scholarly journals Phosphorylation, Mg-ADP, and Inhibitors Differentially Shape the Conformational Dynamics of the A-Loop of Aurora-A

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 567
Author(s):  
Zahra Musavizadeh ◽  
Alessandro Grottesi ◽  
Giulia Guarguaglini ◽  
Alessandro Paiardini
Keyword(s):  
Protein Kinases ◽  
Kinase Inhibitors ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Kinase Domain ◽  
General Description ◽  
Aurora A ◽  
Post Translational Modification ◽  
The Stability

The conformational state of the activation loop (A-loop) is pivotal for the activity of most protein kinases. Hence, the characterization of the conformational dynamics of the A-loop is important to increase our understanding of the molecular processes related to diseases and to support the discovery of small molecule kinase inhibitors. Here, we carry out a combination of molecular dynamics (MD) and essential dynamics (ED) analyses to fully map the effects of phosphorylation, ADP, and conformation disrupting (CD) inhibitors (i.e., CD532 and MLN8054) on the dynamics of the A-loop of Aurora-A. MD revealed that the stability of the A-loop in an open conformation is enhanced by single phospho-Thr-288, while paradoxically, the presence of a second phosphorylation at Thr-287 decreases such stability and renders the A-loop more fluctuant in time and space. Moreover, we found that this post-translational modification has a significant effect on the direction of the A-loop motions. ED analysis suggests that the presence of the phosphate moiety induces the dynamics of Aurora-A to sample two distinct energy minima, instead of a single large minimum, as in unphosphorylated Aurora-A states. This observation indicates that the conformational distributions of Aurora-A with both single and double phospho-threonine modifications are remarkably different from the unphosphorylated state. In the closed states, binding of CD532 and MLN8054 inhibitors has the effect of increasing the distance of the N- and C-lobes of the kinase domain of Aurora-A, and the angle analysis between those two lobes during MD simulations showed that the N- and C-lobes are kept more open in presence of CD532, compared to MLN8054. As the A-loop is a common feature of Aurora protein kinases, our studies provide a general description of the conformational dynamics of this structure upon phosphorylation and different ligands binding.

scholarly journals Characterization of an activated human ros gene.

1986 ◽  
Vol 6 (9) ◽  
pp. 3109-3116 ◽  
Author(s):  
C Birchmeier ◽  
D Birnbaum ◽  
G Waitches ◽  
O Fasano ◽  
M Wigler
Keyword(s):  
Gene Transfer ◽  
Protein Kinases ◽  
Transmembrane Domain ◽  
Extracellular Domain ◽  
Kinase Domain ◽  
Specific Protein ◽  
Normal Human

A human oncogene, mcf3, previously detected by a combination of DNA-mediated gene transfer and a tumorigenicity assay, derives from a human homology of the avian v-ros oncogene. Both v-ros and mcf3 can encode a protein with homology to tyrosine-specific protein kinases, and both mcf3 and v-ros encode a potential transmembrane domain N terminal to the kinase domain. mcf3 probably arose during gene transfer from a normal human ros gene by the loss of a putative extracellular domain. There do not appear to be any other gross rearrangements in the structure of mcf3.

scholarly journals Effects of CD4 Binding on Conformational Dynamics, Molecular Motions, and Thermodynamics of HIV-1 gp120

2019 ◽  
Vol 20 (2) ◽  
pp. 260 ◽  
Author(s):  
Yi Li ◽  
Lei Deng ◽  
Li-Quan Yang ◽  
Peng Sang ◽  
Shu-Qun Liu
Keyword(s):  
Free Energy ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Cell Receptor ◽  
Molecular Motions ◽  
Host Cell Receptor ◽  
Gp120 Core ◽  
Glycoprotein Gp120 ◽  
The Stability ◽  
Hiv 1

Human immunodeficiency virus type-1 (HIV-1) infection is triggered by its envelope (Env) glycoprotein gp120 binding to the host-cell receptor CD4. Although structures of Env/gp120 in the liganded state are known, detailed information about dynamics of the liganded gp120 has remained elusive. Two structural models, the CD4-free gp120 and the gp120-CD4 complex, were subjected to µs-scale multiple-replica molecular dynamics (MD) simulations to probe the effects of CD4 binding on the conformational dynamics, molecular motions, and thermodynamics of gp120. Comparative analyses of MD trajectories in terms of structural deviation and conformational flexibility reveal that CD4 binding effectively suppresses the overall conformational fluctuations of gp120. Despite the largest fluctuation amplitude of the V1/V2 region in both forms of gp120, the presence of CD4 prevents it from approaching the gp120 core. Comparison of the constructed free energy landscapes (FELs) shows that CD4 binding reduces the conformational entropy and conformational diversity while enhancing the stability of gp120. Further comparison of the representative structures extracted from free energy basins/minima of FELs reveals that CD4 binding weakens the reorientation ability of V1/V2 and hence hinders gp120 from transitioning out of the liganded state to the unliganded state. Therefore, locking gp120 conformation via restraining V1/V2 reorientation with small molecules seems to be a promising strategy to control HIV-1 infection. Our computer simulation results support the conformational selection mechanism for CD4 binding to gp120 and facilitate the understanding of HIV-1 immune evasion mechanisms.

Synthesis and characterization of pyrazoleanthrone derivatives as Aurora A kinase inhibitors

2013 ◽  
Vol 29 (6) ◽  
pp. 1098-1103
Author(s):  
Xiao-xiao Sun ◽  
Tao Sun ◽  
Tai-yi Wang ◽  
Yan Zhang ◽  
Hui-juan Liu ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Synthesis And Characterization ◽  
Aurora A Kinase ◽  
Aurora A

scholarly journals Synthesis and Characterization of 5′-p-fluorosulfonylbenzoyl-2′ (or 3′)-(biotinyl)adenosine as an Activity-Based Probe for Protein Kinases

2006 ◽  
Vol 12 (1) ◽  
pp. 126-132 ◽  
Author(s):  
Steven J. Ratcliffe ◽  
Tracey Yi ◽  
Sanjay S. Khandekar
Keyword(s):  
Clinical Trials ◽  
Binding Site ◽  
Protein Kinases ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Synthesis And Characterization ◽  
Atp Binding Site ◽  
Western Blot Method ◽  
Therapeutic Uses

Most of the kinase inhibitors that are approved for therapeutic uses or that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. 5'-Fluorosulfonylbenzoyl 5′-adenosine (FSBA) is an activitybased probe (ABP) that covalently modifies a conserved lysine present in the nucleotide binding site of most kinases. Here the authors describe synthesis of FSBA derivatives, 2′-biotinyl-FSBA and 3′-biotinyl-FSBA as kinase ABPs, and delineate a Western blot method to screen and validate ATP competitive protein kinase inhibitors using biotinyl-FSBA as a nonselective activity-based probe for protein kinases.

scholarly journals Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms

2019 ◽  
Author(s):  
Daniel P. Duarte ◽  
Allan J. Lamontanara ◽  
Giuseppina La Sala ◽  
Sukyo Jeong ◽  
Yoo-Kyoung Sohn ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Kinase Inhibitors ◽  
Md Simulations ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Loss Of Function ◽  
Kinase Activation ◽  
Allosteric Interactions ◽  
B Cell Maturation

ABSTRACTBruton’s tyrosine kinase (Btk) is a key component for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, constitutive Btk signaling drives several B-cell neoplasms, which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the molecular mechanism by which a subset of XLA mutations in the SH2 domain strongly perturbs Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain to which multiple XLA mutations map and which is required for Btk activation. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the Btk SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevented activation of wild-type and TKI-resistant Btk, inhibited Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.

scholarly journals Dynamics of human protein kinases linked to drug selectivity

2018 ◽  
Author(s):  
Warintra Pitsawong ◽  
Vanessa Buosi ◽  
Renee Otten ◽  
Roman V. Agafonov ◽  
Adelajda Zorba ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Protein Kinases ◽  
Conformational Changes ◽  
Drug Targets ◽  
Active Sites ◽  
Kinase Inhibitors ◽  
Drug Binding ◽  
Inhibition Mechanism ◽  
Aurora A ◽  
Drug Selectivity

AbstractProtein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.eLife digestThe Ser/Thr kinase Aurora A is an important target for the development of new anticancer therapies. A longstanding question is how to specifically and effectively inhibit only this kinase in a background of over 550 protein kinases with very similar structures. To this end, understanding the inhibition mechanism of Aurora A by different drugs is essential. Here, we characterize the kinetic mechanism of three distinct kinase drugs, Gleevec (Imatinib), Danusertib (PHA739358) and AT9283 (Pyrazol-4-yl Urea) for Aurora A. We show that inhibitor affinities do not rely exclusively on the recognition of a specific conformation of the Asp-Phe-Gly loop of the kinase. Our quantitative kinetics data put forward an opposing mechanism in which a slow conformational change after drug binding (i.e., induced-fit step) dictates drug affinity.

scholarly journals A moving target: structure and disorder in pursuit of Myc inhibitors

2017 ◽  
Vol 45 (3) ◽  
pp. 709-717 ◽  
Author(s):  
Richard Bayliss ◽  
Selena G. Burgess ◽  
Eoin Leen ◽  
Mark W. Richards
Keyword(s):  
Protein Interaction ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Transactivation Domain ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Molecular Scaffold ◽  
Protein Protein Interaction ◽  
Intrinsically Disordered ◽  
And Function

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.

scholarly journals Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states

2018 ◽  
Vol 115 (51) ◽  
pp. E11894-E11903 ◽  
Author(s):  
Eric W. Lake ◽  
Joseph M. Muretta ◽  
Andrew R. Thompson ◽  
Damien M. Rasmussen ◽  
Abir Majumdar ◽  
...  
Keyword(s):  
Large Scale ◽  
Kinase Inhibitors ◽  
Conformational Dynamics ◽  
Aurora Kinase ◽  
Activation Loop ◽  
Aurora A ◽  
Kinase Activation ◽  
Mitotic Kinase ◽  
Conformational Preferences ◽  
Wide Range

Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states. Profiling a panel of clinically relevant Aurora kinase inhibitors against the mitotic kinase Aurora A revealed a wide range of conformational preferences, with all inhibitors promoting either the active DFG-in state or the inactive DFG-out state, but to widely differing extents. Remarkably, these conformational preferences explain broad patterns of inhibitor selectivity across different activation states of Aurora A, with DFG-out inhibitors preferentially binding Aurora A activated by phosphorylation on the activation loop, which dynamically samples the DFG-out state, and DFG-in inhibitors binding preferentially to Aurora A constrained in the DFG-in state by its allosteric activator Tpx2. The results suggest that many inhibitors currently in clinical development may be capable of differentiating between Aurora A signaling pathways implicated in normal mitotic control and in melanoma, neuroblastoma, and prostate cancer. The technology is applicable to a wide range of clinically important kinases and could provide a wealth of valuable structure–activity information for the development of inhibitors that exploit differences in conformational dynamics to achieve enhanced selectivity.

The Most Common Dasatinib-Resistant BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia Are Sensitive to VX-680: Rationale for Early Combination Kinase Inhibitor Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2175-2175 ◽  
Author(s):  
Neil P. Shah ◽  
Brian Skaggs ◽  
Susan Branford ◽  
Timothy P. Hughes ◽  
John M. Nicoll ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Aurora A ◽  
Abl Kinase ◽  
Micromolar Concentration ◽  
T315i Mutation ◽  
Contact Residues ◽  
Kinase Domain Mutations

Abstract Selection for CML cells with BCR-ABL kinase domain mutations represents the predominant molecular mechanism responsible for loss of response to imatinib. Similarly, we have found acquired resistance to dasatinib to be associated with kinase domain mutations in 100% of cases (n=15). However, unlike the multitude of imatinib resistant mutations, which to a large extent occur at non-contact residues and destabilize the inactive confirmation to which imatinib binds, only two mutations appear to be responsible for nearly all cases of dasatinib resistance, T315I and V299L (Shah et al, submitted, ASH 2006). Both of these mutations occur at critical contact residues between the ABL kinase domain and dasatinib. Successful treatment of dasatinib-resistant cases will therefore require strategies to successfully eliminate cells that harbor these mutations. Use of a combination of kinase inhibitors with the ability to collectively suppress all BCR-ABL kinase domain mutants would be predicted to lead to profoundly minimize disease resistance and relapse on targeted therapy. Although the combination of imatinib and dasatinib may prevent selection of the dasatinib-resistant V299L mutation, these agents share many targets, and their combination may therefore result in substantial toxicity. Moreover, the combination of imatinib and dasatinib is not predicted to effectively inhibit the growth of cells harboring BCR-ABL/T315I. We previously have shown that the Aurora kinase inhibitor VX-680 can bind to the ABL kinase domain and inhibit the kinase activity of the T315I mutation at low micromolar concentration. VX-680 is showing early signs of efficacy in CML cases associated with the T315I mutation. Interestingly, the co-crystal structure of VX-680 reveals that V299 is one of 14 contact residues within the ABL kinase domain. Substitution of leucine at this residue might therefore be expected to diminish the potential affinity of VX-680 for BCR-ABL/V299L. However, analysis of the amino acid sequence of Aurora-A revealed divergence from native BCR-ABL at this the corresponding amino acid position due to the presence of a leucine in Aurora-A. BCR-ABL/V299L is therefore a mimetic of Aurora-A, and as a result, predicted to retain sensitivity to VX-680. We therefore assessed VX-680 for its ability to inhibit the kinase activity of BCR-ABL/V299L in Ba/F3 cells and found effective inhibition of the kinase activity at low micromolar concentration. Consistent with predictions based upon structural considerations, the V299L mutation is somewhat more sensitive to VX-680 than BCR-ABL/T315I. We confirmed these results in an analysis of primary human PBMCs obtained from a dasatinib-resistant patient who had evolved the V299L mutation on therapy. Our findings suggest that early combination therapy with two kinase inhibitors, dasatinib and VX-680, may successfully suppress resistant disease by collectively eliminating BCR-ABL kinase domain mutation as a mechanism of resistance, and thereby achieve effective long-term disease control in the vast majority of patients.

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