent inhibitor with micromolar activity. In our present study we have replaced the N-terminal Arginine using different heterocyclic isosteres capable of interactions similar to critical amino acids of the parent peptide and the triazole. In order to probe the structure-activity relationship, we synthesized pyrazole, furan, pyrrole and thiazole systems and explored various substitutions of the phenyl ring. A number of Ncaps were then ligated to the tetra peptide, RLIF using solid phase synthesis, purified by reverse phase HPLC and characterized by MS. In vitro binding and functional assays were performed in order to study the inhibitory effect of compounds on CDK2/Cyclin A prior to further evaluation in cell viability assays to determine anti-tumor effects. On the basis of the results, further high throughput docking of potential heterocyclic fragments was carried out to identify N-capping groups of varying chemical diversity for synthesis and in vitro testing. The results presented demonstrate the utility of the REPLACE method in the development of protein-protein interaction inhibitors and non-ATP competitive CDK2 inhibitors as anti-tumor therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2011-1351