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Analytica Chimica Acta Oct 2014Abelson tyrosine-protein kinase 1 (ABL1) catalysed phosphorylation involves the addition of a phosphate group from ATP to the tyrosine residue on the substrate abltide....
Abelson tyrosine-protein kinase 1 (ABL1) catalysed phosphorylation involves the addition of a phosphate group from ATP to the tyrosine residue on the substrate abltide. The phosphorylation reactions were carried out by incubating ABL1, ATP and the substrate abltide. Adsorption at the glassy carbon electrode surface in either reaction mixtures or control solutions, followed by differential pulse voltammetry in buffer allowed detection of the variation of abltide tyrosine residue oxidation peak reflecting the occurrence of the phosphorylation reaction. The effect of abltide, ATP and ABL1 concentrations as well as the time course of the phosphorylation reaction were studied. The influence of co-adsorption of ABL1, ATP and phosphorylated abltide was evaluated and the conditions for the electrochemical detection of ABL1-catalysed phosphorylation optimised. The Michaelis-Menten constant for abltide binding KM∼4.5 μM, turnover number kcat∼11 s(-1) and enzyme efficiency kcat/KM∼2.3 s(-1) μM(-1) were calculated. The inhibition of ABL1 by imatinib mesylate and danusertib was also electrochemically investigated and IC50 values of 0.53 and 0.08 μM determined.
Topics: Benzamides; Biocatalysis; Dose-Response Relationship, Drug; Electrochemical Techniques; Fusion Proteins, bcr-abl; Imatinib Mesylate; Molecular Structure; Phosphorylation; Piperazines; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Structure-Activity Relationship
PubMed: 25201268
DOI: 10.1016/j.aca.2014.06.025 -
Journal of Alzheimer's Disease : JAD 2014An unbiased screen for compounds that block amyloid-β protein precursor (AβPP) caspase cleavage identified ADDN-1351, which reduced AβPP-C31 by 90%. Target...
An unbiased screen for compounds that block amyloid-β protein precursor (AβPP) caspase cleavage identified ADDN-1351, which reduced AβPP-C31 by 90%. Target identification studies showed that ADDN-1351 is a TrkA inhibitor, and, in complementary studies, TrkA overexpression increased AβPP-C31 and cell death. TrkA was shown to interact with AβPP and suppress AβPP-mediated transcriptional activation. Moreover, treatment of PDAPP transgenic mice with the known TrkA inhibitor GW441756 increased sAβPPα and the sAβPPα to Aβ ratio. These results suggest TrkA inhibition-rather than NGF activation-as a novel therapeutic approach, and raise the possibility that such an approach may counteract the hyperactive signaling resulting from the accumulation of active NGF-TrkA complexes due to reduced retrograde transport. The results also suggest that one component of an optimal therapy for Alzheimer's disease may be a TrkA inhibitor.
Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Benzamides; CHO Cells; Cell Death; Cricetulus; Disease Models, Animal; Dose-Response Relationship, Drug; Gene Expression Regulation; HEK293 Cells; Humans; Mice; Mice, Transgenic; Mutation; Nerve Growth Factor; Protein Kinase Inhibitors; Pyrazoles; Receptor, trkA; Transfection
PubMed: 24531152
DOI: 10.3233/JAD-130017 -
International Journal of Molecular... Jan 2013Aurora kinases were recently identified as a potential target in anticancer therapy and, amongst their available inhibitors, Tozasertib (VX-680) and Danusertib...
Aurora kinases were recently identified as a potential target in anticancer therapy and, amongst their available inhibitors, Tozasertib (VX-680) and Danusertib (PHA-739358) have been indicated as possible substrates of human flavin-containing monooxygenase 3 (hFMO3). Here we report the in vitro rate of oxidation of these drugs by wild-type hFMO3 and its polymorphic variant V257M. The conversion of Tozasertib and Danusertib to their corresponding metabolites, identified by LC-MS, by the purified wild-type and V257M hFMO3 show significant differences. In the case of Tozasertib, the V257M variant shows a catalytic efficiency, expressed as k(cat)/K(m), similar to the wild-type: 0.39 ± 0.06 min-1µM-1 for V257M compared to 0.33 ± 0.04 min-1µM-1 for the wild type. On the other hand, in the case of Danusertib, V257M shows a 3.4× decrease in catalytic efficiency with k(cat)/K(m) values of 0.05 ± 0.01 min-1µM-1 for V257M and 0.17 ± 0.03 min-1µM-1 for the wild type. These data reveal how a simple V257M substitution ascribed to a single nucleotide polymorphism affects the N-oxidation of relevant anticancer drugs, with important outcome in their therapeutic effects. These findings demonstrate that codon 257 is important for activity of the hFMO3 gene and the codon change V to M has an effect on the catalytic efficiency of this enzyme.
PubMed: 23358255
DOI: 10.3390/ijms14022707 -
Melanoma Research Apr 2013Treatment of metastatic melanoma has long been a challenge because of its resistance to traditional chemotherapeutics, leading to the search for alternative strategies....
Treatment of metastatic melanoma has long been a challenge because of its resistance to traditional chemotherapeutics, leading to the search for alternative strategies. Aurora kinases are key mitotic regulators that are frequently overexpressed in various cancers including melanoma, making them ideal targets for drug development. Several Aurora kinase inhibitors have been developed and tested preclinically and clinically. PHA-739358 is currently one of the most advanced clinical compounds being tested in phase II clinical trials; however, its antitumor effect has not been tested in melanoma. In this study, the antiproliferative and anti-invasive effects of PHA-739358 were investigated in melanoma cell lines. The results demonstrated that PHA-739358 produces a time-dependent and dose-dependent inhibition of cell proliferation, induction of apoptosis, and inhibition of cell migration. Downregulation of matrix metalloproteinase-2 by the inhibition of NFκB-signaling pathway may contribute to PHA-739358-induced inhibition of migration. Furthermore, PHA-739358 enhanced temozolomide and Plx4032-induced apoptosis. This study suggests that Aurora kinase inhibitors may provide a new strategy for the treatment of advanced melanoma.
Topics: Apoptosis; Aurora Kinases; Benzamides; Cell Culture Techniques; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Humans; Melanoma; Protein Kinase Inhibitors; Pyrazoles; Transfection
PubMed: 23344158
DOI: 10.1097/CMR.0b013e32835df5e4 -
PloS One 2013In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest...
In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.
Topics: Animals; Apoptosis; Benzamides; Blotting, Western; Cell Line, Tumor; Cluster Analysis; Drug Resistance, Neoplasm; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Models, Biological; Neoplasm Proteins; Piperazines; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proteomics; Pyrimidines; Signal Transduction
PubMed: 23326482
DOI: 10.1371/journal.pone.0053668 -
Nature Chemical Biology Nov 2012Occurrence of the BCR-ABL(T315I) gatekeeper mutation is among the most pressing challenges in the therapy of chronic myeloid leukemia (CML). Several BCR-ABL inhibitors...
Occurrence of the BCR-ABL(T315I) gatekeeper mutation is among the most pressing challenges in the therapy of chronic myeloid leukemia (CML). Several BCR-ABL inhibitors have multiple targets and pleiotropic effects that could be exploited for their synergistic potential. Testing combinations of such kinase inhibitors identified a strong synergy between danusertib and bosutinib that exclusively affected CML cells harboring BCR-ABL(T315I). To elucidate the underlying mechanisms, we applied a systems-level approach comprising phosphoproteomics, transcriptomics and chemical proteomics. Data integration revealed that both compounds targeted Mapk pathways downstream of BCR-ABL, resulting in impaired activity of c-Myc. Using pharmacological validation, we assessed that the relative contributions of danusertib and bosutinib could be mimicked individually by Mapk inhibitors and collectively by downregulation of c-Myc through Brd4 inhibition. Thus, integration of genome- and proteome-wide technologies enabled the elucidation of the mechanism by which a new drug synergy targets the dependency of BCR-ABL(T315I) CML cells on c-Myc through nonobvious off targets.
Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Fusion Proteins, bcr-abl; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Nitriles; Piperazines; Protein Kinase Inhibitors; Proteomics; Proto-Oncogene Proteins c-myc; Pyrazoles; Pyrimidines; Quinolines; Structure-Activity Relationship; Systems Biology
PubMed: 23023260
DOI: 10.1038/nchembio.1085 -
BJU International Jan 2013To determine the efficacy and toxicity of danusertib (formerly PHA-739358) administered i.v. over two different dosing schedules with equivalent dose intensity in... (Comparative Study)
Comparative Study Randomized Controlled Trial
OBJECTIVE
To determine the efficacy and toxicity of danusertib (formerly PHA-739358) administered i.v. over two different dosing schedules with equivalent dose intensity in patients with metastatic castration-resistant prostate cancer with progressive disease after docetaxel-based treatment.
PATIENTS AND METHODS
In this open-label, multicentre phase II trial 88 patients were randomly assigned (1:1 ratio) to receive either danusertib 330 mg/m(2) over 6 h i.v. on days 1, 8 and 15 (arm A, n = 43) or 500 mg/m(2) over 24 h i.v. on days 1 and 15 (arm B, n = 38), every 4 weeks. The primary endpoint chosen for this exploratory study was PSA response rate at 3 months.
RESULTS
Sixty patients (31/43 in arm A and 29/38 in arm B) were evaluable for the primary endpoint. Median progression-free survival was 12 weeks in both arms. PSA response occurred in one patient in each arm; best overall response was stable disease in eight (18.6%) and 13 (34.2%) patients in arms A and B, respectively. Eleven out of 81 (13.6%) treated patients had stable disease for ≥6 months. Danusertib was generally well tolerated; the most common grade 3 and 4 drug-related adverse event was neutropenia which occurred in 37.2% (arm A) and 15.8% (arm B) of the patients.
CONCLUSION
Danusertib monotherapy shows minimal efficacy in patients with castration-resistant prostate cancer. Further studies are required to establish specific biomarkers predictive for either response or prolonged disease stabilization.
Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Aurora Kinases; Benzamides; Bone Neoplasms; Castration; Disease-Free Survival; Docetaxel; Drug Administration Schedule; Drug Resistance, Neoplasm; Humans; Infusions, Intravenous; Kaplan-Meier Estimate; Male; Middle Aged; Neutropenia; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Pyrazoles; Taxoids; Treatment Failure
PubMed: 22928785
DOI: 10.1111/j.1464-410X.2012.11404.x -
European Journal of Medicinal Chemistry Apr 2013New drugs for neglected tropical diseases such as human African trypanosomiasis (HAT) are needed, yet drug discovery efforts are not often focused on this area due to...
New drugs for neglected tropical diseases such as human African trypanosomiasis (HAT) are needed, yet drug discovery efforts are not often focused on this area due to cost. Target repurposing, achieved by the matching of essential parasite enzymes to those human enzymes that have been successfully inhibited by small molecule drugs, provides an attractive means by which new drug optimization programs can be pragmatically initiated. In this report we describe our results in repurposing an established class of human Aurora kinase inhibitors, typified by danusertib (1), which we have observed to be an inhibitor of trypanosomal Aurora kinase 1 (TbAUK1) and effective in parasite killing in vitro. Informed by homology modeling and docking, a series of analogs of 1 were prepared that explored the scope of the chemotype and provided a nearly 25-fold improvement in cellular selectivity for parasite cells over human cells.
Topics: Aurora Kinases; Benzamides; Crystallography, X-Ray; Dose-Response Relationship, Drug; Drug Discovery; Humans; Models, Molecular; Molecular Structure; Parasitic Sensitivity Tests; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Structure-Activity Relationship; Trypanocidal Agents; Trypanosoma; Trypanosomiasis
PubMed: 22889561
DOI: 10.1016/j.ejmech.2012.07.038 -
Clinical Cancer Research : An Official... Sep 2012Aurora kinases play a crucial role in cell-cycle control. Uncontrolled expression of aurora kinases causes aneuploidy and tumor growth. As conservative treatment options...
Targeting aurora kinases with danusertib (PHA-739358) inhibits growth of liver metastases from gastroenteropancreatic neuroendocrine tumors in an orthotopic xenograft model.
PURPOSE
Aurora kinases play a crucial role in cell-cycle control. Uncontrolled expression of aurora kinases causes aneuploidy and tumor growth. As conservative treatment options for advanced gastroenteropancreatic neuroendocrine tumors (GEP-NET) are disappointing, aurora kinases may be an interesting target for novel therapeutic strategies.
EXPERIMENTAL DESIGN
Human GEP-NETs were tested for aurora kinase expression. The efficacy of the new aurora kinase inhibitor danusertib was evaluated in two human GEP-NET cell lines (BON1 and QGP) in vitro and in vivo.
RESULTS
The majority of ten insulinomas and all 33 nonfunctional pancreatic or midgut GEP-NETs expressed aurora A despite a mostly high degree of cell differentiation. Both human GEP-NET cell lines expressed aurora kinase A and B, and high Ser10 phosphorylation of histone H3 revealed increased aurora B activity. Remarkably, danusertib led to cell-cycle arrest and completely inhibited cell proliferation of the GEP-NET cells in vitro. Decreased phosphorylation of histone H3 indicated effective aurora B inhibition. In a subcutaneous murine xenograft model, danusertib significantly reduced tumor growth in vivo compared with controls or mice treated with streptozotocine/5-fluorouracil. As a consequence, decreased levels of tumor marker chromogranin A were found in mouse serum samples. In a newly developed orthotopic model for GEP-NET liver metastases by intrasplenic tumor cell transplantation, dynamic MRI proved significant growth inhibition of BON1- and QGP-derived liver metastases.
CONCLUSIONS
These results show that danusertib may impose a new therapeutic strategy for aurora kinase expressing metastasized GEP-NETs.
Topics: Animals; Aurora Kinase A; Aurora Kinase B; Aurora Kinases; Benzamides; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Chromogranin A; Gastrointestinal Neoplasms; Histones; Humans; Liver Neoplasms; Mice; Molecular Targeted Therapy; Neuroendocrine Tumors; Protein Serine-Threonine Kinases; Pyrazoles; Transplantation, Heterologous
PubMed: 22753592
DOI: 10.1158/1078-0432.CCR-11-2968 -
Molecular Cancer Jun 2012Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemias (Ph-positive ALL) with clinically approved inhibitors of the Bcr/Abl tyrosine kinase...
BACKGROUND
Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemias (Ph-positive ALL) with clinically approved inhibitors of the Bcr/Abl tyrosine kinase frequently results in the emergence of a leukemic clone carrying the T315I mutation in Bcr/Abl, which confers resistance to these drugs. PHA-739358, an Aurora kinase inhibitor, was reported to inhibit the Bcr/Abl T315I mutant in CML cells but no preclinical studies have examined this in detail in human ALL.
RESULTS
We compared the sensitivity of human Bcr/Abl T315I, Bcr/Abl wild type and non-Bcr/Abl ALL cells to this drug. PHA-739358 inhibited proliferation and induced apoptosis independently of Bcr/Abl, the T315I mutation, or presence of the tumor suppressor p53, but the degree of effectiveness varied between different ALL samples. Since short-term treatment with a single dose of drug only transiently inhibited proliferation, we tested combination treatments of PHA-739358 with the farnesyltransferase inhibitor Lonafarnib, with vincristine and with dasatinib. All combinations reduced viability and cell numbers compared to treatment with a single drug. Clonogenic assays showed that 25 nM PHA-739358 significantly reduced the colony growth potential of Ph-positive ALL cells, and combined treatment with a second drug abrogated colony growth in this assay. PHA-739358 further effectively blocked Bcr/Abl tyrosine kinase activity and Aurora kinase B in vivo, and mice transplanted with human Bcr/Abl T315I ALL cells treated with a 3x 7-day cycle of PHA-739358 as mono-treatment had significantly longer survival.
CONCLUSIONS
PHA-739358 represents an alternative drug for the treatment of both Ph-positive and negative ALL, although combined treatment with a second drug may be needed to eradicate the leukemic cells.
Topics: Animals; Antineoplastic Agents; Apoptosis; Aurora Kinase B; Aurora Kinases; Benzamides; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Enzyme Activation; Fusion Proteins, bcr-abl; Humans; Mice; Mutation; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Vincristine
PubMed: 22721004
DOI: 10.1186/1476-4598-11-42