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Variants of the oncogenic EML4‐ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4‐ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4‐ALK V3 but not V1 protein re‐localises to microtubules, an effect recapitulated in a catalytically inactive EML4‐ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild‐type and catalytically inactive EML4‐ALK V3 proteins, but not a Lys‐Glu salt bridge mutant. We propose that EML4‐ALK foci formation occurs as a result of transient association of stable EML4‐ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4‐ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors., EML4‐ALK fusion proteins phase separate into foci that contain oncogenic signaling factors, through a mechanism that depends on an active ALK kinase conformation. Variants have distinct dynamics determined by a specific region of EML4.

[Image: see text] A combination of focused library and virtual screening, hit expansion, and rational design has resulted in the development of a series of inhibitors of RET(V804M) kinase, the anticipated drug-resistant mutant of RET kinase. These agents do not inhibit the wild type (wt) isoforms of RET or KDR and therefore offer a potential adjunct to RET inhibitors currently undergoing clinical evaluation.

Amplification of MYCN is the driving oncogenic change in a subset of high-risk neuroblastomas. The MYCN protein and the Aurora-A kinase form a complex during the S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in the S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription–replication conflicts and activates ataxia telangiectasia and Rad3-related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR kinases induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription–replication conflicts is an effective therapy for MYCN-driven neuroblastoma. Eilers and colleagues report that Aurora-A suppresses transcription–replication conflicts in MYCN-driven neuroblastoma, a vulnerability that can be targeted with a combination of Aurora-A and ATR kinase inhibitors.

Oncogenic fusions involving tyrosine kinases are common drivers of non-small cell lung cancer (NSCLC). There are at least 15 different variants of the EML4-ALK fusion, all of which have a similar portion of ALK that includes the kinase domain, but different portions of EML4. Targeted treatment with ALK tyrosine kinase inhibitors (TKIs) has proven effective but patient outcomes are variable. Here, we focus on one common variant, EML4-ALK V3, which drives an aggressive form of the disease. EML4-ALK V3 protein forms cytoplasmic liquid droplets that contain the signalling proteins GRB2 and SOS1. The TKIs ceritinib and lorlatinib dissolve these droplets and the EML4-ALK V3 protein re-localises to microtubules, an effect recapitulated by an inactivating mutation in the ALK catalytic site. Mutations that promote a constitutively active ALK stabilise the liquid droplets even in the presence of TKIs, indicating that droplets do not depend on kinase activity per se. Uniquely, the TKI alectinib promotes droplet formation of both the wild-type and catalytically inactive EML4-ALK V3 mutant, but not in a mutant that disrupts a hallmark of the kinase activity, the Lys-Glu salt-bridge. We propose that EML4-ALK V3 liquid droplet formation occurs through transient dimerization of the ALK kinase domain in its active conformation in the context of stable EML4-ALK trimers. Our results provide insights into the relationship between ALK activity, conformational state and the sub-cellular localisation of EML4-ALK V3 protein, and reveal the different effects of structurally divergent ALK TKIs on these properties.

Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers., 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 [IC50 0.15 μM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 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 [IC50 0.06 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.

Background Anaplastic lymphoma kinase (ALK) inhibition using crizotinib has become the standard of care in advancedALK-rearranged non-small cell lung cancer (NSCLC), but the treatment outcomes and duration of response vary widely. Echinoderm microtubule-associated protein-like 4 (EML4)-ALK is the most common translocation, and the fusion variants show different sensitivity to crizotinibin vitro. However, there are only limited data on the specificEML4-ALK variants and clinical responses of patients to various ALK inhibitors. Patients and methods By multiplex reverse-transcriptase PCR, which detects 12 variants of knownEML4-ALK rearrangements, we retrospectively determinedALK fusion variants in 54 advancedALK rearrangement-positive NSCLCs. We subdivided the patients into two groups (variants 1/2/others and variants 3a/b) by protein stability and evaluated correlations of the variant status with clinical responses to crizotinib, alectinib, or ceritinib. Moreover, we established the EML4-ALK variant-expressing system and analyzed patterns of sensitivity of the variants to ALK inhibitors. Results Of the 54 tumors analyzed,EML4-ALK variants 3a/b (44.4%) was the most common type, followed by variants 1 (33.3%) and 2 (11.1%). The 2-year progression-free survival (PFS) rate was 76.0% [95% confidence interval (CI) 56.8–100] in groupEML4-ALK variants 1/2/others versus 26.4% (95% CI 10.5–66.6) in group variants 3a/b (P = 0.034) among crizotinib-treated patients. Meanwhile, the 2-year PFS rate was 69.0% (95% CI 49.9–95.4) in group variants 1/2/others versus 32.7% (95% CI 15.6–68.4) in group variants 3a/b (P = 0.108) among all crizotinib-, alectinib-, and ceritinib-treated patients. Variant 3a- or 5a-harboring cells were resistant to ALK inhibitors with >10-fold higher half maximal inhibitory concentrationin vitro. Conclusion Our findings show that groupEML4-ALK variants 3a/b may be a major source of ALK inhibitor resistance in the clinic. The variant-specific genotype of theEML4-ALK fusion allows for more precise stratification of patients with advanced NSCLC.

EML4–ALK is an oncogenic fusion present in ∼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4–ALK with different patient outcomes. Here, we show that, in cell models, EML4–ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4–ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4–ALK patients. Hence, we propose that EML4–ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4–ALK lung cancer.

Myc family proteins promote cancer by inducing widespread changes in gene expression. Their rapid turnover by the ubiquitin–proteasome pathway is regulated through phosphorylation of Myc Box I and ubiquitination by the E3 ubiquitin ligase SCFFbxW7. However, N-Myc protein (the product of the MYCN oncogene) is stabilized in neuroblastoma by the protein kinase Aurora-A in a manner that is sensitive to certain Aurora-A–selective inhibitors. Here we identify a direct interaction between the catalytic domain of Aurora-A and a site flanking Myc Box I that also binds SCFFbxW7. We determined the crystal structure of the complex between Aurora-A and this region of N-Myc to 1.72-A resolution. The structure indicates that the conformation of Aurora-A induced by compounds such as alisertib and CD532 is not compatible with the binding of N-Myc, explaining the activity of these compounds in neuroblastoma cells and providing a rational basis for the design of cancer therapeutics optimized for destabilization of the complex. We also propose a model for the stabilization mechanism in which binding to Aurora-A alters how N-Myc interacts with SCFFbxW7 to disfavor the generation of Lys48-linked polyubiquitin chains.

EML4-ALK is an oncogenic fusion present in ∼5% lung adenocarcinomas. However, distinct EML4-ALK variants differ in the length of the EML4 microtubule-associated protein encoded within the fusion and are associated with a poorly understood variability in disease progression and therapeutic response. Here, we show that EML4-ALK variant 3, which is linked to accelerated metastatic spread and worse patient outcome, causes microtubule stabilization, formation of extended cytoplasmic protrusions, loss of cell polarity and increased cell migration. Strikingly, this is dependent upon the NEK9 kinase that interacts with the N-terminal region of EML4. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates cell migration in a manner that requires the downstream kinase NEK7 but not ALK activity. Moreover, elevated NEK9 is associated in patients with EML4-ALK V3 expression, as well as reduced progression-free and overall survival. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through recruitment of NEK9 and NEK7 to increase cell migration and that this represents a novel actionable pathway that drives disease progression in lung cancer.

EML4 is a microtubule-associated protein that promotes microtubule stability. We show here that EML4 is distributed as punctate foci along the microtubule lattice in interphase but exhibits reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy and 3D reconstruction reveal that EML4 binds via its basic N-terminal domain to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases Nek6 and Nek7 can phosphorylate EML4 N-terminal domain at S144 and S146 in vitro, and depletion of these kinases leads to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only binds inappropriately to mitotic microtubules but also interferes with chromosome congression. Meanwhile, constitutive activation of Nek6 or Nek7 reduces EML4 association with interphase microtubules. Together, these data support a model in which Nek6 and Nek7-dependent phosphorylation promotes dissociation of EML4 from microtubules in mitosis thereby altering microtubule dynamics to enable chromosome congression.

Hsp72 is a member of the 70-kD heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr66 located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. Here, we determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr66 is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr66 by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle.

Purpose: The aim of this study was to examine the effect of an alternative surgical technique on endosseous dental implant stability parameters in Class D4 bone. Differences between insertion torque (IT) and implant stability quotient (ISQ) produced by a conventional osteotomy versus a modified stepped osteotomy were examined. Correlations between IT, density in Hounsfield units (HU), and ISQ were also examined. Materials and Methods: Computed tomographic scans were taken of 16 cadaver heads to determine bone density at maxillary implant recipient sites, which ranged from 173.4 to 312.1 HU. Twenty-two implants were placed in sites with Misch Class D4 bone; control sites received 11 conventional osteotomies and test sites 11 modified stepped osteotomies. Maximum IT was recorded with an implant motor and confirmed with a torque driver, and ISQs were measured. Significant differences were analyzed with a Wilcoxon signed rank test, and correlations were analyzed with the Pearson test. Results: The mean IT in the control group was 15.91 Ncm, and in the test group it was 26.82 Ncm. The modified stepped osteotomy had a significantly greater mean IT than the conventional osteotomy, but no significant difference in ISQ was found between groups. Pearson correlations showed a significant positive correlation between IT in the control and test groups. Significant correlations were found between ISQ and HU in the test group. No other significant correlations between HU, IT, and ISQ were found. Conclusion: Within the limits of this cadaver study, the modified stepped osteotomy resulted in significantly greater implant stability in terms of IT than the conventional osteotomy in soft bone. Significant correlations were found between the IT produced in the modified stepped osteotomy and bone density. No significant correlations were found between IT and ISQ.

Hsp72 is a novel mitotic substrate of Nek6, and, together, these proteins play an essential role in assembly of robust mitotic spindles capable of efficient chromosome congression through K-fiber recruitment of the ch-TOG and TACC3 complex., Hsp70 proteins represent a family of chaperones that regulate cellular homeostasis and are required for cancer cell survival. However, their function and regulation in mitosis remain unknown. In this paper, we show that the major inducible cytoplasmic Hsp70 isoform, Hsp72, is required for assembly of a robust bipolar spindle capable of efficient chromosome congression. Mechanistically, Hsp72 associates with the K-fiber–stabilizing proteins, ch-TOG and TACC3, and promotes their interaction with each other and recruitment to spindle microtubules (MTs). Targeting of Hsp72 to the mitotic spindle is dependent on phosphorylation at Thr-66 within its nucleotide-binding domain by the Nek6 kinase. Phosphorylated Hsp72 concentrates on spindle poles and sites of MT–kinetochore attachment. A phosphomimetic Hsp72 mutant rescued defects in K-fiber assembly, ch-TOG/TACC3 recruitment and mitotic progression that also resulted from Nek6 depletion. We therefore propose that Nek6 facilitates association of Hsp72 with the mitotic spindle, where it promotes stable K-fiber assembly through recruitment of the ch-TOG–TACC3 complex.

The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopus kinesin-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.

The Myc proteins comprise a family of ubiquitous regulators of gene expression implicated in over half of all human cancers. They interact with a large number of other proteins, such as transcription factors, chromatin-modifying enzymes and kinases. Remarkably, few of these interactions have been characterized structurally. This is at least in part due to the intrinsically disordered nature of Myc proteins, which adopt a defined conformation only in the presence of binding partners. Owing to this behaviour, crystallographic studies on Myc proteins have been limited to short fragments in complex with other proteins. Most recently, we determined the crystal structure of Aurora-A kinase domain bound to a 28-amino acid fragment of the N-Myc transactivation domain. The structure reveals an α-helical segment within N-Myc capped by two tryptophan residues that recognize the surface of Aurora-A. The kinase domain acts as a molecular scaffold, independently of its catalytic activity, upon which this region of N-Myc becomes ordered. The binding site for N-Myc on Aurora-A is disrupted by certain ATP-competitive inhibitors, such as MLN8237 (alisertib) and CD532, and explains how these kinase inhibitors are able to disrupt the protein–protein interaction to affect Myc destabilization. Structural studies on this and other Myc complexes will lead to the design of protein–protein interaction inhibitors as chemical tools to dissect the complex pathways of Myc regulation and function, which may be developed into Myc inhibitors for the treatment of cancer.

The vast majority of clinically-approved protein kinase inhibitors target the ATP binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized, and there is no clearly preferred approach to generating hit matter. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. However, it is currently unclear how to design such a compound because a small molecule or peptide mimetic of TPX2 would be expected to activate, not inhibit the kinase. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, VNAR single domain nanobody scaffold, IgNARV-D01. Biochemical studies and a crystal structure of the Aurora-A/IgNARV-D01 complex show that the nanobody overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the nanobody stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken, and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how nanobodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.SignificanceProtein kinases are commonly dysregulated in cancer and inhibitors of protein kinases are key therapeutic drugs. However, this strategy is often undermined by a lack of selectivity since the ATP binding pocket that kinase inhibitors usually target is highly conserved. Inhibitors that target allosteric sites are more selective but more difficult to generate. Here we identify a single domain antibody (nanobody) to target an allosteric pocket on the catalytic domain of Aurora-A kinase and demonstrate that the mechanism is antagonistic to a physiologically-relevant allosteric activator, TPX2. This work will enable the development of allosteric Aurora-A inhibitors as potential therapeutics, and provide a model for the development of tools to investigate allosteric modes of kinase inhibition.

A fusion between the EML4 (echinoderm microtubule-associated protein-like) and ALK (anaplastic lymphoma kinase) genes was identified in non-small cell lung cancer (NSCLC) in 2007 and there has been rapid progress in applying this knowledge to the benefit of patients. However, we have a poor understanding of EML4 and ALK biology and there are many challenges to devising the optimal strategy for treating EML4-ALK NSCLC patients. In this review, we describe the biology of EML4 and ALK, explain the main features of EML4-ALK fusion proteins and outline the therapies that target EML4-ALK. In particular, we highlight the recent advances in our understanding of the structures of EML proteins, describe the molecular mechanisms of resistance to ALK inhibitors and assess current thinking about combinations of ALK drugs with inhibitors that target other kinases or Hsp90.

Objectives To evaluate changes to the dental and dentoalveolar (WALA Ridge) arch widths with preformed and customized archwires during orthodontic treatment. Methods 20 patients treated with preformed archwires and 20 treated with customized archwires were recruited. Pre-treatment (T1) and post-treatment (T2) mandibular casts were used to determine the changes in dental and dentoalveolar arch width measured at the canine, premolar and molar areas. Ratios of transverse dental to dentoalveolar movements were also calculated. Results were compared to an untreated control group with mandibular casts taken at two comparable time points. Data were analyzed using ANOVA and t -test. Results Significant changes in dental and dentoalveolar arch width were found with the preformed archwire group when compared to the control ( p Conclusions The WALA Ridge is a stable landmark when archwires are customized or shaped to the WALA Ridge. Changes in the WALA Ridge are expected when preformed archwires are used which do not conform to the patient's dentoalveolar arch width defined by the WALA Ridge. This is probably accounted for by the different types of tipping combined with extrusion that both methods employ.

Cellular stress in early mitosis activates the antephase checkpoint, resulting in the decondensation of chromosomes and delayed mitotic progression. Checkpoint with forkhead-associated and RING domains (CHFR) is central to this checkpoint, and its activity is ablated in many tumors and cancer cell lines through promoter hypermethylation or mutation. The interaction between the PAR-binding zinc finger (PBZ) of CHFR and poly(ADP-ribose) (PAR) is crucial for a functional antephase checkpoint. We determined the crystal structure of the cysteine-rich region of human CHFR (amino acids 425-664) to 1.9 Å resolution, which revealed a multizinc binding domain of elaborate topology within which the PBZ is embedded. The PBZ of CHFR closely resembles the analogous motifs from aprataxin-like factor and CG1218-PA, which lie within unstructured regions of their respective proteins. Based on co-crystal structures of CHFR bound to several different PAR-like ligands (adenosine 5'-diphosphoribose, adenosine monophosphate, and P(1)P(2)-diadenosine 5'-pyrophosphate), we made a model of the CHFR-PAR interaction, which we validated using site-specific mutagenesis and surface plasmon resonance. The PBZ motif of CHFR recognizes two adenine-containing subunits of PAR and the phosphate backbone that connects them. More generally, PBZ motifs may recognize different numbers of PAR subunits as required to carry out their functions.

The production of selective protein kinase inhibitors is often frustrated by the similarity of the enzyme active sites. For this reason, it is challenging to design inhibitors that discriminate between the three Aurora kinases, which are important targets in cancer drug discovery. We have used a triple-point mutant of Aurora-A (AurAx3) which mimics the active site of Aurora-B to investigate the structural basis of MLN8054 selectivity. The bias toward Aurora-A inhibition by MLN8054 is fully recapitulated by AurAx3in vitro. X-ray crystal structures of the complex suggest that the basis for the discrimination is electrostatic repulsion due to the T217E substitution, which we have confirmed using a single-point mutant. The activation loop of Aurora-A in the AurAx3–MLN8054 complex exhibits an unusual conformation in which Asp274 and Phe275 side chains point into the interior of the protein. There is to our knowledge no documented precedent for this conformation, which we have termed DFG-up. The sequence requirements of the DFG-up conformation suggest that it might be accessible to only a fraction of kinases. MLN8054 thus circumvents the problem of highly homologous active sites. Binding of MLN8054 to Aurora-A switches the character of a pocket within the active site from polar to a hydrophobic pocket, similar to what is observed in the structure of Aurora-A bound to a compound that induces DFG-out. We propose that targeting this pocket may be a productive route in the design of selective kinase inhibitors and describe the structural basis for the rational design of these compounds.

Mcph1 is mutated in autosomal recessive primary microcephaly and premature chromosome condensation (PCC) syndrome. Increased chromosome condensation is a common feature of cells isolated from patients afflicted with either disease. Normal cells depleted of Mcph1 also exhibit PCC phenotype. Human Mcph1 contains three BRCA1-carboxyl terminal (BRCT) domains, the first of which (Mcph1N) is necessary for the prevention of PCC. The only known disease-associated missense mutation in Mcph1 resides in this domain (T27R). We have determined the X-ray crystal structure of human Mcph1N to 1.6 A resolution. Compared with other BRCT domain structures, the most striking differences are an elongated, ordered beta1-alpha1 loop and an adjacent hydrophobic pocket. This pocket is in the equivalent structural position to the phosphate binding site of BRCT domains that recognize phospho-proteins, although the phosphate-binding residues are absent in Mcph1N. Mutations in the pocket abrogate the ability of full-length Mcph1 to rescue the PCC phenotype of Mcph1(-/-) mouse embryonic fibroblast cells, suggesting that it forms an essential part of a protein-protein interaction site necessary to prevent PCC.

Summary Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the αC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

There is a varying degree of hand torque abilities using finger drivers among clinicians. Calibrating one's own abilities requires complicated instruments not readily available. This study evaluated a simple-to-use method that allows dental practitioners to have a quantifiable clinical assessment of relative torque ability using finger drivers to torque down dental implant components. A typodont that includes dental implants was mounted in a mannequin placed in a patient-reclined position. The subjects were asked to torque as tightly as they could a new healing abutment to an implant secured firmly in resin within the typodont. All participants wore moistened gloves when using a finger driver. The healing abutment was countertorqued using a certified precalibrated precision torque measurement device. The reading on the torque driver was recorded when the healing abutment disengaged. An average of torque values of dentists and dental students was calculated. Fifty subjects had an average maximum torque ability of 24 Ncm (male dentists: 28 Ncm; students: 22 Ncm; male students: 24 Ncm; female students: 19 Ncm). Maximum torque values for all participants ranged from 11 Ncm to 38 Ncm. There was no significant difference between groups. This study showed a varying degree of hand torquing abilities using a finger driver. Clinicians should regularly calibrate their ability to torque implant components to more predictably perform implant dentistry. Dental implant manufacturers should more precisely instruct clinicians as to maximum torque, as opposed to “finger tighten only.”

Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

All auxiliary α 2 δ subunits of voltage-gated Ca 2+ (Ca V ) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in α 2 δ-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the α 2 δ-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant α 2 δ-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT α 2 δ-2, the MIDAS mutant α 2 δ-2 subunit did not enhance and, in some cases, further diminished Ca V 1.2, -2.1, and -2.2 currents coexpressed with β1b by using either Ba 2+ or Na + as a permeant ion. Furthermore, expression of the MIDAS mutant α 2 δ-2 reduced surface expression and strongly increased the perinuclear retention of Ca V α1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108–15 cells. Despite the presence of endogenous α 2 δ subunits, heterologous expression of α 2 δ-2 in differentiated NG108–15 cells further enhanced the endogenous high-threshold Ca 2+ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that α 2 δ subunits normally interact with the Ca V α1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the α 2 δ subunit is required to promote trafficking of the α1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains.

Activating gene fusions in the RET receptor tyrosine kinase have been found to drive 1-2% of lung adenocarcinomas and therefore offer an attractive target for targeted therapy. Whilst non-selective tyrosine kinase inhibitors with RET activity are efficacious in this setting, their use is generally limited by dose limiting toxicity associated with their more potent activity versus other targets, specifically KDR (VEGFR2) in the case of cabozantinib and vandetanib. Given this limitation, there is considerable interest in developing more selective inhibitors of RET kinase. Tyrosine kinase inhibitors are prone to early clinical failure due to mutations in the kinase ATPase binding domain, which render the kinase catalytically active but no longer sensitive to drug treatment. Such mutations often occur in the so-called “gatekeeper” region and in this specific case, resistance is predicted to arise from a Val-Met or Val-Leu mutation at residue 804. Through a combination of computational methods, structural biology and drug design, we have identified and further optimized a series of inhibitors of the V804M mutant RET kinase which show sub-micromolar cellular activity in cells driven by V804M RET. Moreover, these agents show excellent selectivity against the wtRET kinase and KDR. As such, these agents may offer valuable start-points for second-generation RET inhibitors for use in patents who relapse after treatment with first generation selective RET inhibitors. Citation Format: Allan M. Jordan, Rebecca Newton, Bohdan Waszkowycz, Richard Bayliss, Habiba Begum, Daniel Burschowsky, Aude Echalier, Samantha Hitchin, Colin Hutton, Shaun Johns, Stuart Jones, Li-Ying Lin, Mark Richards, Chitra Seewooruthun, Alex Stowell, Ian Waddell, Mandy Watson, Donald Ogilvie. Delivering selective and cell-active inhibitors of V804M mutant RET kinase through structure-guided drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2017-3236

Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-A crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners.

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.)

The microwave processing procedure has been shown to be an efficient and accurate method of polymerizing poly(methyl methacrylate) resin for the fabrication of dentures. This article describes procedures to expand the uses of the microwave technique. With standard laboratory procedures, the microwave technique can improve and simplify denture relines, repairs, and rebasing of partial dentures.

Electronic shade-matching instruments are available for clinical use; however, their accuracy has not been established. This study evaluated a new electronic method of clinical shade matching compared to the standard visual method. The Vita Easyshade system, an electronic method of shade matching, was used on 40 subjects to measure the central region of each patient's maxillary left central incisor (tooth No. 9). Two visual evaluators with predetermined visual shade matching abilities selected a shade from the same area of tooth No. 9 for all subjects. Student's t-test, using a 95% score confidence interval (CI), was used to compare the two methods. The Vita Easyshade system was accurate 85% of the time in this in vivo study; however, the instrument was predictably accurate only 68-91% of the time at the 95% CI.

The Ca(V)beta subunits of voltage-gated calcium channels regulate the trafficking and biophysical properties of these channels. We have taken advantage of mutations in the tyrosine residue within the alpha interaction domain (AID) in the I-II linker of Ca(V)2.2 which reduce, but do not abolish, the binding of beta1b to the AID of Ca(V)2.2. We have found that the mutation Y388S decreased the affinity of Ca(V)beta1b binding to the Ca(V)2.2 I-II linker from 14 to 329 nm. However, the Y388S mutation had no effect on current density and cell surface expression of Ca(V)2.2/alpha2delta-2/beta1b channels expressed in human embryonic kidney tsA-201 cells, when equivalent proportions of cDNA were used. Furthermore, despite the 24-fold reduced affinity of Ca(V)beta1b for the Y388S I-II linker of Ca(V)2.2, all the key features of modulation as well as trafficking by Ca(V)beta subunits remained intact. This is in contrast to the much more marked effect of the W391A mutation, which abolished interaction with the Ca(V)2.2 I-II linker, and very markedly affected the trafficking of the channels. However, using the Xenopus oocyte expression system, where expression levels can be accurately titrated, when Ca(V)beta1b cDNA was diluted 50-fold, all evidence of interaction with Ca(V)2.2 Y388S was lost, although wild-type Ca(V)2.2 was still normally modulated by the reduced concentration of beta1b. These results indicate that high affinity interaction with the alpha1 subunit is not necessary for any of the modulatory effects of Ca(V)beta subunits, but occupancy of the interaction site is important, and this will occur, despite the reduced affinity, if the Ca(V)beta subunit is present in sufficient excess.

It has taken 17 years from the first identification of a voltage-gated Ca2+ channel (CaV) beta-subunit as a band on a gel following purification of skeletal muscle dihydropyridine (DHP) receptors in 1987 to the publication of key information on the structures of Ca2+ channel beta-subunits. Three recent X-ray crystallographic studies have now solved the structures of the core domains of three Ca2+ channel beta-subunits. In this article, the properties of these cytoplasmic auxiliary subunits will first be summarized. Then the CaVbeta structures and the information they provide regarding how these proteins interact with the CaValpha1 subunit will be discussed and the possible implications of these new data for G-protein modulation of Ca2+ channels will be examined.

Purpose Using 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. Materials and Methods An acrylic resin master cast was fabricated with embedded reference points to enable both horizontal and vertical measurements. Four commercially available polyvinyl siloxane impression putties with light body washes were evaluated: 1) Extrude (Kerr), 2) Cutter (Coltere), 3) Express (3M), and 4) Reprosil (Caulk). Each putty was mixed by hand for 30 seconds, placed in a plastic stock tray, and seated on the master cast at 0, 30, 60, 90, and 120 seconds after mixing (N = 100, n = 5). The impressions were poured in improved stone, and vertical and horizontal measurements were made to 0.001 mm between reference points on recovered casts using an optical traveling microscope. ANOVA (p .05) was used to compare vertical and horizontal measurements to master cast dimensions. Results No statistically significant difference was found among casts for all materials and time periods tested. When horizontal measurement ranges were evaluated, Extrude casts exhibited the widest range of measurement, especially when the trays were seated at 90 and 120 seconds. When vertical measurement ranges were evaluated, Express casts at 120 seconds exhibited the widest range. Conclusions A high range of variability in vertical and horizontal dimensions occurred at the later time periods, especially for Extrude and Express casts. As a result of rapid polymerization and poor flow properties of putty materials, the impression tray should be seated within 60 seconds after putty mixing to reduce dimensional variability of recovered casts.

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 t test. Repeatability for both tests was determined with a paired t test. 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.)

Methods to facilitate the seating of artificial crowns can improve the gingival margins and ensure longevity. This study examined the effects of venting and different luting agents on the seating accuracy and compressive strength of Dicor ceramic crowns. Vented and unaltered anterior restorations were tested using zinc phosphate cement (ZnPO4), glass ionomer, and composite resin cement. Rexillium metal dies of a tooth preparation of a maxillary canine were provided and standardized crowns were fabricated. The artificial crowns were evaluated for adaptation of the finish line before and after cementation, then were compressively loaded to failure. Under the conditions of this study, neither the design of the artificial crown nor the luting agent had a significant effect on the compressive strength. Composite resinous cement appeared to enhance seating of the crown, whereas ZnPO4 cement inhibited seating.

The indications and features of the new Denar Combi articulator are reviewed. The Combi articulator was designed primarily as a set path instrument, but can also be used as a semiadjustable or fully adjustable stereographic articulator. The articulator can be programmed for any of these modes, but the Combi model is limited when used as a stereographic instrument according to the manufacturer's instructions, with the instrument-precipitated geometric errors. These errors were identified mathematically, and an accurate technique for the instrument is described.

Summary The Nek family of serine/threonine kinases regulates centrosome and cilia function; in addition, several of its members are potential targets for drug discovery. Nek2 is dimeric, is cell cycle regulated and functions in the separation of centrosomes at G2/M. Here, we report the crystal structures of wild-type human Nek2 kinase domain bound to ADP at 1.55-Å resolution and T175A mutant in apo form as well as that bound to a non-hydrolyzable ATP analog. These show that regions of the Nek2 structure around the nucleotide-binding site can adopt several different but well-defined conformations. None of the conformations was the same as that observed for the previously reported inhibitor-bound structure, and the two nucleotides stabilized two conformations. The structures suggest mechanisms for the auto-inhibition of Nek2 that we have tested by mutagenesis. Comparison of the structures with Aurora-A and Cdk2 gives insight into the structural mechanism of Nek2 activation. The production of specific inhibitors that target individual kinases of the human genome is an urgent challenge in drug discovery, and Nek2 is especially promising as a cancer target. We not only identify potential challenges to the task of producing Nek2 inhibitors but also propose that the conformational variability provides an opportunity for the design of Nek2 selective inhibitors because one of the conformations may provide a unique target.