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Laboratory of Molecular Biophysics
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(Collaborators: Prof. D. Barford, ICR ,London, Prof. J. Harper, Houston, U.S., AstraZeneca pharmaceuticals, members of the Newcastle University Anticancer Drug Discovery Initiative, Prof. L. Johnson, Oxford and Dr. J. Endicott, Oxford).
Sequential activation of members of the cyclin-dependent protein kinase family (CDKs) orders the events required for DNA replication and cell division. Both the CDK and cyclin families have multiple members, but only CDKs 1, 2, 4 and 6, when bound to their cognate cyclins, appear to have major roles in controlling cell cycle progression. These complexes are either pivotal to the integration of signal transduction pathways into the cell cycle or they are the targets of cell cycle checkpoint pathways. They are regulated through mechanisms that include phosphorylation (both activatory and inhibitory), association with additional regulatory molecules (particularly members of the cyclin-dependent kinase inhibitor families (CKIs)), and subcellular localisation [5].
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Cyclin-dependent kinase 2 (CDK2) is an important target for structure-based design of anti-tumour agents. Monomeric CDK2 is inactive. Activation requires rearrangements to key structural elements of the enzyme's active site which accompany cyclin binding and phosphorylation. To assess the validity of using monomeric CDK2 as a model for the active kinase in structure-based drug design, we have solved the structure of the inhibitor indirubin-5-sulphonate (E226) complexed with phospho CDK2-cyclin A and compared it with the structure of E226 bound to inactive, monomeric CDK2 . Activation of monomeric CDK2 leads to a rotation of its N-terminal domain relative to the C-terminal lobe. The accompanying change in position of E226 follows that of the N- terminal domain and its interactions with residues forming part of the adenine binding pocket are conserved. The environment of the ATP-ribose site, not explored by E226, is significantly different in the binary complex compared to the monomeric complex due to movement of the glycine loop (Figure 4). Conformational changes also result in subtle differences in hydrogen bonding and electrostatic interactions between E226's sulphonate and CDK2's phosphate binding site. Affinities calculated by LUDI for the interaction of E226 with active or inactive CDK2 differ by a factor of approximately 10. The accuracy of monomeric CDK2 as an inhibitor design template is restricted to the adenine binding site. The general flexibility observed for the glycine loop, and subtle changes to the phosphate binding site, suggest a need to study interactions between inhibitors and active CDK2 in structure-based drug design programs.
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Recruitment of inhibitors and substrates to complexes of A-, E-, and D-type cyclins, is achieved through binding of a Cy-motif to a conserved hydrophobic pocket on the N-terminal cyclin box fold [6]. The Cy-motif generally has the amino-acid sequence RXLFG, where X can be any amino acid. Various biochemical studies have shown that there is significant redundancy within this sequence, so that sequences as diverged as RRLDLE (from the substrate E2F), and KKLMFK (from the substrate pRb) can act as recruitment sequences. Peptides derived from the canonical Cy motif are effective both in vitro and in vivo as inhibitors of CDK/cyclin activities, suggesting a novel route to the development of specific inhibitors. Such inhibitors might be used as probes of CDK function, or as therapeutic agents.
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