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.

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.

scholarly journals 7-(2-Anilinopyrimidin-4-yl)-1-benzazepin-2-ones Designed by a “Cut and Glue” Strategy Are Dual Aurora A/VEGF-R Kinase Inhibitors

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1611
Author(s):  
Mehmet Karatas ◽  
Apirat Chaikuad ◽  
Bianca Berger ◽  
Michael H. G. Kubbutat ◽  
Frank Totzke ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitors ◽  
Vegf Receptor ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Single Digit ◽  
Wide Range

Although overexpression and hyperactivity of protein kinases are causative for a wide range of human cancers, protein kinase inhibitors currently approved as cancer drugs address only a limited number of these enzymes. To identify new chemotypes addressing alternative protein kinases, the basic structure of a known PLK1/VEGF-R2 inhibitor class was formally dissected and reassembled. The resulting 7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones were synthesized and proved to be dual inhibitors of Aurora A kinase and VEGF receptor kinases. Crystal structures of two representatives of the new chemotype in complex with Aurora A showed the ligand orientation in the ATP binding pocket and provided the basis for rational structural modifications. Congeners with attached sulfamide substituents retained Aurora A inhibitory activity. In vitro screening of two members of the new kinase inhibitor family against the cancer cell line panel of the National Cancer Institute (NCI) showed antiproliferative activity in the single-digit micromolar concentration range in the majority of the cell lines.

Benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one derivatives as Aurora A kinase inhibitors: LQTA-QSAR analysis and detailed systematic validation of the developed model

2015 ◽  
Vol 19 (4) ◽  
pp. 965-974 ◽  
Author(s):  
Ashish M. Kanhed ◽  
Radha Charan Dash ◽  
Nishant Parmar ◽  
Tarun Kumar Das ◽  
Rajani Giridhar ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Qsar Analysis

ChemInform Abstract: Single Step Synthesis of New Fused Pyrimidine Derivatives and Their Evaluation as Potent Aurora-A Kinase Inhibitors.

ChemInform ◽  
2011 ◽  
Vol 43 (4) ◽  
pp. no-no
Author(s):  
Mohamed R. Shaaban ◽  
Tamer S. Saleh ◽  
Abdelrahman S. Mayhoub ◽  
Ahmad M. Farag
Keyword(s):  
Kinase Inhibitors ◽  
Single Step ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Pyrimidine Derivatives

scholarly journals Structure-Based Discovery and Bioactivity Evaluation of Novel Aurora-A Kinase Inhibitors as Anticancer Agents via Docking-Based Comparative Intermolecular Contacts Analysis (dbCICA)

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 6003
Author(s):  
Majd S. Hijjawi ◽  
Reem Fawaz Abutayeh ◽  
Mutasem O. Taha
Keyword(s):  
Anticancer Agents ◽  
Cell Cycle Progression ◽  
Kinase Inhibitors ◽  
Epithelial Mesenchymal Transition ◽  
Resonance Energy ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Characteristic Analysis ◽  
Mesenchymal Transition ◽  
Intermolecular Contacts

Aurora-A kinase plays a central role in mitosis, where aberrant activation contributes to cancer by promoting cell cycle progression, genomic instability, epithelial-mesenchymal transition, and cancer stemness. Aurora-A kinase inhibitors have shown encouraging results in clinical trials but have not gained Food and Drug Administration (FDA) approval. An innovative computational workflow named Docking-based Comparative Intermolecular Contacts Analysis (dbCICA) was applied—aiming to identify novel Aurora-A kinase inhibitors—using seventy-nine reported Aurora-A kinase inhibitors to specify the best possible docking settings needed to fit into the active-site binding pocket of Aurora-A kinase crystal structure, in a process that only potent ligands contact critical binding-site spots, distinct from those occupied by less-active ligands. Optimal dbCICA models were transformed into two corresponding pharmacophores. The optimal one, in capturing active hits and discarding inactive ones, validated by receiver operating characteristic analysis, was used as a virtual in-silico search query for screening new molecules from the National Cancer Institute database. A fluorescence resonance energy transfer (FRET)-based assay was used to assess the activity of captured molecules and five promising Aurora-A kinase inhibitors were identified. The activity was next validated using a cell culture anti-proliferative assay (MTT) and revealed a most potent lead 85(NCI 14040) molecule after 72 h of incubation, scoring IC50 values of 3.5–11.0 μM against PANC1 (pancreas), PC-3 (prostate), T-47D and MDA-MB-231 (breast)cancer cells, and showing favorable safety profiles (27.5 μM IC50 on fibroblasts). Our results provide new clues for further development of Aurora-A kinase inhibitors as anticancer molecules.

scholarly journals Switching Aurora-A kinase on and off at an allosteric site

FEBS Journal ◽  
2017 ◽  
Vol 284 (18) ◽  
pp. 2947-2954 ◽  
Author(s):  
Richard Bayliss ◽  
Selena G. Burgess ◽  
Patrick J. McIntyre
Keyword(s):  
Aurora A Kinase ◽  
Aurora A ◽  
Allosteric Site

scholarly journals Human TPX2 is required for targeting Aurora-A kinase to the spindle

2002 ◽  
Vol 158 (4) ◽  
pp. 617-623 ◽  
Author(s):  
Thomas A. Kufer ◽  
Herman H.W. Silljé ◽  
Roman Körner ◽  
Oliver J. Gruss ◽  
Patrick Meraldi ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Major Protein ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Binding Studies ◽  
Serine Threonine Kinase ◽  
Cell Extracts ◽  
Prominent Component ◽  
Spindle Apparatus

Aurora-A is a serine-threonine kinase implicated in the assembly and maintenance of the mitotic spindle. Here we show that human Aurora-A binds to TPX2, a prominent component of the spindle apparatus. TPX2 was identified by mass spectrometry as a major protein coimmunoprecipitating specifically with Aurora-A from mitotic HeLa cell extracts. Conversely, Aurora-A could be detected in TPX2 immunoprecipitates. This indicates that subpopulations of these two proteins undergo complex formation in vivo. Binding studies demonstrated that the NH2 terminus of TPX2 can directly interact with the COOH-terminal catalytic domain of Aurora-A. Although kinase activity was not required for this interaction, TPX2 was readily phosphorylated by Aurora-A. Upon siRNA-mediated elimination of TPX2 from cells, the association of Aurora-A with the spindle microtubules was abolished, although its association with spindle poles was unaffected. Conversely, depletion of Aurora-A by siRNA had no detectable influence on the localization of TPX2. We propose that human TPX2 is required for targeting Aurora-A kinase to the spindle apparatus. In turn, Aurora-A might regulate the function of TPX2 during spindle assembly.

scholarly journals Development of a Fluorescence Intensity Assay for the Mitotic Serine/Threonine Protein Kinase Aurora-A

2012 ◽  
Vol 18 (2) ◽  
pp. 219-225 ◽  
Author(s):  
Andrew F. Slatter ◽  
Spencer Campbell ◽  
Richard M. Angell
Keyword(s):  
Fluorescence Intensity ◽  
Kinase Inhibitors ◽  
Binding Assay ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Aurora Kinases ◽  
Intramolecular Quenching ◽  
Labeled Probe ◽  
Key Steps ◽  
Tumor Types

The Aurora kinases are a group of serine/threonine protein kinases that regulate key steps during mitosis, and deregulation of these proteins (e.g., by gene amplification or overexpression) has been linked to a wide variety of tumor types. Thus, Aurora-A and Aurora-B have been intensely studied as targets for anticancer therapy and are now clinically validated targets. Here we report on the development of a novel fluorescence intensity binding assay for Aurora-A kinase inhibitors using a fluorescently labeled probe compound that shows intramolecular quenching when unbound but exhibits a dramatic increase in fluorescence when bound to Aurora-A.

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