Structural basis of reversine selectivity in inhibiting Mps1 more potently than aurora B kinase

Hiruma, Yoshitaka; Koch, Andrè; Dharadhar, Shreya; Joosten, Robbie P.; Perrakis, Anastassis

doi:10.1002/prot.25174

Cited by 29 publications

(27 citation statements)

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“…The first example is the match between the Sorafenib‐bound MAPK14 protein kinase (PDB ID: http://www.rcsb.org/pdb/explore/explore.do?structureId=3HEG) and the Ergotamine‐bound 5‐HT 2B receptor (PDB ID: http://www.rcsb.org/pdb/explore/explore.do?structureId=4IB4), consistent with studies showing that the FDA‐approved kinase inhibitor Sorafenib has nanomolar affinity for 5‐HT 2B R . The second example is the match between Reversine‐bound TTK protein kinase (PDB ID: http://www.rcsb.org/pdb/explore/explore.do?structureId=5LJJ) and the triazolecarboximidamide‐bound A 2A receptor (PDB ID: http://www.rcsb.org/pdb/explore/explore.do?structureId=5UIG). Reversine shows weak binding affinity for the adenosine A 2A receptor, and has sub‐micromolar affinity for the homologous adenosine A 3 receptor …”

Section: Resultssupporting

confidence: 68%

3D‐e‐Chem: Structural Cheminformatics Workflows for Computer‐Aided Drug Discovery

et al. 2018

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AbstracteScience technologies are needed to process the information available in many heterogeneous types of protein–ligand interaction data and to capture these data into models that enable the design of efficacious and safe medicines. Here we present scientific KNIME tools and workflows that enable the integration of chemical, pharmacological, and structural information for: i) structure‐based bioactivity data mapping, ii) structure‐based identification of scaffold replacement strategies for ligand design, iii) ligand‐based target prediction, iv) protein sequence‐based binding site identification and ligand repurposing, and v) structure‐based pharmacophore comparison for ligand repurposing across protein families. The modular setup of the workflows and the use of well‐established standards allows the re‐use of these protocols and facilitates the design of customized computer‐aided drug discovery workflows.

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Section: Resultssupporting

confidence: 68%

3D‐e‐Chem: Structural Cheminformatics Workflows for Computer‐Aided Drug Discovery

et al. 2018

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“…Comparison of the structure of the C604Y mutant bound to reversine (PDB: 5LJJ, [23]) clearly shows that there is no steric hindrance with the Tyr604 side chain, explaining why C604Y, and by deduction C604W, are non-resistant to reversine as previously reported [11,12].…”

Section: Cc-by-nc-ndmentioning

confidence: 63%

“…The site-specific mutant of C604W was generated using the QuickChange (Stratagene). The Mps1 kinase domain C604Y/W variants were produced as previously described [23].…”

Section: Protein Productionmentioning

confidence: 99%

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Understanding inhibitor resistance in Mps1 kinase through novel biophysical assays and structures

Hiruma

Koch

Hazraty

et al. 2017

Preprint

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Monopolar spindle 1 (Mps1/TTK) is a protein kinase essential in mitotic checkpoint signalling, preventing anaphase until all chromosomes are properly attached to spindle microtubules. Mps1 has emerged as a potential target for cancer therapy, and a variety of compounds have been developed to inhibit its kinase activity. Mutations in the catalytic domain of Mps1 that give rise to inhibitor resistance, but retain catalytic activity and do not display cross-resistance to other Mps1 inhibitors, have been described. Here we characterize the interactions of two such mutants, Mps1 C604Y and C604W, which raise resistance to two closely related compounds, NMS-P715 and its derivative Cpd-5, but not to the well-characterised Mps1 inhibitor, reversine. We show that estimates of the IC50 (employing a novel specific and efficient assay that utilizes a fluorescently labelled substrate) and of the binding affinity (KD) indicate that in both mutants, Cpd-5 should be better tolerated than the closely related NMS-P715. To gain further insight, we determined the crystal structure of the Mps1 kinase mutants bound to Cpd-5 and NMS-P715, and compare the binding modes of Cpd-5, NMS-P715 and reversine. The difference in steric hindrance between Tyr/Trp604 and the trifluoromethoxy moiety of NMS-P715, the methoxy moiety of Cpd-5, and complete absence of such a group in reversine, account for differences we observe in vitro. Our analysis enforces the notion that inhibitors targeting Mps1 drug-resistant mutations can emerge as a feasible intervention strategy based on existing scaffolds, if the clinical need arises.Summary statementThe inhibition of specific Mps1 kinase inhibitors towards the wild-type protein and inhibitor-resistant mutants is explained by a novel specific activity assay, biophysical characterisation, and X-ray structures.AbbreviationsATPadenosine triphosphateBub1/Bub3budding uninhibited by benzimidazoles 1 / budding uninhibited by benzimidazoles 3Cpd-5Compound-5 (N-(2,6-diethylphenyl)-8-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide)FPfluorescence polarizationGSTGlutathione S-transferaseIC50half maximal inhibitory concentrationKDdissociation constantΔGcalccalculated Gibbs energy difference for ligand bindingKNL1kinetochore null protein 1KPi(inorganic) potassium phosphateMps1monopolar spindle 1MSTmicroscale thermophoresisNMS-P715N-(2,6-diethylphenyl)-1-methyl-8-({4-[(1-methylpiperidin-4-yl)carbamoyl]-2-(trifluoromethoxy)phenyl}amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamideTMRtetramethylrhodamineWTwild-type

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“…Cellular mechanisms sensitive to reversine include mitochondria [22], caspases [5, 22], Monopolar spindle 1 (Mps1) kinase [27-29], aurora kinase A and B [17, 25, 27, 28], and Akt/ mTOR [3, 30]. …”

Section: Introductionmentioning

confidence: 99%

Inhibition of Suicidal Erythrocyte Death by Reversine

Jèmaà

Fezai

Läng³

2017

Cell Physiol Biochem

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Background/Aims: The A3 adenosine receptor antagonist reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine) influences cellular differentiation, inhibits cell proliferation, induces cell-cycle arrest, triggers apoptosis, causes cell swelling with polyploidy and stimulates autophagy. The effect on apoptosis involves mitochondria and caspases. Erythrocytes are lacking mitochondria but express caspases and are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), energy depletion and oxidative stress. The present study explored, whether reversine influences eryptosis. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), Ca²⁺ loading (30 minutes treatment with 1 µM Ca²⁺ ionophore ionomycin), or oxidative stress (15 min exposure to 0.3 mM tert-butylhydroperoxide). Results: A 48 hours exposure of human erythrocytes to reversine (1-10 µM) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, Ca²⁺ loading, and oxidative stress were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of each, Ca²⁺ loading, energy depletion and oxidative stress on annexin-V-binding were significantly blunted in the presence of reversine (1-10 µM). The effect of ionomycin, but not the effects of energy depletion and oxidative stress on forward scatter were again significantly blunted in the presence of reversine (≥1 µM]. Conclusions: Reversine is a powerful inhibitor of cell membrane scrambling following energy depletion, Ca²⁺ loading and oxidative stress.

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Structural basis of reversine selectivity in inhibiting Mps1 more potently than aurora B kinase

Cited by 29 publications

References 29 publications

3D‐e‐Chem: Structural Cheminformatics Workflows for Computer‐Aided Drug Discovery

3D‐e‐Chem: Structural Cheminformatics Workflows for Computer‐Aided Drug Discovery

Understanding inhibitor resistance in Mps1 kinase through novel biophysical assays and structures

Inhibition of Suicidal Erythrocyte Death by Reversine

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