Abstract
Nek2 (NIMA-related kinase 2) is a cell cycle-dependent serine/threonine protein kinase that regulates centrosome separation at the onset of mitosis. Overexpression of Nek2 is common in human cancers and suppression can restrict tumor cell growth and promote apoptosis. Nek2 inhibition with small molecules, therefore, offers the prospect of a new therapy for cancer. To achieve this goal, a better understanding of the requirements for selective-inhibition of Nek2 is required. 6-Alkoxypurines were identified as ATP-competitive inhibitors of Nek2 and CDK2. Comparison with CDK2-inhibitor structures indicated that judicious modification of the 6-alkoxy and 2-arylamino substituents could achieve discrimination between Nek2 and CDK2. In this study, a library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)-N,N-dimethylbenzamide (CDK2 IC50 = 7.0 μM; Nek2 IC50 = 0.62 μM) with >10-fold selectivity. Deletion of the 6-substituent abrogated activity against both Nek2 and CDK2. Nine compounds containing an (E)-dialkylaminovinyl substituent at C-6, all showed selectivity for Nek2, e.g. (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine (CDK2 IC50 = 2.70 μM; Nek2 IC50 = 0.27 μM). Structural biology of selected compounds enabled a partial rationalization of the observed structure activity relationships and mechanism of Nek2 activation. This showed that carboxamide 11 is the first reported inhibitor of Nek2 in the DFG-in conformation.
Highlights
A library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)N,N-dimethylbenzamide (CDK2 IC50 = 7.0 μM; Nek2 IC50 = 0.62 μM) with >10-fold selectivity
Abnormalities in centrosome number and function are common in many cancers, indicating that loss of centrosome cycle regulation may be a major factor in tumor progression [1]
Nek2 (NIMA-related kinase 2) is a human cell cycle-dependent serine/threonine protein kinase that localizes to the centrosome [2]
Summary
Abnormalities in centrosome number and function are common in many cancers, indicating that loss of centrosome cycle regulation may be a major factor in tumor progression [1]. To address the modest ligand efficiency (LE) of 2, a hybrid class of compounds was generated by combining the core aminopyrazine moiety of the initial series with side-chains from the benzimidazole series Optimisation of this new class of inhibitors improved potency, LE and kinase selectivity against a panel of cell cycle kinases, and culminated in the identification of 3 (Nek IC50 = 0.022 μM) [17]. This paper describes extensive structure-guided design, synthesis and structure-activity relationship (SAR) studies conducted with the purine scaffold and directed towards the development of potent and selective reversible Nek inhibitors These tools have provided an initial insight in to some of the key requirements for selective inhibition of Nek over CDK2 using purinebased inhibitors
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