Dynamics of human protein kinase Aurora A linked to 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.

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Basis for drug selectivity of plasmepsin IX and X inhibition for Plasmodium falciparum and vivax

10.21203/rs.3.rs-944781/v1 ◽

2021 ◽

Author(s):

Anthony N. Hodder ◽

Stephen Scally ◽

Tony Triglia ◽

Anna Ngo ◽

Richard W. Birkinshaw ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Crystal Structures ◽

Drug Targets ◽

Active Sites ◽

Drug Binding ◽

Dual Inhibitor ◽

3 Dimensional ◽

Protein Substrates ◽

Binding Cleft ◽

Parasite Egress

Abstract Plasmepsin IX (PMIX) and X (PMX) are aspartyl proteases of Plasmodium spp. that play essential roles in parasite egress, invasion and development. Consequently, they are important drug targets for Plasmodium falciparum and P. vivax. WM4 and WM382 are potent inhibitors of PMIX and PMX that block invasion of liver and blood stages and transmission to mosquitoes. WM4 specifically inhibits PMX whilst WM382 is a dual inhibitor of PMIX and PMX. To understand the function of PMIX and PMX proteases we identified new protein substrates in P. falciparum and together with detailed kinetic analyses and structural analyses identified key molecular interactions in the active site responsible for the specificity of WM4 and WM382 inhibition. The crystal structures of PMX apo enzyme and the protease/drug complexes of PMX/WM382 and PMX/WM4 for P. falciparum and P. vivax have been solved. We show PMIX and PMX have similar substrate selectivity, however, there are distinct differences for both peptide and full-length protein substrates through differences in localised 3-dimensional structures for the enzyme substrate-binding cleft and substrate interface. The differences in affinities of WM4 and WM382 binding for PMIX and PMX map to variations in surface interactions with each protease in the S' region of the active sites. Crystal structures of PMX reveal interactions and mechanistic detail on the selectivity of drug binding which will be important for further development of clinical candidates against these important molecular targets.

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Cryo-EM structure of a dimeric B-Raf:14-3-3 complex reveals asymmetry in the active sites of B-Raf kinases

Science ◽

10.1126/science.aay0543 ◽

2019 ◽

Vol 366 (6461) ◽

pp. 109-115 ◽

Cited By ~ 28

Author(s):

Yasushi Kondo ◽

Jana Ognjenović ◽

Saikat Banerjee ◽

Deepti Karandur ◽

Alan Merk ◽

...

Keyword(s):

Drug Targets ◽

Active Sites ◽

Kinase Inhibitors ◽

Distal Segment ◽

Kinase Domain ◽

Terminal Segment ◽

Cancer Drug ◽

Active Conformation ◽

Raf Kinase ◽

Cryo Electron Microscopy

Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo–electron microscopy structural analysis of a phosphorylated B-Raf kinase domain dimer in complex with dimeric 14-3-3, at a resolution of ~3.9 angstroms, shows an asymmetric arrangement in which one kinase is in a canonical “active” conformation. The distal segment of the C-terminal tail of this kinase interacts with, and blocks, the active site of the cognate kinase in this asymmetric arrangement. Deletion of the C-terminal segment reduces Raf activity. The unexpected asymmetric quaternary architecture illustrates how the paradoxical activation of Raf by kinase inhibitors reflects an innate mechanism, with 14-3-3 facilitating inhibition of one kinase while maintaining activity of the other. Conformational modulation of these contacts may provide new opportunities for Raf inhibitor development.

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Identification of Potential Drug Targets and Prediction of the Potential Impact of High Risk Non Synonymous Single Nucleotide Polymorphism in SARS-CoV-2 3C like Proteinase (3CLpro): A Computational Approach

10.26434/chemrxiv.12235181.v1 ◽

2020 ◽

Author(s):

Sahar Elbager ◽

abdelrahman hamza ◽

Afra M. Al Bkrye ◽

Asia M. Alrashied ◽

Entisar N. M. Ali ◽

...

Keyword(s):

Protein Interactions ◽

Drug Targets ◽

Drug Binding ◽

Snp Analysis ◽

Potential Drug ◽

Full Genome Sequencing ◽

Drug Binding Site ◽

Adme Properties ◽

Approved Drugs ◽

And Function

On January 2020, a new coronavirus (officially named SARS-CoV-2) was associated with alarming outbreak of a pneumonia-like illness, which was later named by the WHO as COVID-19, originating from Wuhan City, China. Although many clinical studies involving antiviral and immunomodulatory drug treatments for SARS-CoV-2 all without reported results, no approved drugs have been found to effectively inhibit the virus so far. Full genome sequencing of the virus was done, and uploaded to be freely available for the world scientists to explore. A promising target for SARS-CoV-2 drug design is a chymotrypsin-like cysteine protease (3CLpro), a main protease responsible for the replication and maturation of functional proteins in the life cycle of the SARS coronavirus. Here we aim to explore SARS-CoV-2 3CLpro as possible drug targets based on ligand- protein interactions. In addition, ADME properties of the ligands were also analyzed to predict their drug likeliness. The results revealed Out of 9 ligands, 8 ligands (JFM, X77, RZG, HWH, T8A, 0EN, PEPTIDE and DMS) showed best ADME properties. These findings suggest that these ligands can be used as potential molecules for developing potent inhibitors against SARS-CoV-2 3CLpro, which could be helpful in inhibiting the propagation of the COVID-19. Furthermore, 10 potential amino acids residues were recognized as potential drug binding site (THR25, HIS41, GLY143, SER144, CYS145, MET165, GLU166, GLN189, ASP295 and ARG298). All those amino acid residues were subjected to missense SNP analysis were recognized to affect the structure and function of the protein. These characteristics provide them the promising to be target sites for the fresh generation inhibitors to work with and overcome drug resistance. These findings would be beneficial for the drug development for inhibiting SARS-CoV-2 3CLpro hence assisting the pharmacogenomics effort to manage the infection. of SARS-CoV-2.

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Extending native mass spectrometry approaches to integral membrane proteins

Biological Chemistry ◽

10.1515/hsz-2015-0136 ◽

2015 ◽

Vol 396 (9-10) ◽

pp. 991-1002 ◽

Cited By ~ 16

Author(s):

Albert Konijnenberg ◽

Jeroen F. van Dyck ◽

Lyn L. Kailing ◽

Frank Sobott

Keyword(s):

Mass Spectrometry ◽

Membrane Proteins ◽

Gas Phase ◽

Conformational Changes ◽

Drug Targets ◽

Native Mass Spectrometry ◽

Lipid Binding ◽

Drug Binding ◽

Integral Membrane Proteins ◽

Oligomeric State

Abstract Recent developments in native mass spectrometry and ion mobility have made it possible to analyze the composition and structure of membrane protein complexes in the gas-phase. In this short review we discuss the experimental strategies that allow to elucidate aspects of the dynamic structure of these important drug targets, such as the structural effects of lipid binding or detection of co-populated conformational and assembly states during gating on an ion channel. As native mass spectrometry relies on nano-electrospray of natively reconstituted proteins, a number of commonly used lipid- and detergent-based reconstitution systems have been evaluated for their compatibility with this approach, and parameters for the release of intact, native-like folded membrane proteins studied in the gas-phase. The strategy thus developed can be employed for the investigation of the subunit composition and stoichiometry, oligomeric state, conformational changes, and lipid and drug binding of integral membrane proteins.

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Compilation of Potential Protein Targets for SARS-CoV-2: Preparation of Homology Model and Active Site Determination for Future Rational Antiviral Design

10.26434/chemrxiv.12084468.v1 ◽

2020 ◽

Author(s):

Sourav Pal ◽

Dr. Arindam Talukdar

Keyword(s):

Active Site ◽

Drug Targets ◽

Active Sites ◽

Structural Alignment ◽

Homology Model ◽

Active Site Residues ◽

Protein Targets ◽

Rational Development ◽

Novel Coronavirus ◽

Approved Drugs

<p>The recent pandemic due to the novel coronavirus SARS-CoV-2 (COVID-19) is causing significant mortality worldwide. However, there is a lack of specific drugs which can either prevent or treat the patient suffering from COVID-19. To understand the SARS-CoV-2 receptor recognition causing infectivity and pathogenesis, we have compiled a list of 20 probable drug targets on host and virus based on viral life cycle along with their PDB IDs for the rational development of future antivirals. We have prepared nine homology model for vital proteins for which no crystal structure is reported, which includes protein from host, viral membrane proteins and essential non-structural proteins (NSPs) of virus. The generated models were validated followed by Ramachandran plot along with their sequence and structural alignment. The active site residues of all the protein models are calculated by utilizing COACH meta-server and also cross verified with the CASTp webservers. All the active sites of the homology build proteins were evaluated after superimposition of the closely related X-ray crystallized structure bound with the co-crystal ligands. These information present in the manuscript can be used for the discovery effort towards new antivirals as well as repurposing FDA approved drugs against SARS-CoV-2.</p><br>

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Basis for drug selectivity of plasmepsin IX and X inhibition for Plasmodium falciparum and vivax

10.21203/rs.3.rs-944781/v2 ◽

2021 ◽

Author(s):

Alan Cowman ◽

Anthony Hodder ◽

Janni Christensen ◽

Stephen Scally ◽

Tony Triglia ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Crystal Structures ◽

Drug Targets ◽

Active Sites ◽

Drug Binding ◽

Dual Inhibitor ◽

3 Dimensional ◽

Protein Substrates ◽

Binding Cleft ◽

Parasite Egress

Abstract Plasmepsin IX (PMIX) and X (PMX) are aspartyl proteases of Plasmodium spp. that play essential roles in parasite egress, invasion and development. Consequently, they are important drug targets for Plasmodium falciparum and P. vivax. WM4 and WM382 are potent inhibitors of PMIX and PMX that block invasion of liver and blood stages and transmission to mosquitoes. WM4 specifically inhibits PMX whilst WM382 is a dual inhibitor of PMIX and PMX. To understand the function of PMIX and PMX proteases we identified new protein substrates in P. falciparum and together with detailed kinetic analyses and structural analyses identified key molecular interactions in the active site responsible for the specificity of WM4 and WM382 inhibition. The crystal structures of PMX apo enzyme and the protease/drug complexes of PMX/WM382 and PMX/WM4 for P. falciparum and P. vivax have been solved. We show PMIX and PMX have similar substrate selectivity, however, there are distinct differences for both peptide and full-length protein substrates through differences in localised 3-dimensional structures for the enzyme substrate-binding cleft and substrate interface. The differences in affinities of WM4 and WM382 binding for PMIX and PMX map to variations in surface interactions with each protease in the S' region of the active sites. Crystal structures of PMX reveal interactions and mechanistic detail on the selectivity of drug binding which will be important for further development of clinical candidates against these important molecular targets.

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Crystal structures of human MGST2 reveal synchronized conformational changes regulating catalysis

Nature Communications ◽

10.1038/s41467-021-21924-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Madhuranayaki Thulasingam ◽

Laura Orellana ◽

Emmanuel Nji ◽

Shabbir Ahmad ◽

Agnes Rinaldo-Matthis ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Drug Targets ◽

Active Sites ◽

Oxidative Dna Damage ◽

Glutathione S Transferase ◽

Concerted Mechanism ◽

Related Drug ◽

Stress Oxidative ◽

And Control

AbstractMicrosomal glutathione S-transferase 2 (MGST2) produces leukotriene C4, key for intracrine signaling of endoplasmic reticulum (ER) stress, oxidative DNA damage and cell death. MGST2 trimer restricts catalysis to only one out of three active sites at a time, but the molecular basis is unknown. Here, we present crystal structures of human MGST2 combined with biochemical and computational evidence for a concerted mechanism, involving local unfolding coupled to global conformational changes that regulate catalysis. Furthermore, synchronized changes in the biconical central pore modulate the hydrophobicity and control solvent influx to optimize reaction conditions at the active site. These unique mechanistic insights pertain to other, structurally related, drug targets.

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

Biochemical Journal ◽

10.1042/bj20091530 ◽

2010 ◽

Vol 427 (1) ◽

pp. 19-28 ◽

Cited By ~ 60

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.

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Identification of Potential Drug Targets and Prediction of the Potential Impact of High Risk Non Synonymous Single Nucleotide Polymorphism in SARS-CoV-2 3C like Proteinase (3CLpro): A Computational Approach

10.26434/chemrxiv.12235181 ◽

2020 ◽

Author(s):

Sahar Elbager ◽

abdelrahman hamza ◽

Afra M. Al Bkrye ◽

Asia M. Alrashied ◽

Entisar N. M. Ali ◽

...

Keyword(s):

Protein Interactions ◽

Drug Targets ◽

Drug Binding ◽

Snp Analysis ◽

Potential Drug ◽

Full Genome Sequencing ◽

Drug Binding Site ◽

Adme Properties ◽

Approved Drugs ◽

And Function

On January 2020, a new coronavirus (officially named SARS-CoV-2) was associated with alarming outbreak of a pneumonia-like illness, which was later named by the WHO as COVID-19, originating from Wuhan City, China. Although many clinical studies involving antiviral and immunomodulatory drug treatments for SARS-CoV-2 all without reported results, no approved drugs have been found to effectively inhibit the virus so far. Full genome sequencing of the virus was done, and uploaded to be freely available for the world scientists to explore. A promising target for SARS-CoV-2 drug design is a chymotrypsin-like cysteine protease (3CLpro), a main protease responsible for the replication and maturation of functional proteins in the life cycle of the SARS coronavirus. Here we aim to explore SARS-CoV-2 3CLpro as possible drug targets based on ligand- protein interactions. In addition, ADME properties of the ligands were also analyzed to predict their drug likeliness. The results revealed Out of 9 ligands, 8 ligands (JFM, X77, RZG, HWH, T8A, 0EN, PEPTIDE and DMS) showed best ADME properties. These findings suggest that these ligands can be used as potential molecules for developing potent inhibitors against SARS-CoV-2 3CLpro, which could be helpful in inhibiting the propagation of the COVID-19. Furthermore, 10 potential amino acids residues were recognized as potential drug binding site (THR25, HIS41, GLY143, SER144, CYS145, MET165, GLU166, GLN189, ASP295 and ARG298). All those amino acid residues were subjected to missense SNP analysis were recognized to affect the structure and function of the protein. These characteristics provide them the promising to be target sites for the fresh generation inhibitors to work with and overcome drug resistance. These findings would be beneficial for the drug development for inhibiting SARS-CoV-2 3CLpro hence assisting the pharmacogenomics effort to manage the infection. of SARS-CoV-2.

Download Full-text