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Title: Chemical and biological studies with Nek2 kinase inhibitors
Author: Matheson, Christopher
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2012
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The aim of modern cancer chemotherapy is to develop targeted drugs designed to exploit pharmacological differences between tumour cells and healthy tissues. One focus of this effort has been the identification of protein kinases that are expressed at elevated levels or in mutated forms, indicating a reliance of the tumour on specific kinase function. Nek2 is a human serine/threonine protein kinase related to the fungal protein NIMA, a critical mediator of mitosis. Interestingly, Nek2 is found to be upregulated in a variety of tumour cell lines derived from breast, cervical and prostate carcinomas, as well as lymphomas. Human Nek2 is implicated in the regulation of the centrosome and formation of a bipolar spindle, a framework that is vital for correct separation of sister chromatids during mitosis. It is proposed that Nek2 may complex with, and phosphorylate, proteins accumulated at the centrosome, possibly playing a role in intercentriolar linker cleavage during the centrosome cycle. Abnormalities in centrosome number and function are common in many cancers, indicating that loss of centrosome cycle regulation may be a major contributing factor in tumour progression. Overexpression of Nek2 may result in premature centrosome disjunction, and deregulation of this tightly controlled mitotic machinery leads to chromatid segregation errors, aneuploidy and chromosomal instability, common genetic abnormalities observed in tumour cells. This indicates a role for abnormal Nek2 function in tumourigenesis, and Nek2 depletion in a number of tumour cell lines has been shown to cause growth suppression and apoptosis. Nek2 is thus a potentially attractive cancer therapeutic target for small-molecule kinase inhibitors. Previous studies identified substituted purine derivatives as modest inhibitors of Nek2, leading to the discovery of two distinct inhibitor classes, exhibiting ATP-competitive and irreversible inhibition of the kinase, respectively. Purine-based compounds bearing substituents at the 8-position have emerged as modest competitive inhibitors of Nek2 that occupy the kinase ATP-domain through an unusual binding orientation (45; IC = 51.8 M). Additional possible interactions within the ATP- 50 binding site available to inhibitors of this class were explored, with the objective of developing tight-binding type II reversible inhibitors of Nek2. Structure-activity relationship studies resulted in a 10-fold improvement in activity over the initial hit compounds and a substantial improvement in drug-like properties (e.g. 129; IC = 5.1 M)). However, all 50 efforts to improve the potency of this series were unsuccessful. 6-Ethynylpurines have been identified as irreversible inhibitors of Nek2 through covalent modification of an active-site cysteine residue. The initial hit compound (147; IC = 0.14 50 M) was found to be a potent and selective inhibitor of the kinase in vitro, but with poor cellular activity attributed to limited permeability. Extensive structural modification of the 2- arylamino side-chain of this series afforded cell permeable analogues with improved potency, both in vitro and in vivo (e.g. 177; IC = 0.062 ± 0.01 M). 50 Biochemical studies using 177 suggested that inhibition of Nek2 resulted in an increase in mitotic abnormalities and a delay in mitotic progression, despite poor cellular growth inhibition being observed in initial tumour cell lines. Further cellular growth inhibition and cytotoxicity studies with selected compounds identified several sensitive tumour cell lines. However, kinase-inactive control compounds essentially devoid of Nek-inhibitory activity (e.g. 425; IC > 100 M) retained growth-inhibitory activity, indicating an alternative locus 50 of activity for the 6-ethynylpurine chemotype.
Supervisor: Not available Sponsor: Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available