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Zhonghua Yi Xue Za Zhi Jan 2012To explore the molecular mechanisms of Aurora kinase inhibitor PHA739358 in inhibited proliferation and in vitro induced apoptosis of breast cancer cells.
OBJECTIVE
To explore the molecular mechanisms of Aurora kinase inhibitor PHA739358 in inhibited proliferation and in vitro induced apoptosis of breast cancer cells.
METHODS
The in vitro cultured T47D cells in logarithmic growth phase were used. The effects of PHA739358 on cell proliferation were examined by MTT (methyl thiazolyl tetrazolium) assay. The variety of nuclear and spindle morphologies was examined by immunofluorescence. And G2/M arrest was determined by flow cytometry. The morphological changes of apoptotic cells were observed by fluorescent microscope. The levels of Aurora kinase relative protein expression phosphonate-AuroraA (p-AuroraA), AuroraA, phosphonate-histone H3 (p-histone H3), histone H3, cell cycle-specific protein expression CyclinB1, cell cycle regulative protein expression Cdc2, Cdc25c, phosphonate-Cdc2 (p-Cdc2), phosphonate-Cdc25c (p-Cdc25c) and apoptosis relative protein expression PARP, Bcl-2 and Bax were detected by Western blot. The apoptotic rate was examined by flow cytometer.
RESULTS
PHA739358 obviously inhibited the proliferation of T47D cells after a 24 h or 48 h treatment in a dose-dependent and time-dependent manner. Their IC50 values were (3.44 ± 0.54) and (0.21 ± 0.67) µmol/L respectively. Flow cytometry showed that G2/M arrested in a dose-dependent manner. PHA739358 dose-dependently and time-dependently promoted the dissection of PARP (poly (ADP-ribose) polymerase); down-regulated the expressions of Bcl-2, p-AuroraA, p-histone H3, CyclinB1 and up-regulated the levels of Bax, p-Cdc25c, p-Cdc2, P21 and P53 protein. PHA739358 had no significant effects on the expressions of AuroraA and histone H3. Flow cytometry and fluorescence microscope showed that PHA739358 significantly induced apoptosis. Flow cytometry showed the rate of apoptosis significantly increased from 0.31% ± 0.03% to 40.6% ± 0.81%.
CONCLUSION
The proliferation-inhibiting and apoptosis-inducing effects of PHA739358 may provide new therapeutic approaches of breast cancer.
Topics: Apoptosis; Benzamides; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Female; Humans; Pyrazoles
PubMed: 22490658
DOI: No ID Found -
Expert Opinion on Investigational Drugs Mar 2012Drugs that interfere with the normal progression of mitosis belong to the most successful cytotoxic agents currently used for anticancer treatment. Aurora kinases are... (Review)
Review
INTRODUCTION
Drugs that interfere with the normal progression of mitosis belong to the most successful cytotoxic agents currently used for anticancer treatment. Aurora kinases are serine/threonine kinases that function as key regulators of mitosis and are frequently overexpressed in human cancers. The use of several small molecule aurora kinase inhibitors as potential anticancer therapeutic is being investigated. Danusertib (formerly PHA-739358) is a small ATP competitive molecule that inhibits aurora A, B and C kinases. Interestingly, danusertib also inhibits several receptor tyrosine kinases such as Abl, Ret, FGFR-1 and TrkA. These tyrosine kinases are involved in the pathogenesis of a variety of malignancies and the observed multi-target inhibition may increase the antitumor activity resulting in extending the indication. Danusertib was one of the first aurora kinase inhibitors to enter the clinic and has been studied in Phase I and II trials.
AREAS COVERED
This review provides an updated summary of preclinical and clinical experience with danusertib up to July 2011.
EXPERT OPINION
Future studies with danusertib should focus on the possibility of combining this agent with other targeted anticancer agents, chemotherapy or radiotherapy. As a single agent, danusertib may show more promise in the treatment of leukemias than in solid tumors.
Topics: Animals; Antineoplastic Agents; Aurora Kinases; Benzamides; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Drug Delivery Systems; Drug Design; Humans; Neoplasms; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyrazoles
PubMed: 22242557
DOI: 10.1517/13543784.2012.652303 -
Biochemical Pharmacology Feb 2012Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal drug-metabolizing monooxygenase that catalyzes the NADPH-dependent oxygenation of a wide range of drugs...
Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal drug-metabolizing monooxygenase that catalyzes the NADPH-dependent oxygenation of a wide range of drugs and xenobiotics which contain a soft-nucleophiles, usually sulfur or nitrogen. As the release from the microsomal membranes can facilitate the in vitro experimental determination of drug metabolism by hFMO3, in this work we identified and eliminated the membrane anchoring sequence without affecting the activity of the enzyme and producing a soluble active enzyme. The truncated hFMO3 carrying a C-terminal deletion of 17 amino acids (tr-hFMO3) was expressed and purified from the cytosolic fraction. The tr-hFMO3 proves to be detached from the membrane, properly folded and fully active towards well-known marker substrates such as benzydamine and sulindac sulfide with measured apparent K(m) values of 45 ± 8 μM and 25 ± 4 μM, respectively. Its activity was further tested with newly discovered Aurora kinase inhibitors, Tozasertib and Danusertib, and compared to those of the wild type enzyme. The use of this soluble form of the hFMO3 enzyme as opposed to the usual microsomal preparations is advantageous for in vitro drug metabolism studies that are a requirement in the early phases of drug development by pharmaceutical industry.
Topics: Benzamides; Benzydamine; Binding Sites; Cell-Free System; Humans; Models, Molecular; Molecular Structure; Oxygenases; Piperazines; Protein Conformation; Pyrazoles; Sulindac
PubMed: 22177984
DOI: 10.1016/j.bcp.2011.11.029 -
Postepy Higieny I Medycyny... Dec 2011Recently novel treatment modalities has focused on targeted therapies. Tyrosine kinases represent a good target for cancer treatment since they are involved in... (Review)
Review
Recently novel treatment modalities has focused on targeted therapies. Tyrosine kinases represent a good target for cancer treatment since they are involved in transferring phosphate groups from ATP to tyrosine residues in specific substrate proteins transducing intracellular signals engaged in the many mechanisms, playing an important role in the modulation of growth factors signaling that are strongly related to carcinogenesis. Deregulation of tyrosine kinases activity was also found in hematological malignancies, particularly overexpression of tyrosine kinases was observed in chronic myeloid leukemia or acute lymphoblastic leukemia. Herein we show that tyrosine kinase inhibitors have revolutionized hematology malignancies therapy in a very short period of time and they still remain one of the most interesting anticancer compounds that could give a hope for cure and not only long-lasting complete remission. This manuscript summarizes current view on the first generation tyrosine kinase inhibititor--imatinib, second generation--dasatinib, nilotinib and bosutnib as well as new generation tyrosine kinase inhibititors--ponatinib and danusertib in hematooncology.
Topics: Aniline Compounds; Antineoplastic Agents; Benzamides; Dasatinib; Hematologic Neoplasms; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nitriles; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein-Tyrosine Kinases; Pyrimidines; Quinolines; Thiazoles
PubMed: 22173446
DOI: 10.5604/17322693.968778 -
Gene Feb 2012Aurora kinases represent an appealing target for anticancer therapies and several Aurora inhibitors are in clinical development, including the potent pan-Aurora...
Aurora kinases represent an appealing target for anticancer therapies and several Aurora inhibitors are in clinical development, including the potent pan-Aurora inhibitor Danusertib. Treatment with Aurora inhibitors has been shown to induce diverse biological responses in different tumor cells, in part depending on TP53 status. To characterize the effects of Danusertib at the transcriptional level we carried out gene expression profiling of wt and TP53 mutant tumor cells showing differential cell cycle response upon drug treatment. We found that treatment with Danusertib induces a strong transcriptional response only in TP53 wt cells, with an overlapping pattern of expression of TP53-dependent genes among the three cell lines tested, while a prevalent signature could not be identified in the two TP53 mutant cells, suggesting that TP53 status is a key determinant for the observed transcriptional effects. This work led to the identification of a number of genes consistently modulated by Aurora treatment in TP53 cells. One of these is GDF15, a secreted protein belonging to the TGF-β superfamily, for which we found a potential role in resistance to Danusertib, and which could represent a potential biomarker for Danusertib treatment in TP53 WT tumors and in surrogate tissues such as blood or skin.
Topics: Aurora Kinases; Benzamides; Cell Cycle; Cell Line; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Profiling; Genes, p53; Genetic Markers; Humans; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Transcription, Genetic
PubMed: 21914463
DOI: 10.1016/j.gene.2011.08.014 -
Molecular Cancer Therapeutics Oct 2011Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant...
Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant phenotype and results from the chromosomal translocation t(11;22)(q24;q12). Poor overall survival and pronounced long-term side effects associated with traditional chemotherapy necessitate the development of novel, targeted, therapeutic strategies. We therefore conducted a focused viability screen with 200 small molecule kinase inhibitors in 2 different Ewing's sarcoma cell lines. This resulted in the identification of several potential molecular intervention points. Most notably, tozasertib (VX-680, MK-0457) displayed unique nanomolar efficacy, which extended to other cell lines, but was specific for Ewing's sarcoma. Furthermore, tozasertib showed strong synergies with the chemotherapeutic drugs etoposide and doxorubicin, the current standard agents for Ewing's sarcoma. To identify the relevant targets underlying the specific vulnerability toward tozasertib, we determined its cellular target profile by chemical proteomics. We identified 20 known and unknown serine/threonine and tyrosine protein kinase targets. Additional target deconvolution and functional validation by RNAi showed simultaneous inhibition of Aurora kinases A and B to be responsible for the observed tozasertib sensitivity, thereby revealing a new mechanism for targeting Ewing's sarcoma. We further corroborated our cellular observations with xenograft mouse models. In summary, the multilayered chemical biology approach presented here identified a specific vulnerability of Ewing's sarcoma to concomitant inhibition of Aurora kinases A and B by tozasertib and danusertib, which has the potential to become a new therapeutic option.
Topics: Animals; Apoptosis; Aurora Kinases; Bone Neoplasms; Cell Cycle; Down-Regulation; Drug Synergism; Gene Knockdown Techniques; Humans; Mice; Mice, SCID; Oncogene Proteins, Fusion; Piperazines; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Protein c-fli-1; RNA Interference; RNA-Binding Protein EWS; Random Allocation; Sarcoma, Ewing; Xenograft Model Antitumor Assays
PubMed: 21768330
DOI: 10.1158/1535-7163.MCT-11-0100 -
PloS One Apr 2011The success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic...
The success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic stem cells. Danusertib (formerly PHA-739358) is a potent pan-aurora and ABL kinase inhibitor with activity against known BCR-ABL mutations, including T315I. Here, the individual contribution of both signaling pathways to the therapeutic effect of Danusertib as well as mechanisms underlying the development of resistance and, as a consequence, strategies to overcome resistance to Danusertib were investigated. Starting at low concentrations, a dose-dependent inhibition of BCR-ABL activity was observed, whereas inhibition of aurora kinase activity required higher concentrations, pointing to a therapeutic window between the two effects. Interestingly, the emergence of resistant clones during Danusertib exposure in vitro occurred considerably less frequently than with comparable concentrations of IM. In addition, Danusertib-resistant clones had no mutations in BCR-ABL or aurora kinase domains and remained IM-sensitive. Overexpression of Abcg2 efflux transporter was identified and functionally validated as the predominant mechanism of acquired Danusertib resistance in vitro. Finally, the combined treatment with IM and Danusertib significantly reduced the emergence of drug resistance in vitro, raising hope that this drug combination may also achieve more durable disease control in vivo.
Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Apoptosis; Aurora Kinases; Benzamides; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Clone Cells; Drug Resistance, Neoplasm; Drug Synergism; Fusion Proteins, bcr-abl; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutation; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Piperazines; Polyploidy; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Pyrimidines; Signal Transduction
PubMed: 21541334
DOI: 10.1371/journal.pone.0019164 -
Current Hematologic Malignancy Reports Jun 2011The introduction of tyrosine kinase inhibitors (TKIs) has changed the landscape of therapy for chronic myelogenous leukemia (CML). Once considered an incurable... (Review)
Review
The introduction of tyrosine kinase inhibitors (TKIs) has changed the landscape of therapy for chronic myelogenous leukemia (CML). Once considered an incurable malignancy, CML now has become a manageable chronic condition. Despite the great advances that imatinib has brought to the treatment of CML, some patients still develop resistance to imatinib and other TKIs, such as dasatinib and nilotinib. Furthermore, none of the clinically available TKIs is capable of eradicating leukemia stem cells and therefore curing CML. Several new compounds have been developed in recent years in an attempt to manage TKI-resistant CML. These include third-generation TKIs (ponatinib, danusertib) and even old compounds such as omacetaxine, which were developed before imatinib and now find a possible niche in the treatment of imatinib-resistant CML. We review the current preclinical and clinical data on the most promising new compounds for the treatment of CML.
Topics: Benzamides; Dasatinib; Drug Resistance, Neoplasm; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Imidazoles; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Piperazines; Protein Kinase Inhibitors; Pyridazines; Pyrimidines; Thiazoles
PubMed: 21327563
DOI: 10.1007/s11899-011-0079-9 -
Journal of Computer-aided Molecular... Feb 2011Aurora kinase family is one of the emerging targets in oncology drug discovery and several small molecules targeting aurora kinases have been discovered and evaluated...
Aurora kinase family is one of the emerging targets in oncology drug discovery and several small molecules targeting aurora kinases have been discovered and evaluated under early phase I/II trials. Among them, PHA-739358 (compound 1r) is a 3-aminopyrazole derivative with strong activity against Aurora A under early phase II trial. Inhibitory potency of compound 1r (the benzylic substituent at the pro-R position) is 30 times over that of compound 1s (the benzylic substituent at the pro-S position). In present study, the mechanism of how different configurations influence the binding affinity was investigated using molecular dynamics (MD) simulations, free energy calculations and free energy decomposition analysis. The predicted binding free energies of these two complexes are consistent with the experimental data. The analysis of the individual energy terms indicates that although the van der Waals contribution is important for distinguishing the binding affinities of these two inhibitors, the electrostatic contribution plays a more crucial role in that. Moreover, it is observed that different configurations of the benzylic substituent could form different binding patterns with protein, thus leading to variant inhibitory potency of compounds 1r and 1s. The combination of different molecular modeling techniques is an efficient way to interpret the chirality effects of inhibitors and our work gives valuable information for the chiral drug design in the near future.
Topics: Aurora Kinases; Benzamides; Drug Design; Hydrogen Bonding; Molecular Dynamics Simulation; Protein Binding; Protein Conformation; Protein Interaction Mapping; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Stereoisomerism; Structure-Activity Relationship
PubMed: 21222017
DOI: 10.1007/s10822-010-9408-7 -
Cancer Research Dec 2010In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a...
In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis, making them attractive targets for anticancer therapy. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser(10), a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes.
Topics: Adult; Animals; Aurora Kinase A; Aurora Kinase B; Aurora Kinases; Benzamides; Cell Line, Tumor; Cell Proliferation; Clinical Trials as Topic; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Female; HCT116 Cells; HeLa Cells; Histones; Humans; Mice; Mice, Nude; Mutation; Neoplasms; Organophosphates; Paclitaxel; Phosphorylation; Phthalazines; Piperazines; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Quinazolines; Xenograft Model Antitumor Assays
PubMed: 20935223
DOI: 10.1158/0008-5472.CAN-10-3001