Abstract #LB-78: Structural analysis of the CDK4/cyclin D1 substrate recognition site and basis for inhibitor selectivity
Authors: Shu Liu, Lindsay Kirkland, Campbell McInnes
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

An alternative strategy for inhibition of the cyclin dependent kinases in anti-tumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. This approach offers the potential of generating cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7, 8 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anti-cancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. As no experimental structural information is yet available for the CDK4/cyclin D1 complex, we have generated a protein homology structure for cyclin D1 modeled on available crystal structures for other human cyclins. Based on reported peptide inhibitors of CDK4/cyclin D mediated pRb phosphorylation, peptide-cyclin complexes have been generated with cyclin A and cyclin D and used to validate the homology structure and to correlate predicted affinity with known structure-activity relationships. The results for this study reveal that a single amino acid variation in the cyclin D1 groove has a significant impact on the potency of cyclin A inhibitors and could potentially be exploited in the design of compounds with greater affinity and selectivity for D1. Exchange of Ile214 in cyclin A for valine leads to decreased interaction with the Leu-Phe motif of cyclin A inhibitory compounds. In addition, differences in charged residues between the two cyclins leads to lesser impact of positively charged inhibitory peptides in the cyclin D1 context. These results suggest that modification of appropriate peptide functionality has the potential to significantly increase affinity for cyclin D1. Elucidation of the structural basis for peptide inhibition was thus further applied to identify compounds that have increased potency and greater selectivity against CDK4 phosphorylation of the Rb protein. We have synthesized peptidic and non-peptidic compounds that have increased potency against the cyclin D1 groove which to our knowledge are the first reported examples of CDK4 selective inhibitors of substrate recruitment. Such compounds will be useful chemical biology probes to determine the cellular and anti-tumor effects of CDK inhibitors that are cell cycle specific and do not inhibit the transcriptional regulatory effects of other cyclin dependent kinases.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-78.