Structure-guided design of purine-based probes for selective Nek2 inhibition

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.

Bioinformatics Analysis of Global Proteomic and Phosphoproteomic Data Sets Revealed Activation of NEK2 and AURKA in Cancers

“…The activation of NEK2 is through the phosphorylation on its auto-phosphorylation sites such as (T170/S171 and T175) [37]. Carboxamide 11 is one of the best known selective inhibitors of NEK2 [38]. NEK2 has been reported to have a role in the progression of several malignancies, importantly in breast cancer, therefore devising the potential of an anticancer therapy [36,39,40].…”

Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine

“…Dose-response experiments with Nek7 WT and Nek7 SRS demonstrated that a Nek2 inhibitor, compound 51 [21], successfully inhibited both enzymes (Figure 2b,c). The apparent IC 50 values calculated in these experiments are 79.01 mM and 40.13 mM for Nek7 WT and Nek7 SRS , respectively.…”

“…Compound 51 inhibits Nek2 with an IC 50 of 1.42 mM, ∼50-fold more potently than Nek7 SRS [21]. To gain insights into this difference, we aligned the protein sequences (Figure 6a) and determined the crystal structure of Nek2 catalytic domain bound to compound 51 to a resolution of 2.0 Å (PDB code 6SK9; Figure 6b).…”

“…Studies on the cellular functions of protein kinases are underpinned by the availability of kinase inhibitors [16]. Unfortunately, the Nek family of kinases suffers from a shortage of such tools and, with the exception of Nek2, there are no published attempts to develop selective and potent inhibitors of Neks [17][18][19][20][21]. This situation is likely to change over the next few years because the important biological roles of this family have been identified.…”

“…(b) Compound 51 inhibits both Nek7 SRS (circles) and Nek7 WT (squares). (c) The chemical structure of compound 51[21].…”