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Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC500.15 μM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC500.14 μM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC500.06 μM (Nek2); GI50(SKBR3) 2.2 μM] which exhibited >5–10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 μM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 μM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.

The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopuskinesin-like protein 2) are overexpressed in cancers, and it has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for activating Aurora-A during mitosis, ensuring proper cell division. Disruption of the interface with TPX2 is therefore a potential target for novel anticancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. Here, we investigate the interface using coprecipitation assays and isothermal titration calorimetry to quantify the energetic contribution of individual residues of TPX2. Residues Tyr8, Tyr10, Phe16, and Trp34 of TPX2 are shown to be crucial for robust complex formation, suggesting that the interaction could be abrogated through blocking any of the three pockets on Aurora-A that complement these residues. Phosphorylation of Aurora-A on Thr288 is also necessary for high-affinity binding, and here we identify arginine residues that communicate the phosphorylation of Thr288 to the TPX2 binding site. With these findings in mind, we conducted a high-throughput X-ray crystallography-based screen of 1255 fragments against Aurora-A and identified 59 hits. Over three-quarters of these hits bound to the pockets described above, both validating our identification of hotspots and demonstrating the druggability of this protein–protein interaction. Our study exemplifies the potential of high-throughput crystallography facilities such as XChem to aid drug discovery. These results will accelerate the development of chemical inhibitors of the Aurora-A/TPX2 interaction.

CaVβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3)-domain and a guanylate kinase-like (GK)-domain. The SH3-domain is split, with its final (5th) β-strand separated from the rest of the domain by an intervening sequence termed the HOOK-domain, whose sequence varies between CaVβ subunits. Here we have been guided by the recent structural studies of CaVβ subunits in the design of specific truncated constructs, with the goal of investigating the role of the HOOK-domain of CaVβ subunits in the modulation of inactivation of N-type calcium channels. We have co-expressed the β subunit constructs with CaV2.2 and α2δ-2, using the N-terminally palmitoylated β2a subunit, because it supports very little voltage-dependent inactivation, and making comparisons with β1b domains. Deletion of the variable region of the β2a HOOK-domain resulted in currents with a rapidly inactivating component, and additional mutation of the β2a palmitoylation motif further enhanced inactivation. The isolated GK-domain of β2a alone enhanced current amplitude, but the currents were rapidly and completely inactivating. When the β2a-GK-domain construct was extended proximally, by including the HOOK-domain and the ε-strand of the SH3-domain, inactivation was about 4 fold slower than in the absence of the HOOK domain. When the SH3-domain of β2a truncated prior to the HOOK-domain was co-expressed with the (HOOK+εSH3+GK)-domain of β2a, all the properties of β2a were restored, in terms of loss of inactivation. Furthermore, removal of the HOOK sequence from the (HOOK+εSH3+GK)-β2a construct increased inactivation. Together, these results provide evidence that the HOOK domain is an important determinant of inactivation.

PurposeUsing the one‐step polyvinyl siloxane impression technique, this study compared the effect of putty material working time on the dimensional accuracy of recovered improved stone casts.

Statement of problem.When 2 implants are used to support a removable overdenture, optimal stress distribution to minimize forces to the implants is desired. Purpose.This study used photoelastic analysis to compare the stress patterns generated around implants with 2 retention mechanisms. Material And Methods.Two root form implants were anchored in a photoelastic mandible and a removable overdenture was fabricated. The denture was lined with a resilient layer of impression material to simulate oral mucosa. Attachment of the denture to the implants was varied by using ball/O-ring and bar-clip attachments. Vertical and oblique occlusal forces from 10 to 200 lb were applied unilaterally to the right and left first molars. Resultant stress distributions in the photoelastic mandible were photographed and evaluated. Results.With vertical forces, the ball/O-ring attachment transferred minimal stress to both implants. Vertical forces applied to the bar-clip overdenture created immediate stress patterns of greater magnitude and concentration on both implants. A posterior oblique load resulted in similar stress patterns to both systems with slightly greater stress to the bar-clip overdenture. Conclusions.Within the limits of this study, ball/O-ring attachments transferred less stress to implants than the bar-clip attachments when the photoelastic model was subjected to a posterior vertical load. (J Prosthet Dent 1998;80:559-64.)

Statement of problem.Visual color matching to determine shades in dentistry is inconsistent and unreliable. If accurate, instrumental measurement of tooth color would provide objective, quantified data to match natural teeth to clinical shade guides. Purpose.This study evaluated and compared the ability of a new computerized colorimeter and a simple visual test to match ceramic shade guide teeth. Material and methods.Thirty-one (n = 31) observers with normal color vision were allowed unlimited time to match one set of Vita Lumin shade guide teeth to the corresponding shade guide teeth of a second Vita Lumin shade guide. The same test was administered to 14 of the observers several months later to determine within-subject variability. A computerized colorimeter (Colortron II) equipped with a positioning guide was used to measure the middle third of each shade guide tooth. Through a “match tool” present in the computer’s software, readings from one shade guide were matched with readings of the other shade guide by using CIELAB measurements and ΔE values. The mean number of correct matches by the colorimeter and of correct matches in visual test were compared with a 1-tailed ttest. Repeatability for both tests was determined with a paired ttest. Results.The Colortron II instrument correctly matched 8 of the 16 tabs (50% correct), whereas visual matching by examiners averaged 7.7 of 16 correct matches (48% correct) (standard deviation 2.7). No statistically significant differences existed between the 2 methods. The colorimeter demonstrated 100% repeatability and the visual test demonstrated fair repeatability (correlation coefficient r = .60). Conclusions.Shade determination by visual means was inconsistent. Accuracy of a new colorimeter in matching porcelain shade guide teeth was only slightly better. (J Prosthet Dent 1998;80:642-8.)

NEK family kinases target EML4 to ensure faithful chromosome alignment during metaphase.

Phosphorylation uncouples the functions of the chaperone Hsp72 during mitosis.