scholarly journals Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors

2016 ◽  
Vol 113 (48) ◽  
pp. 13726-13731 ◽  
Author(s):  
Mark W. Richards ◽  
Selena G. Burgess ◽  
Evon Poon ◽  
Anne Carstensen ◽  
Martin Eilers ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Neuroblastoma Cells ◽  
Direct Interaction ◽  
Cancer Therapeutics ◽  
Structural Basis ◽  
Aurora A ◽  
Polyubiquitin Chains ◽  
Ubiquitin Proteasome ◽  
A Site

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 theMYCNoncogene) 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-Å 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 SCFFbxW7to disfavor the generation of Lys48-linked polyubiquitin chains.

scholarly journals HSP27 Is a Ubiquitin-Binding Protein Involved in I-κBα Proteasomal Degradation

2003 ◽  
Vol 23 (16) ◽  
pp. 5790-5802 ◽  
Author(s):  
Arnaud Parcellier ◽  
Elise Schmitt ◽  
Sandeep Gurbuxani ◽  
Daphné Seigneurin-Berny ◽  
Alena Pance ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Cell Types ◽  
26S Proteasome ◽  
Necrosis Factor Alpha ◽  
Polyubiquitin Chains ◽  
Ubiquitinated Proteins ◽  
Activation Of Transcription ◽  
Ubiquitin Proteasome

ABSTRACT HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-α). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-κB by degrading its main inhibitor, I-κBα. HSP27 overexpression increases NF-κB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, ΤNF-α, and interleukin 1β. HSP27 does not affect I-κBα phosphorylation but enhances the degradation of phosphorylated I-κBα by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-κBα. A protein complex that includes HSP27, phosphorylated I-κBα, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-κBα. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-κB activity.

scholarly journals Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain

Open Biology ◽  
2016 ◽  
Vol 6 (7) ◽  
pp. 160089 ◽  
Author(s):  
Selena G. Burgess ◽  
Arkadiusz Oleksy ◽  
Tommaso Cavazza ◽  
Mark W. Richards ◽  
Isabelle Vernos ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Salt Bridge ◽  
Binding Pocket ◽  
Protein Kinase Inhibitors ◽  
Single Domain ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Allosteric Site ◽  
Kinase Catalytic Domain

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. 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. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain 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 single domain antibodies 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.

Crystal structure of an Aurora-A mutant that mimics Aurora-B bound to MLN8054: insights into selectivity and drug design

2010 ◽  
Vol 427 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Charlotte A. Dodson ◽  
Magda Kosmopoulou ◽  
Mark W. Richards ◽  
Butrus Atrash ◽  
Vassilios Bavetsias ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Rational Design ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Structural Basis ◽  
Cancer Drug ◽  
Aurora B ◽  
Aurora A ◽  
Point Mutant

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.

scholarly journals Allosteric inhibition of Aurora-A kinase by a synthetic VNAR nanobody

2016 ◽  
Author(s):  
Selena G. Burgess ◽  
Arkadiusz Oleksy ◽  
Tommaso Cavazza ◽  
Mark W. Richards ◽  
Isabelle Vernos ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Binding Pocket ◽  
Protein Kinase Inhibitors ◽  
Single Domain ◽  
Atp Binding ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Allosteric Site

AbstractThe 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.

Src Kinase Inhibitors: Promising Cancer Therapeutics?

2012 ◽  
Vol 17 (2) ◽  
pp. 145-159 ◽  
Author(s):  
Helen Creedon ◽  
Valerie G . Brunton
Keyword(s):  
Kinase Inhibitors ◽  
Src Kinase ◽  
Cancer Therapeutics

scholarly journals Gastrointestinal Stromal Tumors (GISTs): Novel Therapeutic Strategies with Immunotherapy and Small Molecules

2021 ◽  
Vol 22 (2) ◽  
pp. 493
Author(s):  
Christos Vallilas ◽  
Panagiotis Sarantis ◽  
Anastasios Kyriazoglou ◽  
Evangelos Koustas ◽  
Stamatios Theocharis ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Gastrointestinal Stromal Tumors ◽  
Kinase Inhibitors ◽  
Checkpoint Inhibitors ◽  
Survival Rates ◽  
Cancer Therapeutics ◽  
Gastrointestinal Neoplasms ◽  
Mesenchymal Tumors ◽  
Stromal Tumors

Gastrointestinal stromal tumors (GISTs) are the most common types of malignant mesenchymal tumors in the gastrointestinal tract, with an estimated incidence of 1.5/100.000 per year and 1–2% of gastrointestinal neoplasms. About 75–80% of patients have mutations in the KIT gene in exons 9, 11, 13, 14, 17, and 5–10% of patients have mutations in the platelet-derived growth factor receptor a (PDGFRA) gene in exons 12, 14, 18. Moreover, 10–15% of patients have no mutations and are classified as wild type GIST. The treatment for metastatic or unresectable GISTs includes imatinib, sunitinib, and regorafenib. So far, GIST therapies have raised great expectations and offered patients a better quality of life, but increased pharmacological resistance to tyrosine kinase inhibitors is often observed. New treatment options have emerged, with ripretinib, avapritinib, and cabozantinib getting approvals for these tumors. Nowadays, immune checkpoint inhibitors form a new landscape in cancer therapeutics and have already shown remarkable responses in various tumors. Studies in melanoma, non-small-cell lung cancer, and renal cell carcinoma are very encouraging as these inhibitors have increased survival rates. The purpose of this review is to present alternative approaches for the treatment of the GIST patients, such as combinations of immunotherapy and novel inhibitors with traditional therapies (tyrosine kinase inhibitors).

scholarly journals Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 576
Author(s):  
Sofia Giacosa ◽  
Catherine Pillet ◽  
Irinka Séraudie ◽  
Laurent Guyon ◽  
Yann Wallez ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Renal Carcinoma ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Cancer Therapeutics ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Cell Renal Cell Carcinoma ◽  
Von Hippel Lindau ◽  
Renal Carcinoma Cells

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL− cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug–gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

scholarly journals Anti-EGFR antibody 528 binds to domain III of EGFR at a site shifted from the cetuximab epitope

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Koki Makabe ◽  
Takeshi Yokoyama ◽  
Shiro Uehara ◽  
Tomomi Uchikubo-Kamo ◽  
Mikako Shirouzu ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Growth Factor Receptor ◽  
Cancer Therapeutics ◽  
Antibody Engineering ◽  
Egfr Mutations ◽  
Particle Analysis ◽  
Extracellular Region ◽  
Domain Iii ◽  
A Site ◽  
Egfr Antibody

AbstractAntibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Epidermal growth factor receptor (EGFR) is a promising target for the cancer therapeutics, against which several antibody clones have been developed and brought into therapeutic use. Another antibody clone, 528, is an antagonistic anti-EGFR antibody, which has been the focus of our antibody engineering studies to develop cancer drugs. In this study, we explored the interaction of 528 with the extracellular region of EGFR (sEGFR) via binding analyses and structural studies. Dot blotting experiments with heat treated sEGFR and surface plasmon resonance binding experiments revealed that 528 recognizes the tertiary structure of sEGFR and exhibits competitive binding to sEGFR with EGF and cetuximab. Single particle analysis of the sEGFR–528 Fab complex via electron microscopy clearly showed the binding of 528 to domain III of sEGFR, the domain to which EGF and cetuximab bind, explaining its antagonistic activity. Comparison between the two-dimensional class average and the cetuximab/sEGFR crystal structure revealed that 528 binds to a site that is shifted from, rather than identical to, the cetuximab epitope, and may exclude known drug-resistant EGFR mutations.

scholarly journals Crystallization of and selenomethionine phasing strategy for a SETMAR–DNA complex

2016 ◽  
Vol 72 (9) ◽  
pp. 713-719 ◽  
Author(s):  
Qiujia Chen ◽  
Millie Georgiadis
Keyword(s):  
Dna Binding ◽  
Catalytic Domain ◽  
Specific Binding ◽  
Critical Role ◽  
Binding Activity ◽  
Structural Basis ◽  
Unexpected Finding ◽  
Terminal Inverted Repeat ◽  
Dna Binding Activity ◽  
New Genes

Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of theHsmar1transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestralHsmar1terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD–DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.

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