Structure-based design of cyclin-dependent kinase inhibitors

Abstract

The eukaryotic cell cycle is tightly regulated by the sequential activation and deactivation of the cyclin-dependent kinases (CDKs). Aberrant CDK activity is a common defect in human tumours, and clinically, it often confers a poor prognosis. The strong genetic link between CDKs and the molecular pathology of cancer has provided the rationale for developing small-molecule inhibitors of these kinases. X-ray crystallography recently has revealed the molecular details of CDK regulation by cyclin binding and phosphorylation, and by complex formation with endogenous inhibitors. Knowledge of the structure of CDK2 has been key in driving the design and development of a large number of ATP competitive inhibitors. Crystallography has revealed that the ATP-binding site of CDK2 can accommodate a number of diverse molecular frameworks, exploiting various sites of interaction. In addition, residues outside the main ATP-binding cleft have been identified that could be targeted to increase specificity and potency. These results suggest that it may be possible to design pharmacologically relevant ligands that act as specific and potent inhibitors of CDK activity. We provide a review of the current state of the field, along with an overview of our current inhibitor design studies.

Introduction

Attention is increasingly being focussed on the cell cycle as a potential target for therapeutic intervention in several proliferative diseases, including cancer. Timely and coordinated progression through the cell cycle is controlled by the sequential activation and deactivation of members of the cyclin-dependent kinase (CDK) family (Morgan, 1997). Disrupted cell-cycle control due to aberrant CDK activity has been linked directly to the molecular pathology of cancer Kamb, 1995, Vogt & Reed, 1998, Pavletich, 1999. For example, loss of function of endogenous inhibitors such as p16^INK4A, over-expression of cyclin D1 and CDK4, and alterations to CDK substrates such as retinoblastoma gene product (pRb) are frequently observed in human tumours Shapiro & Harper, 1999, Senderowicz & Sausville, 2000.

Members of the CDK family are active at distinct points in the cell cycle (Morgan, 1997). The G₀/G₁ transition is characterised by increased transcription of cyclin D, giving rise to activation of CDK4 and CDK6, whose primary target is pRb. In its hypophosphorylated state, pRb binds to the E2F transcription factor, repressing its activity at promoter sites. pRb hyperphosphorylation disrupts these complexes, permitting the transcription of several genes whose products are associated with S-phase progression. General targets include cyclin E, dihydrofolate reductase, and DNA polymerases Dyson, 1998, Zhang et al., 1999, Harbour & Dean, 2000. Alterations in the level of CDK4 or CDK6 activity in malignant cells have suggested these kinases as important targets for therapeutic intervention. However, inhibition of CDK4/6 by p16^INK4A in cells lacking pRb does not lead to cell-cycle arrest (Shapiro & Harper, 1999).

CDK2 is essential for G₁ progression and S-phase entry. Complexed with cyclin E, it sustains pRb hyperphosphorylation to support progression through G₁ into S-phase. Early in S-phase, CDK2 associates with cyclin A, where it plays a role in inactivating E2F, and it is required for the completion of S-phase. Persistence of E2F activity during S-phase leads to apoptosis Krek et al., 1995, Shapiro & Harper, 1999, and, therefore, selective inhibition of CDK2-cyclin A may achieve cytotoxicity instead of cell-cycle arrest. Inhibition in S-phase may also be a strategy to which transformed cells are particularly sensitive due to their higher levels of E2F (Chen, 1999).

These studies have provided the rationale for developing small-molecule CDK inhibitors as anticancer agents (Hall & Peters, 1996). Interfering with CDK catalytic activity through ATP-competitive ligands has proved a successful strategy. A disadvantage of this approach is the large number of protein kinases found within the cell Consortium, 2001, Venter, 2001, most of which share a high degree of sequence similarity within the active site. Nevertheless, highly potent and selective ATP-competitive inhibitors have been identified for a number of other kinases. The epidermal growth factor receptor tyrosine kinase inhibitor PD 153035 (Fry et al., 1994) and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 (Lee et al., 1999) are examples.