Abstract LB-198: Design and synthesis of non-ATP competitive inhibitors of CDK4/Cyclin D using REPLACE

Abstract

Abstract CDK4/Cyclin D is an established anticancer drug target closely associated with breast and non-small cell lung cancers. Conventionally, the ATP binding site of CDKs has been the focus of inhibitor design. Here we present an alternative strategy to target CDK4 which allows us to design non-ATP competitive inhibitors by exploring the cyclin binding groove (CBG) present in Cyclin D, its positive regulatory subunit. The CBG of Cyclin D recognizes a cyclin binding motif (CBM) possessed by both natural inhibitors and substrates of CDK4/Cyclin D and is composed of primary and secondary hydrophobic pockets separated by a polar subsite; In addition, we recently described a hydrophobic extension to the major pocket and which is unique to Cyclin D1. The REPLACE method has been previously applied, through which determinants in known potent peptide inhibitors (binding in an extended conformation) are replaced with non-peptidic surrogates. In this study, a new series of non-peptidic surrogates to replace the original C-terminal residues (C-capping groups) that are key to binding to the primary hydrophobic pocket have been ligated to truncated peptides. Also using this approach, additional series of N-capping groups, (surrogates for N-terminal residues) which are important for the binding to the secondary hydrophobic pocket have been ligated to peptides. Both N- and C-capping groups have been selected to obtain more potent binding to their specific subsite. In addition, through guided docking of a small set of compounds into the hydrophobic extension unique to the cyclin D1 CBG, we have identified compounds with high predicted affinity to cyclin D, suggesting that appropriate modification of an inhibitor could lead to an increase in potency and selectivity. In order to do this, a virtual library composed of low molecular weight fragments has been modeled. Fragments were selected based on several criteria: 1) Log P ≤ 3; 2) Rotational bonds ≤ 3; 3) MW between 170 and 280. After docking, formation and energy minimization of the protein-fragment peptide complex enabled the prediction of capping groups with the highest potential for binding to the Cyclin D pocket. In order to evaluate these compounds in a facile and straightforward manner and to eliminate ATP competitive inhibitors, a competitive binding assay based on fluorescence polarization, has been developed for the first time for cyclin D1. The FP assay for Cyclin D provided insights into the binding of peptidic inhibitors to cyclin D1, as well as SAR for both N- and C-capping groups. By virtue of the establishment of computational, synthetic and in vitro testing work, the prediction of more potent capping groups will be facilitated. These experiments will serve as a useful guide for future direction of drug development for the design of inhibitors with selectivity towards Cyclin D and with potential 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 LB-198. doi:10.1158/1538-7445.AM2011-LB-198