Structural and Functional Analysis of Cyclin D1 Reveals p27 and Substrate Inhibitor Binding Requirements

Abstract

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. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential of generating highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 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. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27KIP1, with cyclin D1. This information has been used shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 and which can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and non-peptidic compounds have been synthesized in order to explore structure-activity relationship for binding to the cyclin D1 groove which to date has not been carried out in a systematic fashion. Collectively, the data presented provides new insights into how compounds can be developed that function as chemical biology probes to determine the cellular and anti-tumor effects of CDK inhibition. Furthermore, such compounds will serve as templates for structure-guided efforts to develop potential therapeutics based on selective inhibition of CDK4/cyclin D activity.

Fluorescent Biosensor for Detection of the R248Q Aggregation‐Prone Mutant of p53

“…Although most tumour‐suppressor genes are deleted or truncated in human cancers, the TP53 gene frequently undergoes missense mutations, resulting in single amino acid substitutions, which cause loss of wild‐type (WT) function, but may also exert a dominant negative effect through binding and inhibition of WT p53 . Moreover, several mutations result in gain of function, which endows p53 mutants with activities that contribute to hyperproliferation; increased tumourigenicity; anchorage‐independent cell growth, metastasis and invasiveness; decreased sensitivity to chemotherapeutic drugs; disruption of the spindle checkpoint; activated topoisomerase I activity and induction of gene amplification . For instance, the R175H gain‐of‐function mutation promotes activation of the multidrug resistance gene MDR1 .…”

Optimization of Non-ATP Competitive CDK/Cyclin Groove Inhibitors through REPLACE-Mediated Fragment Assembly

“…Previously it has been shown in SAR studies that peptide affinity can be significantly impacted through substitution of the N-terminal residues contacting the secondary hydrophobic pocket and acidic region in both cyclin A2 and D1 contexts and also that this can lead to preferential binding to one cyclin over the other 20 . In order to explore this further and to optimize inhibitor binding, an extensive SAR was generated for the phenylheterocyclic capping groups described.…”

The other side of the coin: The tumor-suppressive aspect of oncogenes and the oncogenic aspect of tumor-suppressive genes, such as those along the CCND–CDK4/6–RB axis

“…Since currently there has not yet been an approach available for clinical use that can specifically decrease CCND1 or CDK4/6 protein, some peers are considering designing peptides or compounds that block other functional, such as the substrate-binding, domains of CDK4/6 proteins. 241,242 Whether these alternatives, alone and in combination with PD0332991 or other ATP site blockers, are more effective is an intriguing question.…”