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Sample records for kinase inhibitor suppresses

  1. Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A.

    PubMed Central

    Hu, D E; Fan, T P

    1995-01-01

    1. Vascular endothelial growth factor (VEGF) is a heparin-binding angiogenic factor which specifically acts on endothelial cells via distinct membrane-spanning tyrosine kinase receptors. Here we used the rat sponge implant model to test the hypothesis that the angiogenic activity of VEGF can be suppressed by protein tyrosine kinase (PTK) inhibitors. 2. Neovascular responses in subcutaneous sponge implants were determined by measurements of relative sponge blood flow by use of a 133Xe clearance technique, and confirmed by histological studies and morphometric analysis. 3. Daily local administration of 250 ng VEGF165 accelerated the rate of 133Xe clearance from the sponges and induced an intense neovascularisation. This VEGF165-induced angiogenesis was inhibited by daily co-administration of the selective PTK inhibitor, lavendustin A (10 micrograms), but not its negative control, lavendustin B (10 micrograms). Blood flow measurements and morphometric analysis of 8-day-old sponges showed that lavendustin A reduced the 133Xe clearance of VEGF165-treated sponges from 32.9 +/- 1.5% to 20.9 +/- 1.6% and the total fibrovascular growth area from 62.4 +/- 6.1% to 21.6 +/- 6.8% (n = 12, P < 0.05). 4. Co-injection of suramin (3 mg), an inhibitor of heparin-binding growth factors, also suppressed the VEGF165-elicited neovascular response. In contrast, neither lavendustin A nor suramin produced any effect on the basal sponge-induced angiogenesis. 5. When given alone, low doses of VEGF165 (25 ng) or basic fibroblast growth factor (bFGF; 10 ng) did not modify the basal sponge-induced neovascularisation.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2 Figure 2 PMID:7533611

  2. Met tyrosine kinase inhibitor, PF-2341066, suppresses growth and invasion of nasopharyngeal carcinoma

    PubMed Central

    Zhao, Yuanyuan; Zhang, Jing; Tian, Ying; Xue, Cong; Hu, Zhihuang; Zhang, Li

    2015-01-01

    Purpose We explored the effect of hepatocyte growth factor (HGF)/Met signaling pathway on nasopharyngeal carcinoma (NPC) cells in vitro and in vivo, and investigated the ability of Met tyrosine kinase inhibitor (TKI) to block HGF-induced biological signaling. Experimental design Met TKI inhibitor PF-2341066 alone, or in combination with cisplatin, was investigated for its ability to block HGF-induced signaling and biological effects in vitro and in vivo. HGF/Met expression and activation of signaling in NPC cells were detected by using Western blot and immunohistochemistry. Biological evaluation, including wound healing, cell proliferation, and invasion of NPC cells, was also examined, and the correlation between HGF/Met expression of primary and metastatic tumor in NPC patients and clinical prognosis were also analyzed. Results Met TKI inhibitor, PF-2341066, inhibited growth of NPC cells in vivo with half maximal inhibitory concentration of 0.79±0.21 μmol/L, and suppressed invasion and migration of NPC cells; also, the inhibition of PF-2341066 was synergized with cisplatin treatment. Compared with the control group, Met TKI inhibited metastasis of transplanted NPC in nude mice (the number of live metastases [mean ± SD]: 5.8±2.2 versus 11.8±2.2, P=0.03; the number of lung metastases: 2.3±1.5 versus 5.3±0.9, P=0.06). HGF was widely expressed in both primary and metastatic lesions while Met expression of metastatic lesions was higher than that of primary lesions (primary lesions: 24.7%; liver metastases: 40%; lung metastases: 29%; lymph node metastases: 29%, P<0.05), and overall survival of NPC patients with higher expression of Met was shorter (P=0.13). Conclusion Our results demonstrated that HGF/Met signaling promoted NPC growth, further resulting in metastasis and poor prognosis. Met TKI, PF-2341066, showed potent antitumor activity in vivo and in vitro which was enhanced by combination with cisplatin. Our study implied that HGF/Met signaling was the

  3. Dual Suppression of the Cyclin-Dependent Kinase Inhibitors CDKN2C and CDKN1A in Human Melanoma

    PubMed Central

    2012-01-01

    Resistance to BRAFV600E inhibitors is associated with reactivation of mitogen-activated protein kinase (MAPK) signaling at different levels in melanoma. To identify downstream effectors of MAPK signaling that could be used as potential additional therapeutic targets for BRAFV600E inhibitors, we used hTERT/CDK4R24C/p53DD-immortalized primary human melanocytes genetically modified to ectopically express BRAF V600E or NRAS G12D and observed induction of the AP-1 transcription factor family member c-Jun. Using a dominant negative approach, in vitro cell proliferation assays, western blots, and flow cytometry showed that MAPK signaling via BRAFV600E promotes melanoma cell proliferation at G1 through AP-1-mediated negative regulation of the INK4 family member, cyclin-dependent kinase inhibitor 2C (CDKN2C), and the CIP/KIP family member, cyclin-dependent kinase inhibitor 1A (CDKN1A). These effects were antagonized by pharmacological inhibition of CDKN2C and CDKN1A targets CDK2 and CDK4 in vitro. In contrast to BRAF V600E or NRAS G12D-expressing melanocytes, melanoma cells have an inherent resistance to suppression of AP-1 activity by BRAFV600E- or MEK-inhibitors. Here, CDK2/4 inhibition statistically significantly augmented the effects of BRAFV600E- or MEK-inhibitors on melanoma cell viability in vitro and growth in athymic nude Foxn1 nu mice (P = .03 when mean tumor volume at day 13 was compared for BRAFV600E inhibitor vs BRAFV600E inhibitor plus CDK2/4 inhibition; P = .02 when mean tumor volume was compared for MEK inhibitor vs MEK inhibitor plus CDK2/4 inhibition; P values were calculated by a two-sided Welch t test; n = 4–8 mice per group). PMID:22997239

  4. AT9283, a novel aurora kinase inhibitor, suppresses tumor growth in aggressive B-cell lymphomas.

    PubMed

    Qi, Wenqing; Liu, Xiaobing; Cooke, Laurence S; Persky, Daniel O; Miller, Thomas P; Squires, Matthew; Mahadevan, Daruka

    2012-06-15

    Aurora kinases are oncogenic serine/threonine kinases that play key roles in regulating the mitotic phase of the eukaryotic cell cycle. Auroras are overexpressed in numerous tumors including B-cell non-Hodgkin's lymphomas and are validated oncology targets. AT9283, a pan-aurora inhibitor inhibited growth and survival of multiple solid tumors in vitro and in vivo. In this study, we demonstrated that AT9283 had potent activity against Aurora B in a variety of aggressive B-(non-Hodgkin lymphoma) B-NHL cell lines. Cells treated with AT9283 exhibited endoreduplication confirming the mechanism of action of an Aurora B inhibitor. Also, treatment of B-NHL cell lines with AT9283 induced apoptosis in a dose and time dependent manner and inhibited cell proliferation with an IC(50) < 1 μM. It is well known that inhibition of auroras (A or B) synergistically enhances the effects of microtubule targeting agents such as taxanes and vinca alkaloids to induce antiproliferation and apoptosis. We evaluated whether AT9283 in combination with docetaxel is more efficient in inducing apoptosis than AT9283 or docetaxel alone. At very low doses (5 nM) apoptosis was doubled in the combination (23%) compared to AT9283 or docetaxel alone (10%). A mouse xenograft model of mantle cell lymphoma demonstrated that AT9283 at 15 mg/kg and docetaxel (10 mg/kg) alone had modest anti-tumor activity. However, AT9283 at 20 mg/kg and AT9283 (15 or 20 mg/kg) plus docetaxel (10 mg/kg) demonstrated a statistically significant tumor growth inhibition and enhanced survival. Together, our results suggest that AT9283 plus docetaxel may represent a novel therapeutic strategy in B-cell NHL and warrant early phase clinical trial evaluation. PMID:21796626

  5. Dose-Dependent Suppression of Cytokine production from T cells by a Novel Phosphoinositide 3-Kinase Delta Inhibitor

    PubMed Central

    Way, Emily E.; Trevejo-Nunez, Giraldina; Kane, Lawrence P.; Steiner, Bart H.; Puri, Kamal D.; Kolls, Jay K.; Chen, Kong

    2016-01-01

    There remains a significant need for development of effective small molecules that can inhibit cytokine-mediated inflammation. Phosphoinositide 3 kinase (PI3K) is a direct upstream activator of AKT, and plays a critical role in multiple cell signaling pathways, cell cycle progression, and cell growth, and PI3K inhibitors have been approved or are in clinical development. We examined novel PI3Kdelta inhibitors, which are highly selective for the p110delta isoform of in CD3/CD28 stimulated T-cell cytokine production. In vitro generated CD4+ T effector cells stimulated in the presence of a PI3Kdelta inhibitor demonstrated a dose-dependent suppression of cytokines produced by Th1, Th2, and Th17 cells. This effect was T-cell intrinsic, and we observed similar effects on human PBMCs. Th17 cells expressing a constitutively activated form of AKT were resistant to PI3Kdelta inhibition, suggesting that the inhibitor is acting through AKT signaling pathways. Additionally, PI3Kdelta inhibition decreased IL-17 production in vivo and decreased neutrophil recruitment to the lung in a murine model of acute pulmonary inflammation. These experiments show that targeting PI3Kdelta activity can modulate T-cell cytokine production and reduce inflammation in vivo, suggesting that PI3Kdelta inhibition could have therapeutic potential in treating inflammatory diseases. PMID:27461849

  6. Suppression of cell cycle progression by a fungal lectin: activation of cyclin-dependent kinase inhibitors.

    PubMed

    Liua, W; Ho, J C; Ng, T

    2001-01-01

    The antiproliferative activity of a fungal lectin (VVL) isolated from the mushroom, Volvariella volvacea, was studied using a battery of cultured tumor cell lines. It was revealed that [(3)H]thymidine incorporation into the cell lines was markedly reduced at 0.32 microM VVL. When S180 mouse sarcoma cells were incubated for 48 hr with doses of VVL ranging from 0.32 to 0.8 microM, prominent blebs on the cell surface and large vacuoles in the cytoplasm, but not apoptotic bodies, were observed under a fluorescence microscopy. VVL did not exert ribosome-inactivating activity or induce any changes in the expression of cyclins A, D1, and E. However, it did activate the expression of cyclin kinase inhibitors, namely p21, p27, p53, and Rb, in a dose-dependent manner. Flow cytometric analysis demonstrated an accumulation of cells in the G2/M phase in a time- and dose-dependent manner, indicating that VVL arrested cell proliferation by blocking cell cycle progression in the G2/M phase. PMID:11137706

  7. The Rho-kinase inhibitor HA-1077 suppresses proliferation/migration and induces apoptosis of urothelial cancer cells

    PubMed Central

    2014-01-01

    Background Activation of Rho, one of the small GTPases, and its major downstream target Rho-kinase (ROCK) promotes the development and metastasis of cancer. We previously showed that elevation of Rho and ROCK expression was associated with tumor invasion, metastasis, and an unfavorable prognosis in patients with urothelial cancer of the bladder or upper urinary tract. Methods We investigated the effects of a ROCK inhibitor on the growth, migration, and apoptosis of bladder cancer cells. We also examined phosphorylation of RhoA (RhoA activity) by measuring its GTP-bound active form and assessed the expression of ROCK to explore the underlying molecular mechanisms. Results Lysophosphatidic acid (LPA) and geranylgeraniol (GGOH) induced an increase of cell proliferation and migration in association with promotion of RhoA activity and upregulation of ROCK expression. The ROCK inhibitor fasudil (HA-1077) suppressed cell proliferation and migration, and also induced apoptosis in a dose-dependent manner. HA-1077 dramatically suppressed the expression of ROCK-I and ROCK-II, but did not affect RhoA activity. Conclusions These findings suggest that ROCK could be a potential molecular target for the treatment of urothelial cancer. PMID:24908363

  8. The novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 overcomes acquired BRAF inhibitor resistance through suppression of Mcl-1 and MEK expression

    PubMed Central

    Phadke, Manali S.; Sini, Patrizia; Smalley, Keiran S. M.

    2015-01-01

    Resistance to BRAF inhibitors is a major clinical problem. Here we evaluate BI-847325, an ATP-competitive inhibitor of MEK and Aurora kinases, in treatment-naïve and drug-resistant BRAF-mutant melanoma models. BI-847325 potently inhibited growth and survival of melanoma cell lines that were both BRAF inhibitor naïve and resistant in 2D culture, 3D cell culture conditions and in colony formation assays. Western blot studies showed BI-847325 to reduce expression of phospho-ERK and phospho-histone 3 in multiple models of vemurafenib resistance. Mechanistically, BI-847325 decreased the expression of MEK and Mcl-1 while increasing the expression of the pro-apoptotic protein BIM. Strong suppression of MEK expression was observed after 48 h of treatment, with no recovery following >72 h of washout. siRNA mediated knockdown of Mcl-1 enhanced the effects of BI-847325, whereas Mcl-1 overexpression reversed this in both 2D cell culture and 3D spheroid melanoma models. In vivo, once weekly BI-847325 (70 mg/kg) led to durable regression of BRAF-inhibitor naive xenografts with no regrowth seen (>65 days of treatment). In contrast, treatment with the vemurafenib analog PLX4720 was associated with tumor relapse at >30 days. BI-847325 also suppressed the long-term growth of xenografts with acquired PLX4720 resistance. Analysis of tumor samples revealed BI-847325 to induce apoptosis associated with suppression of phospho-ERK, total MEK, phospho-Histone3 and Mcl-1 expression. Our studies indicate that BI-847325 is effective in overcoming BRAF inhibitor resistance and has long-term inhibitory effects upon BRAF-mutant melanoma in vivo, through a mechanism associated with the decreased expression of both MEK and Mcl-1. PMID:25873592

  9. The Novel ATP-Competitive MEK/Aurora Kinase Inhibitor BI-847325 Overcomes Acquired BRAF Inhibitor Resistance through Suppression of Mcl-1 and MEK Expression.

    PubMed

    Phadke, Manali S; Sini, Patrizia; Smalley, Keiran S M

    2015-06-01

    Resistance to BRAF inhibitors is a major clinical problem. Here, we evaluate BI-847325, an ATP-competitive inhibitor of MEK and Aurora kinases, in treatment-naïve and drug-resistant BRAF-mutant melanoma models. BI-847325 potently inhibited growth and survival of melanoma cell lines that were both BRAF inhibitor naïve and resistant in 2D culture, 3D cell culture conditions, and in colony formation assays. Western blot studies showed BI-847325 to reduce expression of phospho-ERK and phospho-histone 3 in multiple models of vemurafenib resistance. Mechanistically, BI-847325 decreased the expression of MEK and Mcl-1 while increasing the expression of the proapoptotic protein BIM. Strong suppression of MEK expression was observed after 48 hours of treatment, with no recovery following >72 hours of washout. siRNA-mediated knockdown of Mcl-1 enhanced the effects of BI-847325, whereas Mcl-1 overexpression reversed this in both 2D cell culture and 3D spheroid melanoma models. In vivo, once weekly BI-847325 (70 mg/kg) led to durable regression of BRAF-inhibitor naïve xenografts with no regrowth seen (>65 days of treatment). In contrast, treatment with the vemurafenib analog PLX4720 was associated with tumor relapse at >30 days. BI-847325 also suppressed the long-term growth of xenografts with acquired PLX4720 resistance. Analysis of tumor samples revealed BI-847325 to induce apoptosis associated with suppression of phospho-ERK, total MEK, phospho-Histone3, and Mcl-1 expression. Our studies indicate that BI-847325 is effective in overcoming BRAF inhibitor resistance and has long-term inhibitory effects upon BRAF-mutant melanoma in vivo, through a mechanism associated with the decreased expression of both MEK and Mcl-1. PMID:25873592

  10. Dasatinib suppression of medulloblastoma survival and migration is markedly enhanced by combining treatment with the aurora kinase inhibitor AT9283

    PubMed Central

    Petersen, William; Liu, Jingbo; Yuan, Liangping; Zhang, Hongying; Schneiderjan, Matthew; Cho, Yoon-Jae; MacDonald, Tobey J.

    2014-01-01

    Medulloblastoma (MB) expresses Src kinase, while aurora kinase A overexpression correlates with poor survival. We thus investigated novel combination treatment with dasatinib and AT9283, inhibitors of Src and aurora kinase, respectively, on MB growth in vitro and in vivo. Treatment with each drug significantly reduced cell viability and combined treatment markedly potentiated this response. AT9283 induced p53 expression, autophagy, and G2/M cell-cycle arrest, while combined treatment induced S phase arrest. Dasatinib treatment caused tumor regression in vivo. Activated Src was detected in 44% MB analyzed. We conclude that further evaluation of this combination therapy for MB is highly warranted. PMID:25107642

  11. [Suppressive effect of protein kinase C inhibitors on tumor cell function via phosphorylation of p53 protein in mice].

    PubMed

    Nakamura, K; Shinozuka, K; Kunitomo, M

    2000-12-01

    We examined the role of protein kinase C (PKC) in the phosphorylation of a p53 protein. Exposure to a protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), increased the phosphorylation of the wild type p53 protein, whereas exposure to a tumor promoter phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), decreased it in vivo after incubation with mouse epidermal JB6 cells for 3 h. Exposure to a cAMP dependent protein kinase (PKA) activator, forskolin, did not decrease the phosphorylation of p53 protein. In the transient transfection/luciferase reporter transactivation assay, H7 slightly increased the mouse double minute (MDM) 2 reporter transactivation activity of the p53 protein after treatment for 24 h, whereas TPA completely blocked it. Exposure to H7 and a specific PKC inhibitor, bisindolylmaleimide (bis), dose-dependently reduced the lung-colonizing potential of highly metastatic B16-F10 mouse melanoma cells in syngeneic mice. These results suggest that the phosphorylation of the wild type p53 protein is inversely related to PKC activation, and also suggest that the phosphorylation of the p53 protein is involved in the function of its transcription factor. The PKC inhibitor may exhibit a potent anti-metastatic effect through the phosphorylation of wild type p53 protein and the activation of its function. PMID:11193387

  12. [Kinase inhibitors and their resistance].

    PubMed

    Togashi, Yosuke; Nishio, Kazuto

    2015-08-01

    Kinase cascades are involved in all stages of tumorigenesis through modulation of transformation and differentiation, cell-cycle progression, and motility. Advances in molecular targeted drug development allow the design and synthesis of inhibitors targeting cancer-associated signal transduction pathways. Potent selective inhibitors with low toxicity can benefit patients especially with several malignancies harboring an oncogenic driver addictive signal. This article evaluates information on solid tumor-related kinase signals and inhibitors, including receptor tyrosine kinase or serine/threonine kinase signals that lead to successful application in clinical settings. In addition, the resistant mechanisms to the inhibitors is summarized. PMID:26281685

  13. Suppression of PRKAR1A expression enhances anti-proliferative and apoptotic effects of protein kinase inhibitors and chemotherapeutic drugs on cholangiocarcinoma cells.

    PubMed

    Loilome, Watcharin; Juntana, Sirinun; Pinitsoontorn, Chadamas; Namwat, Nisana; Tassaneeyakul, Wichittra; Yongvanit, Puangrat

    2012-01-01

    Suppression of protein kinase A regulatory subunit 1 alpha (PRKAR1A) has been proven to inhibit cholangiocarcinoma (CCA) cell growth and enhance apoptosis. In the present study, we aimed to determine synergistic and/or additive effects of chemotherapeutic agents, including protein kinase inhibitors (i.e. sorafenib, sunitinib, gefitinib, Met inhibitor) and conventional chemotherapeutic drugs (i.e. 5-fluorouracil, doxorubicin, paclitaxel, gemcitabine), in PRKARIA knockdown CCA cell lines. The results revealed that PRKAR1A suppressed CCA cell lines demonstrated enhanced sensitivity to some chemotherapeutic drugs when compared to control cells. Moreover, PRKAR1A knockdown in combination with either sorafenib or 5-fluorouracil increased apoptotic effects on CCA cell lines. Therefore, selective inhibition of PRKAR1A appears to enhance the growth inhibitory effects of chemotherapeutic drugs as well as induce apoptotic cell death. Our findings suggest that additional suppression of PRKAR1A expression may increase the efficacy of conventional CCA chemotherapeutic treatment. Clinical studies in CCA patients now need to be conducted. PMID:23480756

  14. Targeting cancer with kinase inhibitors

    PubMed Central

    Gross, Stefan; Rahal, Rami; Stransky, Nicolas; Lengauer, Christoph; Hoeflich, Klaus P.

    2015-01-01

    Kinase inhibitors have played an increasingly prominent role in the treatment of cancer and other diseases. Currently, more than 25 oncology drugs that target kinases have been approved, and numerous additional therapeutics are in various stages of clinical evaluation. In this Review, we provide an in-depth analysis of activation mechanisms for kinases in cancer, highlight recent successes in drug discovery, and demonstrate the clinical impact of selective kinase inhibitors. We also describe the substantial progress that has been made in designing next-generation inhibitors to circumvent on-target resistance mechanisms, as well as ongoing strategies for combining kinase inhibitors in the clinic. Last, there are numerous prospects for the discovery of novel kinase targets, and we explore cancer immunotherapy as a new and promising research area for studying kinase biology. PMID:25932675

  15. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

    PubMed Central

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  16. Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

    PubMed Central

    Chen, Eleanor Y.; DeRan, Michael T.; Ignatius, Myron S.; Grandinetti, Kathryn Brooke; Clagg, Ryan; McCarthy, Karin M.; Lobbardi, Riadh M.; Brockmann, Jillian; Keller, Charles; Wu, Xu; Langenau, David M.

    2014-01-01

    Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/β-catenin pathway, recombinant WNT3A and stabilized β-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/β-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/β-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS. PMID:24706870

  17. Suppression of CD4+ T lymphocyte activation in vitro and experimental encephalomyelitis in vivo by the phosphatidyl inositol 3-kinase inhibitor PIK-75.

    PubMed

    Acosta, Y Y; Montes-Casado, M; Aragoneses-Fenoll, L; Dianzani, U; Portoles, P; Rojo, J M

    2014-01-01

    Class IA phosphatidyl inositol-3 kinases (PI3-K) are important targets in cancer therapy and are essential to immune responses, particularly through costimulation by CD28 and ICOS. Thus, small PI3-K inhibitors are likely candidates to immune intervention. PIK-75 is an efficient inhibitor of the PI3-K p110alpha catalytic subunits that suppresses tumor growth, and its effects on immune and autoimmune responses should be studied. Here, we describe the effect of PIK-75 on different immune parameters in vitro and in vivo. PIK-75 at concentrations commonly used in vitro (≥0.1 μM) inhibited T and B cell activation by Concanavalin A and LPS, respectively, and survival of non-stimulated spleen cells. In naive CD4+ T lymphocytes, PIK-75 induced apoptosis of resting or activated cells that was prevented by caspase inhibitors. At low nanomolar concentrations (≤10 nM), PIK-75 inhibited naive CD4+ T cell proliferation, and IL-2 and IFN-gamma production induced by anti-CD3 plus anti-CD28. In activated CD4+ T blasts costimulated by ICOS, PIK-75 (less than 10 nM) inhibited IFN-gamma, IL-17A, or IL-21 secretion. Furthermore, PIK-75 (20 mg/kg p.o.) suppressed clinical symptoms in ongoing experimental autoimmune encephalomyelitis (EAE) and inhibited MOG-specific responses in vitro. Thus, PIK-75 is an efficient suppressor of EAE, modulating lymphocyte function and survival. PMID:24674679

  18. Mangiferin, a novel nuclear factor kappa B-inducing kinase inhibitor, suppresses metastasis and tumor growth in a mouse metastatic melanoma model.

    PubMed

    Takeda, Tomoya; Tsubaki, Masanobu; Sakamoto, Kotaro; Ichimura, Eri; Enomoto, Aya; Suzuki, Yuri; Itoh, Tatsuki; Imano, Motohiro; Tanabe, Genzoh; Muraoka, Osamu; Matsuda, Hideaki; Satou, Takao; Nishida, Shozo

    2016-09-01

    Advanced metastatic melanoma, one of the most aggressive malignancies, is currently without reliable therapy. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone and exerts many beneficial biological activities. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we evaluated the effect of mangiferin on metastasis and tumor growth in a mouse metastatic melanoma model. We found that mangiferin inhibited spontaneous metastasis and tumor growth. Furthermore, mangiferin suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated NF-κB-inducing kinase (NIK), inhibitor of kappa B kinase (IKK), and inhibitor of kappa B (IκB) and increases the expression of IκB protein in vivo. In addition, we found that mangiferin inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs) in vivo. Mangiferin treatment also increased the expression of cleaved caspase-3, cleaved Poly ADP ribose polymerase-1 (PARP-1), p53 upregulated modulator of apoptosis (PUMA), p53, and phosphorylated p53 proteins, and decreased the expression of Survivin and Bcl-associated X (Bcl-xL) proteins in vivo. These results indicate that mangiferin selectivity suppresses the NF-κB pathway via inhibition of NIK activation, thereby inhibiting metastasis and tumor growth. Importantly, the number of reported NIK selective inhibitors is limited. Taken together, our data suggest that mangiferin may be a potential therapeutic agent with a new mechanism of targeting NIK for the treatment of metastatic melanoma. PMID:27417526

  19. DNA-dependent protein kinase catalytic subunit inhibitor reverses acquired radioresistance in lung adenocarcinoma by suppressing DNA repair.

    PubMed

    Li, Yong; Li, Hang; Peng, Wen; He, Xin-Yun; Huang, Min; Qiu, Dong; Xue, Ying-Bo; Lu, Liang

    2015-07-01

    The mechanisms underlying lung cancer radioresistance remain to be fully elucidated. The DNA repair pathway is a predominant target of radiotherapy, which is considered to be involved in the acquired radioresistance of cancer cells. The present study aimed to establish a radioresistant cell model using the A549 human lung cancer cell line, and to further investigate the potential mechanisms underlying the radioresistance. The A549R radioresistant lung cancer cell variant was established by exposing the parental A549 cells to repeated γ-ray irradiation at a total dose of 60 Gy. Colony formation assays were then used to determine cell survival following γ-ray exposure. The established radioresistant cells were subsequently treated with or without the NU7026 DNA-PKcs inhibitor. The levels of DNA damage were determined by counting the number of fluorescent γ-H2AX foci in the cells. The cellular capacity for DNA repair was assessed using antibodies for the detection of various DNA repair pathway proteins. The radioresistant sub-clones exhibited significantly decreased survival following NU7026 treatment, compared with the parental cells, as determined by colony formation assays (P<0.05), and this finding was found to be dose-dependent. Treatment with the DNA-dependent protein kinase (DNA-PK) inhibitor significantly reduced γ-H2AX foci formation (P<0.05) following acute radiation exposure in the radioresistant sub-clones, compared with the parental control cells. The decreased levels of γ-H2AX were accompanied by an increase in the percentage of apoptotic cells in the radioresistant cell line following post-radiation treatment with the DNA-PKcs inhibitor. The expression levels of proteins associated with the DNA repair pathway were altered markedly in the cells treated with NU7026. The results of the present study suggested that radioresistance may be associated with enhanced DNA repair following exposure to radiation, resulting in reduced apoptosis. Therefore, the

  20. Tyrosine kinase inhibitor suppresses coronary arteriosclerotic changes and vasospastic responses induced by chronic treatment with interleukin-1 beta in pigs in vivo.

    PubMed Central

    Ito, A; Shimokawa, H; Kadokami, T; Fukumoto, Y; Owada, M K; Shiraishi, T; Nakaike, R; Takayanagi, T; Egashira, K; Takeshita, A

    1995-01-01

    We recently demonstrated that chronic treatment with IL-1 beta induces coronary arteriosclerotic changes and vasospastic responses to autacoids in pigs in vivo and that those responses are importantly mediated by PDGF. The receptors for PDGF and other major growth factors are known to have tyrosine kinase activity. We therefore investigated the effects of a selective tyrosine kinase inhibitor, ST 638, on those responses induced by IL-1 beta in our swine model. Intimal thickening and coronary vasospastic responses to serotonin and histamine were induced at the site of the coronary artery where IL-1 beta was chronically and locally applied. These responses were significantly suppressed in a dose-dependent manner by cotreatment with ST 638. In addition, ST 494, which is an inactive form of ST 638, did not inhibit those responses. The treatment with ST 638 alone did not affect the coronary vasoconstricting responses to the autacoids. Immunoblotting using an antibody to phosphotyrosines confirmed the inhibitory effects of ST 638 on the tyrosine phosphorylations induced by IL-1 beta. These results thus suggest that tyrosine kinase activation may play an important role in mediating the effects of IL-1 beta, while also suggesting that ST 638 has an inhibitory effect on the arteriosclerotic changes and vasospastic responses to autacoids in our swine model in vivo. Images PMID:7657803

  1. Effective growth-suppressive activity of maternal embryonic leucine-zipper kinase (MELK) inhibitor against small cell lung cancer

    PubMed Central

    Inoue, Hiroyuki; Kato, Taigo; Olugbile, Sope; Tamura, Kenji; Chung, Suyoun; Miyamoto, Takashi; Matsuo, Yo; Salgia, Ravi; Nakamura, Yusuke; Park, Jae-Hyun

    2016-01-01

    Maternal embryonic leucine zipper kinase (MELK), that plays a critical role in maintenance of cancer stem cells (CSCs), is predominantly expressed in various types of human cancer including small cell lung cancer (SCLC). SCLC usually acquires resistance to anti-cancer drugs and portends dismal prognosis. We have delineated roles of MELK in development/progression of SCLC and examined anti-tumor efficacy of OTS167, a highly potent MELK inhibitor, against SCLC. MELK expression was highly upregulated in both SCLC cell lines and primary tumors. siRNA-mediated MELK knockdown induced significant growth inhibition in SCLC cell lines. Concordantly, treatment with OTS167 exhibited strong cytotoxicity against eleven SCLC cell lines with IC50 of < 10 nM. As similar to siRNA knockdown, OTS167 treatment induced cytokinetic defects with intercellular bridges, and in some cell lines we observed formation of neuronal protrusions accompanied with increase of a neuronal differentiation marker (CD56), indicating that the compound induced differentiation of cancer cells to neuron-like cells. Furthermore, the MELK inhibition decreased its downstream FOXM1 activity and Akt expression in SCLC cells, and led to apoptotic cell death. OTS167 appeared to be more effective to CSCs as measured by the sphere formation assay, thus MELK inhibition might become a promising treatment modality for SCLC. PMID:26871945

  2. Pantoprazole, an FDA-approved proton-pump inhibitor, suppresses colorectal cancer growth by targeting T-cell-originated protein kinase.

    PubMed

    Zeng, Xiaoyu; Liu, Lin; Zheng, Mengzhu; Sun, Huimin; Xiao, Juanjuan; Lu, Tao; Huang, Guangqian; Chen, Pianpian; Zhang, Jianmin; Zhu, Feng; Li, Hua; Duan, Qiuhong

    2016-04-19

    T-cell-originated protein kinase (TOPK) is highly expressed in several cancer cells and promotes tumorigenesis and progression, and therefore, it is an important target for drug treatment of tumor. Pantoprazole (PPZ) was identified to be a TOPK inhibitor from FDA-approved drug database by structure based virtual ligand screening. Herein, the data indicated that pantoprazole inhibited TOPK activities by directly binding with TOPK in vitro and in vivo. Ex vivo studies showed that pantoprazole inhibited TOPK activities in JB6 Cl41 cells and HCT 116 colorectal cancer cells. Moreover, knockdown of TOPK in HCT 116 cells decreased their sensitivities to pantoprazole. Results of an in vivo study demonstrated that i.p. injection of pantoprazole in HCT 116 colon tumor-bearing mice effectively suppressed cancer growth. The TOPK downstream signaling molecule phospho-histone H3 in tumor tissues was also decreased after pantoprazole treatment. In short, pantoprazole can suppress growth of colorectal cancer cells as a TOPK inhibitor both in vitro and in vivo. PMID:26967058

  3. Pantoprazole, an FDA-approved proton-pump inhibitor, suppresses colorectal cancer growth by targeting T-cell-originated protein kinase

    PubMed Central

    Sun, Huimin; Xiao, Juanjuan; Lu, Tao; Huang, Guangqian; Chen, Pianpian; Zhang, Jianmin; Zhu, Feng; Li, Hua; Duan, Qiuhong

    2016-01-01

    T-cell-originated protein kinase (TOPK) is highly expressed in several cancer cells and promotes tumorigenesis and progression, and therefore, it is an important target for drug treatment of tumor. Pantoprazole (PPZ) was identified to be a TOPK inhibitor from FDA-approved drug database by structure based virtual ligand screening. Herein, the data indicated that pantoprazole inhibited TOPK activities by directly binding with TOPK in vitro and in vivo. Ex vivo studies showed that pantoprazole inhibited TOPK activities in JB6 Cl41 cells and HCT 116 colorectal cancer cells. Moreover, knockdown of TOPK in HCT 116 cells decreased their sensitivities to pantoprazole. Results of an in vivo study demonstrated that i.p. injection of pantoprazole in HCT 116 colon tumor-bearing mice effectively suppressed cancer growth. The TOPK downstream signaling molecule phospho-histone H3 in tumor tissues was also decreased after pantoprazole treatment. In short, pantoprazole can suppress growth of colorectal cancer cells as a TOPK inhibitor both in vitro and in vivo. PMID:26967058

  4. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

    PubMed Central

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  5. ZD6474, a Multitargeted Inhibitor for Receptor Tyrosine Kinases, Suppresses Growth of Gliomas Expressing an Epidermal Growth Factor Receptor Mutant, EGFRvIII, in the Brain

    PubMed Central

    Yiin, Jia-Jean; Hu, Bo; Schornack, Paul A.; Sengar, Raghvendra S.; Liu, Kun-wei; Feng, Haizhong; Lieberman, Frank S.; Chiou, Shih-Hwa; Sarkaria, Jann N.; Wiener, Erik C.; Ma, Hsin-I; Cheng, Shi-Yuan

    2010-01-01

    Epidermal growth factor receptor (EGFR) vIII is a mutated EGFR that is frequently overexpressed in glioblastomas and implicated in response to receptor tyrosine kinase inhibitors. In this study, we investigate the effect of ZD6474 (ZACTIMA, vandetanib), a dual inhibitor for vascular endothelial growth factor receptor 2 and EGFR on growth and angiogenesis of gliomas expressing EGFRvIII. We used two glioma xenograft models, U87MG cells overexpressing EGFRvIII and short-term cultured primary glioma GBM8 cells with EGFRvIII. ZD6474 inhibited tumor growth and angiogenesis and induced cell apoptosis in various brain gliomas. Moreover, significant inhibition of EGFRvIII-expressing U87MG and GBM8 gliomas was observed compared with their controls. Magnetic resonance imaging analysis using the apparent diffusion coefficient and three-dimensional T2*weighed measurements validated ZD6474 inhibition on tumor growth and angiogenesis in EGFRvIII-expressing GBM8 gliomas. Mechanistically, ZD6474 shows better inhibition of cell growth and survival of U87MG/EGFRvIII, GBM6, and GBM8 cells that express EGFRvIII than U87MG or GBM14 cells that have nondetectable EGFRvIII through attenuation of activated phosphorylation of signal transducer and activator of transcription 3, Akt, and Bcl-XL expression. Albeit in lesser extent, ZD6474 also displays suppressions of U87MG/EGFR and GBM12 cells that overexpress wild-type EGFR. Additionally, ZD6474 inhibits activation of extracellular signal-regulated kinase 1/2 in both types of cells, and expression of a constitutively active phosphoinositide 3-kinases partially rescued ZD6474 inhibition in U87MG/EGFRvIII cells. Taken together, these data show that ZD6474 significantly inhibited growth and angiogenesis of gliomas expressing EGFRvIII by specifically blocking EGFRvIII-activated signaling mediators, suggesting a potential application of ZD6474 in treatments for glioblastomas that overexpress EGFRvIII. PMID:20371720

  6. The Stilbenoid Tyrosine Kinase Inhibitor, G6, Suppresses Jak2-V617F-mediated Human Pathological Cell Growth in Vitro and in Vivo*

    PubMed Central

    Kirabo, Annet; Embury, Jennifer; Kiss, Róbert; Polgár, Tímea; Gali, Meghanath; Majumder, Anurima; Bisht, Kirpal S.; Cogle, Christopher R.; Keserű, György M.; Sayeski, Peter P.

    2011-01-01

    Using structure-based virtual screening, we previously identified a novel stilbenoid inhibitor of Jak2 tyrosine kinase named G6. Here, we hypothesized that G6 suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo. We found that G6 inhibited proliferation of the Jak2-V617F expressing human erythroleukemia (HEL) cell line by promoting marked cell cycle arrest and inducing apoptosis. The G6-dependent increase in apoptosis levels was concomitant with increased caspase 3/7 activity and cleavage of PARP. G6 also selectively inhibited phosphorylation of STAT5, a downstream signaling target of Jak2. Using a mouse model of Jak2-V617F-mediated hyperplasia, we found that G6 significantly decreased the percentage of blast cells in the peripheral blood, reduced splenomegaly, and corrected a pathologically low myeloid to erythroid ratio in the bone marrow by eliminating HEL cell engraftment in this tissue. In addition, drug efficacy correlated with the presence of G6 in the plasma, marrow, and spleen. Collectively, these data demonstrate that the stilbenoid compound, G6, suppresses Jak2-V617F-mediated aberrant cell growth. As such, G6 may be a potential therapeutic lead candidate against Jak2-mediated, human disease. PMID:21127060

  7. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  8. Pharmacologic inhibitors of IkappaB kinase suppress growth and migration of mammary carcinosarcoma cells in vitro and prevent osteolytic bone metastasis in vivo.

    PubMed

    Idris, Aymen I; Libouban, Hélène; Nyangoga, Hervé; Landao-Bassonga, Euphemie; Chappard, Daniel; Ralston, Stuart H

    2009-08-01

    The NF-kappaB signaling pathway is known to play an important role in the regulation of osteoclastic bone resorption and cancer cell growth. Previous studies have shown that genetic inactivation of IkappaB kinase (IKK), a key component of NF-kappaB signaling, inhibits osteoclastogenesis, but the effects of pharmacologic IKK inhibitors on osteolytic bone metastasis are unknown. Here, we studied the effects of the IKK inhibitors celastrol, BMS-345541, parthenolide, and wedelolactone on the proliferation and migration of W256 cells in vitro and osteolytic bone destruction in vivo. All compounds tested inhibited the growth and induced apoptosis of W256 cells as evidenced by caspase-3 activation and nuclear morphology. Celastrol, BMS-345541, and parthenolide abolished IL1beta and tumor necrosis factor alpha-induced IkappaB phosphorylation and prevented nuclear translocation of NF-kappaB and DNA binding. Celastrol and parthenolide but not BMS-345541 prevented the activation of both IKKalpha and IKKbeta, and celastrol inhibited IKKalpha/beta activation by preventing the phosphorylation of TAK1, a key receptor-associated factor upstream of IKK. Celastrol and parthenolide markedly reduced the mRNA expression of matrix metalloproteinase 9 and urinary plasminogen activator, and inhibited W256 migration. Administration of celastrol or parthenolide at a dose of 1 mg/kg/day suppressed trabecular bone loss and reduced the number and size of osteolytic bone lesions following W256 injection in rats. Histomorphometric analysis showed that both compounds decreased osteoclast number and inhibited bone resorption. In conclusion, pharmacologic inhibitors of IKK are effective in preventing osteolytic bone metastasis in this model and might represent a promising class of agents to the prevention and treatment of metastatic bone disease associated with breast cancer. PMID:19671767

  9. Suppression of the senescence-associated secretory phenotype (SASP) in human fibroblasts using small molecule inhibitors of p38 MAP kinase and MK2.

    PubMed

    Alimbetov, Dauren; Davis, Terence; Brook, Amy J C; Cox, Lynne S; Faragher, Richard G A; Nurgozhin, Talgat; Zhumadilov, Zhaxybay; Kipling, David

    2016-04-01

    Senescent cells show an altered secretome profile termed the senescence-associated secretory phenotype (SASP). There is an increasing body of evidence that suggests that the accumulation of SASP-positive senescent cells in humans is partially causal in the observed shift to a low-level pro-inflammatory state in aged individuals. This in turn suggests the SASP as a possible therapeutic target to ameliorate inflammatory conditions in the elderly, and thus a better understanding of the signalling pathways underlying the SASP are required. Prior studies using the early generation p38 MAPK inhibitor SB203580 indicated that p38 signalling was required for the SASP. In this study, we extend these observations using two next-generation p38 inhibitors (UR-13756 and BIRB 796) that have markedly improved selectivity and specificity compared to SB203580, to strengthen the evidence that the SASP is p38-dependent in human fibroblasts. BIRB 796 has an efficacy and toxicity profile that has allowed it to reach Phase III clinical trials, suggesting its possible use to suppress the SASP in vivo. We also demonstrate for the first time a requirement for signalling through the p38 downstream MK2 kinase in the regulation of the SASP using two MK2 inhibitors. Finally, we demonstrate that a commercially-available multiplex cytokine assay technology can be used to detect SASP components in the conditioned medium of cultured fibroblasts from both young and elderly donors. This assay is a high-throughput, multiplex microtitre-based assay system that is highly sensitive, with very low sample requirements, allowing it to be used for low-volume human biological fluids. Our initial studies using existing multiplex plates form the basis for a "SASP signature" assay that could be used as a high-throughput system in a clinical study setting. Our findings therefore provide important steps towards the study of, and intervention in, the SASP in human ageing and age-related disease. PMID:26400758

  10. Bruton tyrosine kinase (Btk) suppresses osteoblastic differentiation.

    PubMed

    Kaneshiro, Shoichi; Ebina, Kosuke; Shi, Kenrin; Yoshida, Kiyoshi; Otsuki, Dai; Yoshikawa, Hideki; Higuchi, Chikahisa

    2015-09-01

    The Tec family of nonreceptor tyrosine kinases has been shown to play a key role in inflammation and bone destruction. Bruton tyrosine kinase (Btk) has been the most widely studied because of its critical role in B cells. Furthermore, recent evidence has demonstrated that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. The role of Btk in osteoblastic differentiation has not been well elucidated. In this study, we demonstrated the role of Btk in osteoblastic differentiation and investigated the effects of a Btk inhibitor on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells, primary calvarial osteoblasts, and bone marrow stromal ST2 cells. Btk expression was detected in all three cell lines. Btk inhibition stimulated mRNA expression of osteoblastic markers (alkaline phosphatase, osteocalcin, and osterix) and promoted mineralization of the extracellular matrix. In addition, Btk knockdown caused increased mRNA expression of osteoblastic markers. Furthermore, Btk inhibition suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor kappa B (NFκB), and protein kinase Cα (PKCα). Our results indicate that Btk may regulate osteoblastic differentiation through the MAPK, NFκB, and PKCα signaling pathways. PMID:25230818

  11. Multi-kinase inhibitors, AURKs and cancer.

    PubMed

    Cicenas, Jonas; Cicenas, Erikas

    2016-05-01

    Inhibitors that impact function of kinases are valuable both for the biological research as well as therapy of kinase-associated diseases, such as different cancers. There are quite a number of inhibitors, which are quite specific for certain kinases and several of them are either already approved for the cancer therapy or are in clinical studies of various phases. However, that does not mean that each single kinase inhibitor is suitable for targeted therapy. Some of them are not effective others might be toxic or fail some other criteria for the use in vivo. On the other hand, even in case of successful therapy, many responders eventually develop resistance to the inhibitors. The limitations of various single kinase inhibitors can be fought using compounds which target multiple kinases. This tactics can increase effectiveness of the inhibitors by the synergistic effect or help to diminish the likelihood of drug resistance. To date, several families of kinases are quite popular targets of the inhibition in cancers, such as tyrosine kinases, cycle-dependent kinases, mitogen-activated protein kinases, phosphoinositide 3-kinases as well as their pathway "players" and aurora kinases. Aurora kinases play an important role in the control of the mitosis and are often altered in diverse human cancers. Here, we will describe the most interesting multi-kinase inhibitors which inhibit aurora kinases among other targets and their use in preclinical and clinical cancer studies. PMID:27038473

  12. Activity-based kinase profiling of approved tyrosine kinase inhibitors.

    PubMed

    Kitagawa, Daisuke; Yokota, Koichi; Gouda, Masaki; Narumi, Yugo; Ohmoto, Hiroshi; Nishiwaki, Eiji; Akita, Kensaku; Kirii, Yasuyuki

    2013-02-01

    The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K(m) for ATP. In addition, profiling at a physiological ATP concentration (1 mm) was carried out, and the IC(50) values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C(trough) and C(max) values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K(m) and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors. PMID:23279183

  13. Cinnamon and its Components Suppress Vascular Smooth Muscle Cell Proliferation by Up-Regulating Cyclin-Dependent Kinase Inhibitors.

    PubMed

    Kwon, Hyeeun; Lee, Jung-Jin; Lee, Ji-Hye; Cho, Won-Kyung; Gu, Min Jung; Lee, Kwang Jin; Ma, Jin Yeul

    2015-01-01

    Cinnamomum cassia bark has been used in traditional herbal medicine to treat a variety of cardiovascular diseases. However, the antiproliferative effect of cinnamon extract on vascular smooth muscle cells (VSMCs) and the corresponding restenosis has not been explored. Hence, after examining the effect of cinnamon extract on VSMC proliferation, we investigated the possible involvement of signal transduction pathways associated with early signal and cell cycle analysis, including regulatory proteins. Besides, to identify the active components, we investigated the components of cinnamon extract on VSMC proliferation. Cinnamon extract inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and suppressed the PDGF-stimulated early signal transduction. In addition, cinnamon extract arrested the cell cycle and inhibited positive regulatory proteins. Correspondingly, the protein levels of p21 and p27 not only were increased in the presence of cinnamon extract, also the expression of proliferating cell nuclear antigen (PCNA) was inhibited by cinnamon extract. Besides, among the components of cinnamon extract, cinnamic acid (CA), eugenol (EG) and cinnamyl alcohol significantly inhibited the VSMC proliferation. Overall, the present study demonstrates that cinnamon extract inhibited the PDGF-BB-induced proliferation of VSMCs through a G0/G1 arrest, which down-regulated the expression of cell cycle positive regulatory proteins by up-regulating p21 and p27 expression. PMID:26119954

  14. Raf kinase inhibitor protein suppresses nuclear factor-κB-dependent cancer cell invasion at the level of matrix metalloproteinase expression but not cell migration

    PubMed Central

    Beshir, Anwar B.; Ren, Gang; Magpusao, Anniefer N.; Barone, Lauren M.; Yeung, Kam C.; Fenteany, Gabriel

    2010-01-01

    Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIP’s function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion. PMID:20855151

  15. Suppression of caspase-11 expression by histone deacetylase inhibitors

    SciTech Connect

    Heo, Hyejung; Yoo, Lang; Shin, Ki Soon; Kang, Shin Jung

    2009-01-02

    It has been well documented that histone deacetylase inhibitors suppress inflammatory gene expression. Therefore, we investigated whether histone deacetylase inhibitors modulate the expression of caspase-11 that is known as an inducible caspase regulating both inflammation and apoptosis. In the present study, we show that sodium butyrate and trichostatin A, two structurally unrelated inhibitors of histone deacetylase (HDAC), effectively suppressed the induction of caspase-11 in mouse embryonic fibroblasts stimulated with lipopolysaccharides. Sodium butyrate inhibited the activation of upstream signaling events for the caspase-11 induction such as activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, degradation of inhibitor of {kappa}B, and activation of nuclear factor-{kappa}B. These results suggest that the HDAC inhibitor suppressed cytosolic signaling events for the induction of caspase-11 by inhibiting the deacetylation of non-histone proteins.

  16. From a natural product lead to the identification of potent and selective benzofuran-3-yl-(indol-3-yl)maleimides as glycogen synthase kinase 3beta inhibitors that suppress proliferation and survival of pancreatic cancer cells.

    PubMed

    Gaisina, Irina N; Gallier, Franck; Ougolkov, Andrei V; Kim, Ki H; Kurome, Toru; Guo, Songpo; Holzle, Denise; Luchini, Doris N; Blond, Sylvie Y; Billadeau, Daniel D; Kozikowski, Alan P

    2009-04-01

    Recent studies have demonstrated that glycogen synthase kinase 3beta (GSK-3beta) is overexpressed in human colon and pancreatic carcinomas, contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3beta inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3beta and an enhanced selectivity against cyclin-dependent kinase 2 (CDK-2). Selected GSK-3beta inhibitors were tested in the pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds, namely compounds 5, 6, 11, 20, and 26, demonstrate antiproliferative activity against some or all of the pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of pancreatic cancer cells with GSK-3beta inhibitors 5 and 26 resulted in suppression of GSK-3beta activity and a distinct decrease of the X-linked inhibitor of apoptosis (XIAP) expression, leading to significant apoptosis. The present data suggest a possible role for GSK-3beta inhibitors in cancer therapy, in addition to their more prominent applications in CNS disorders. PMID:19338355

  17. Lipoxygenase inhibitors suppress IL-2 synthesis: relationship with rise of [Ca++]i and the events dependent on protein kinase C activation.

    PubMed

    Dornand, J; Sekkat, C; Mani, J C; Gerber, M

    1987-11-01

    The present study was performed in an attempt to understand the mechanism involved in the inhibition of interleukin 2 (IL-2) synthesis by lipoxygenase (LO) pathway inhibitors. Using the two IL-2-producing lymphoid cell lines, (Jurkat and EL4 cells), we showed first that the inhibitory effect of the phenolic compounds tested (NDGA, BHA and caffeic acid) acted on lymphoid cells themselves and not on eventual monocytic or granulocytic contaminant cells. Secondly, these inhibitors were demonstrated as exerting their effect on two levels: they affected the events controlled by both second messengers implicated in T cell activation, namely rise of intracellular free calcium concentration [( Ca++]i) and protein kinase C (PKC) activation. For this purpose, LO inhibitor effects have been compared: (a) on IL-2 production by the two different lines: Jurkat cells, which need both signals, and EL4 cells, which require only PKC activation for the induction of this production; and (b) on the events induced by the different ways of Jurkat cell activation: PHA (or anti-CD3 monoclonal antibody) versus calcium ionophore. These results are discussed with respect to an eventual involvement of arachidonic acid [AA] derivatives in IL-2 synthesis. PMID:3123378

  18. Ocular Toxicity of Tyrosine Kinase Inhibitors

    PubMed Central

    Davis, Mary Elizabeth

    2016-01-01

    Purpose/Objectives To review common tyrosine kinase inhibitors, as well as their ocular side effects and management. Data Sources A comprehensive literature search was conducted using cINahl®, Pubmed, and cochrane databases for articles published since 2004 with the following search terms: ocular toxicities, tyrosine kinase inhibitors, ophthalmology, adverse events, eye, and vision. Data Synthesis Tyrosine kinase inhibitors can cause significant eye toxicity. Conclusions Given the prevalence of new tyrosine kinase inhibitor therapies and the complexity of possible pathogenesis of ocular pathology, oncology nurses can appreciate the occurrence of ocular toxicities and the role of nursing in the management of these problems. Implications for Nursing Knowledge of the risk factors and etiology of ocular toxicity of targeted cancer therapies can guide nursing assessment, enhance patient education, and improve care management. Including a review of eye symptoms and vision issues in nursing assessment can enhance early detection and treatment of ocular toxicity. PMID:26906134

  19. BI 5700, a Selective Chemical Inhibitor of IκB Kinase 2, Specifically Suppresses Epithelial-Mesenchymal Transition and Metastasis in Mouse Models of Tumor Progression

    PubMed Central

    Huber, Margit A.; Maier, Harald J.; Alacakaptan, Memetcan; Wiedemann, Eva; Braunger, Jürgen; Boehmelt, Guido; Madwed, Jeffrey B.; Young, Erick R.R.; Marshall, Daniel R.; Pehamberger, Hubert; Wirth, Thomas; Kraut, Norbert; Beug, Hartmut

    2010-01-01

    Increasing evidence suggests that processes termed epithelial-mesenchymal transitions (EMTs) play a key role in therapeutic resistance, tumor recurrence, and metastatic progression. NF-κB signaling has been previously identified as an important pathway in the regulation of EMT in a mouse model of tumor progression. However, it remains unclear whether there is a broad requirement for this pathway to govern EMT and what the relative contribution of IKK family members acting as upstream NF-κB activators is toward promoting EMT and metastasis. To address this question, we have used a novel, small-molecule inhibitor of IκB kinase 2 (IKK2/IKKβ), termed BI 5700. We investigated the role of IKK2 in a number of mouse models of EMT, including TGFβ-induced EMT in the mammary epithelial cell line EpRas, CT26 colon carcinoma cells, and 4T1 mammary carcinoma cells. The latter model was also used to evaluate in vivo activities of BI 5700.We found that BI 5700 inhibits IKK2 with an IC50 of 9 nM and was highly selective as compared to other IKK family members (IKK1, IKKε, and TBK1) and other kinases. BI 5700 effectively blocks NF-κB activity in EpRas cells and prevents TGFβ-induced EMT. In addition, BI 5700 reverts EMT in mesenchymal CT26 cells and prevents EMT in the 4T1 model. Oral application of BI 5700 significantly interferes with metastasis after mammary fat-pad injection of 4T1 cells, yielding fewer, smaller, and more differentiated metastases as compared to vehicle-treated control animals. We conclude that IKK2 is a key regulator of both the induction and maintenance of EMT in a panel of mouse tumor progression models and that the IKK2 inhibitor BI 5700 constitutes a promising candidate for the treatment of metastatic cancers. PMID:21779445

  20. A novel p38 mitogen activated protein kinase (MAPK) specific inhibitor suppresses respiratory syncytial virus and influenza A virus replication by inhibiting virus-induced p38 MAPK activation.

    PubMed

    Choi, Myung-Soo; Heo, Jinyuk; Yi, Chae-Min; Ban, Junsu; Lee, Noh-Jin; Lee, Na-Rae; Kim, Sang Won; Kim, Nam-Jung; Inn, Kyung-Soo

    2016-08-26

    Respiratory syncytial virus (RSV) and influenza A virus are leading causes of acute lower respiratory infectious disease. Respiratory diseases caused by RSV and influenza A virus result in serious economic burden and life-threatening disease for immunocompromised people. With the revelation that p38 mitogen-activated protein kinase (MAPK) activity in host cells is crucial for infection and replication of RSV and influenza A virus, inhibition of p38 MAPK activity has been suggested as a potential antiviral therapeutic strategy. However, the low selectivity and high toxicity of the p38 MAPK inhibitors necessitate the development of better inhibitors. Herein, we report the synthesis of a novel p38 MAPK inhibitor, NJK14047, with high kinase selectivity. In this work, it was demonstrated that NJK14047 inhibits RSV- and influenza A-mediated p38 MAPK activation in epithelial cells. Subsequently, NJK14047 treatment resulted in decreased viral replication and viral mRNA synthesis. In addition, secretion of interleukin-6 from infected cells was greatly diminished by NJK14047, suggesting that it can ameliorate immunopathological responses to RSV and influenza A. Collectively, the results suggest that NJK14047 has therapeutic potential to treat respiratory viral infection through the suppression of p38 MAPK activation, which is suggested to be an essential step for respiratory virus infection. PMID:27346133

  1. Spleen tyrosine kinase suppresses osteoblastic differentiation through MAPK and PKCα.

    PubMed

    Yoshida, Kiyoshi; Higuchi, Chikahisa; Nakura, Akio; Yoshikawa, Hideki

    2011-08-12

    Spleen tyrosine kinase (Syk) is a non-receptor protein kinase present in abundance in a wide range of hematopoietic cells. Syk reportedly plays a crucial role in immune signaling in B cells and cells bearing Fcγ-activation receptors. The role of syk in osteoblastic differentiation has not been well elucidated. We report herein the role of syk in osteoblastic differentiation. We investigated the effects of two syk inhibitors on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. Expression of syk was detected in these two cell lines. Two syk inhibitors stimulated mRNA expression of osteoblastic markers (ALP, Runx2, Osterix). Mineralization of extracellular matrix was also promoted by treatment with syk inhibitors. Knockdown of Syk caused increased mRNA expression of osteoblastic markers. In addition, syk inhibitor and knockdown of Syk suppressed phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase Cα (PKCα). Our results indicate that syk might regulate osteoblastic differentiation through MAPK and PKCα. PMID:21782794

  2. Pharmacological inhibitors of cyclin-dependent kinases.

    PubMed

    Knockaert, Marie; Greengard, Paul; Meijer, Laurent

    2002-09-01

    Cyclin-dependent kinases (CDKs) regulate the cell division cycle, apoptosis, transcription and differentiation in addition to functions in the nervous system. Deregulation of CDKs in various diseases has stimulated an intensive search for selective pharmacological inhibitors of these kinases. More than 50 inhibitors have been identified, among which >20 have been co-crystallized with CDK2. These inhibitors all target the ATP-binding pocket of the catalytic site of the kinase. The actual selectivity of most known CDK inhibitors, and thus the underlying mechanism of their cellular effects, is poorly known. Pharmacological inhibitors of CDKs are currently being evaluated for therapeutic use against cancer, alopecia, neurodegenerative disorders (e.g. Alzheimer's disease, amyotrophic lateral sclerosis and stroke), cardiovascular disorders (e.g. atherosclerosis and restenosis), glomerulonephritis, viral infections (e.g. HCMV, HIV and HSV) and parasitic protozoa (Plasmodium sp. and Leishmania sp.). PMID:12237154

  3. Aurora Kinase Inhibitors: Current Status and Outlook

    PubMed Central

    Bavetsias, Vassilios; Linardopoulos, Spiros

    2015-01-01

    The Aurora kinase family comprises of cell cycle-regulated serine/threonine kinases important for mitosis. Their activity and protein expression are cell cycle regulated, peaking during mitosis to orchestrate important mitotic processes including centrosome maturation, chromosome alignment, chromosome segregation, and cytokinesis. In humans, the Aurora kinase family consists of three members; Aurora-A, Aurora-B, and Aurora-C, which each share a conserved C-terminal catalytic domain but differ in their sub-cellular localization, substrate specificity, and function during mitosis. In addition, Aurora-A and Aurora-B have been found to be overexpressed in a wide variety of human tumors. These observations led to a number of programs among academic and pharmaceutical organizations to discovering small molecule Aurora kinase inhibitors as anti-cancer drugs. This review will summarize the known Aurora kinase inhibitors currently in the clinic, and discuss the current and future directions. PMID:26734566

  4. Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function.

    PubMed

    Booth, Laurence; Shuch, Brian; Albers, Thomas; Roberts, Jane L; Tavallai, Mehrad; Proniuk, Stefan; Zukiwski, Alexander; Wang, Dasheng; Chen, Ching-Shih; Bottaro, Don; Ecroyd, Heath; Lebedyeva, Iryna O; Dent, Paul

    2016-03-15

    We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone - chaperone and chaperone - client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 -dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 -induced activation of ER stress signaling and maintained mTOR activity; AR-12 -mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types. PMID:26887051

  5. Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function

    PubMed Central

    Roberts, Jane L.; Tavallai, Mehrad; Proniuk, Stefan; Zukiwski, Alexander; Wang, Dasheng; Chen, Ching-Shih; Bottaro, Don; Ecroyd, Heath; Lebedyeva, Iryna O.; Dent, Paul

    2016-01-01

    We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone – chaperone and chaperone – client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 –dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 –induced activation of ER stress signaling and maintained mTOR activity; AR-12 –mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types. PMID:26887051

  6. Plantar injection of formalin in rats reduces the expression of a potassium chroride cotransporter KCC2 in the spinal cord and a kinase inhibitor suppresses this reduction.

    PubMed

    Tsuruga, Kenkichi; Hashimoto, Toshikazu; Kato, Ryoko; Kato, Rui; Uchida, Yousuke; Hase, Tetsutaro; Morimoto, Yuji

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) is released from activated microglia during neuropathic pain and is hypothesized to downregulate the expression of the potassium chloride cotransporter 2 (KCC2) via the TrkB receptor. Previous studies reported that KCC2 is downregulated 5 min after the plantar injection of formalin in rats; however, the mechanism behind this decrease in KCC2 expression during acute inflammatory pain remains unknown. In this study, we determined whether the TrkB receptor contributes to the expression of KCC2 during the acute pain. Five minutes after the plantar injection of formalin in rats, the ratio of KCC2-immunoreactive area in layer II of the spinal cord significantly decreased on the stimulated side compared to the unaffected side. On the other hand, this response was inhibited by the injection of a kinase inhibitor, K252a, in the subarachnoid space 15 min before the formalin injection. These findings suggest that in acute pain, the TrkB receptor may contribute to the decrease in the expression of KCC2. PMID:27545000

  7. Sphingosine kinase inhibitor suppresses IL-18-induced interferon-gamma production through inhibition of p38 MAPK activation in human NK cells

    SciTech Connect

    Cheon, Soyoung; Song, Seok Bean; Jung, Minkyung; Park, Yoorim; Bang, Jung-Wook; Kim, Tae Sung; Park, Hyunjeong; Kim, Cherl-hyun; Yang, Yool-hee; Bang, Sa Ik; Cho, Daeho

    2008-09-12

    Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-{gamma} inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-{gamma} production, we measured IL-18-induced IFN-{gamma} production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-{gamma} expression was blocked by SKI pre-treatment in both mRNA and protein levels. In addition, the increased IFN-{gamma} production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-{gamma} production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-{gamma} production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-{gamma} production via p38 MAPK.

  8. Down-regulation of the PTTG1 proto-oncogene contributes to the melanoma suppressive effects of the cyclin-dependent kinase inhibitor PHA-848125.

    PubMed

    Caporali, Simona; Alvino, Ester; Levati, Lauretta; Esposito, Alessia I; Ciomei, Marina; Brasca, Maria G; Del Bufalo, Donatella; Desideri, Marianna; Bonmassar, Enzo; Pfeffer, Ulrich; D'Atri, Stefania

    2012-09-01

    We previously demonstrated that PHA-848125, a cyclin-dependent kinase inhibitor presently under Phase II clinical investigation, impairs melanoma cell growth. In this study, gene expression profiling showed that PHA-848125 significantly modulated the expression of 128 genes, predominantly involved in cell cycle control, in the highly drug-sensitive GL-Mel (p53 wild-type) melanoma cells. Up-regulation of 4 selected genes (PDCD4, SESN2, DDIT4, DEPDC6), and down-regulation of 6 selected genes (PTTG1, CDC25A, AURKA, AURKB, PLK1, BIRC5) was confirmed at protein levels. The same protein analysis performed in PHA-848125-treated M10 melanoma cells - p53 mutated and less sensitive to the drug than GL-Mel cells - revealed no DEPDC6 expression and no changes of PTTG1, PDCD4 and BIRC5 levels. Upon PHA-848125 treatment, a marked PTTG1 down-modulation was also observed in A375 cells (p53 wild-type) but not in CN-Mel cells (p53 mutated). PTTG1 silencing significantly inhibited melanoma cell proliferation and induced senescence, with effects less pronounced in p53 mutated cells. PTTG1 silencing increased PHA-848125 sensitivity of p53 mutated cells but not that of A375 or GL-Mel cells. Accordingly, in M10 but not in A375 cells a higher level of senescence was detected in PHA-848125-treated/PTTG1-silenced cells with respect to PHA-848125-treated controls. In A375 and GL-Mel cells, TP53 silencing attenuated PHA-848125-induced down-modulation of PTTG1 and decreased cell sensitivity to the drug. These findings indicate that PHA-848125-induced down-regulation of PTTG1 depends, at least in part, on p53 function and contributes to the antiproliferative activity of the drug. Our study provides further molecular insight into the antitumor mechanism of PHA-848125. PMID:22704958

  9. KIDFamMap: a database of kinase-inhibitor-disease family maps for kinase inhibitor selectivity and binding mechanisms.

    PubMed

    Chiu, Yi-Yuan; Lin, Chih-Ta; Huang, Jhang-Wei; Hsu, Kai-Cheng; Tseng, Jen-Hu; You, Syuan-Ren; Yang, Jinn-Moon

    2013-01-01

    Kinases play central roles in signaling pathways and are promising therapeutic targets for many diseases. Designing selective kinase inhibitors is an emergent and challenging task, because kinases share an evolutionary conserved ATP-binding site. KIDFamMap (http://gemdock.life.nctu.edu.tw/KIDFamMap/) is the first database to explore kinase-inhibitor families (KIFs) and kinase-inhibitor-disease (KID) relationships for kinase inhibitor selectivity and mechanisms. This database includes 1208 KIFs, 962 KIDs, 55 603 kinase-inhibitor interactions (KIIs), 35 788 kinase inhibitors, 399 human protein kinases, 339 diseases and 638 disease allelic variants. Here, a KIF can be defined as follows: (i) the kinases in the KIF with significant sequence similarity, (ii) the inhibitors in the KIF with significant topology similarity and (iii) the KIIs in the KIF with significant interaction similarity. The KIIs within a KIF are often conserved on some consensus KIDFamMap anchors, which represent conserved interactions between the kinase subsites and consensus moieties of their inhibitors. Our experimental results reveal that the members of a KIF often possess similar inhibition profiles. The KIDFamMap anchors can reflect kinase conformations types, kinase functions and kinase inhibitor selectivity. We believe that KIDFamMap provides biological insights into kinase inhibitor selectivity and binding mechanisms. PMID:23193279

  10. KIDFamMap: a database of kinase-inhibitor-disease family maps for kinase inhibitor selectivity and binding mechanisms

    PubMed Central

    Chiu, Yi-Yuan; Lin, Chih-Ta; Huang, Jhang-Wei; Hsu, Kai-Cheng; Tseng, Jen-Hu; You, Syuan-Ren; Yang, Jinn-Moon

    2013-01-01

    Kinases play central roles in signaling pathways and are promising therapeutic targets for many diseases. Designing selective kinase inhibitors is an emergent and challenging task, because kinases share an evolutionary conserved ATP-binding site. KIDFamMap (http://gemdock.life.nctu.edu.tw/KIDFamMap/) is the first database to explore kinase-inhibitor families (KIFs) and kinase-inhibitor-disease (KID) relationships for kinase inhibitor selectivity and mechanisms. This database includes 1208 KIFs, 962 KIDs, 55 603 kinase-inhibitor interactions (KIIs), 35 788 kinase inhibitors, 399 human protein kinases, 339 diseases and 638 disease allelic variants. Here, a KIF can be defined as follows: (i) the kinases in the KIF with significant sequence similarity, (ii) the inhibitors in the KIF with significant topology similarity and (iii) the KIIs in the KIF with significant interaction similarity. The KIIs within a KIF are often conserved on some consensus KIDFamMap anchors, which represent conserved interactions between the kinase subsites and consensus moieties of their inhibitors. Our experimental results reveal that the members of a KIF often possess similar inhibition profiles. The KIDFamMap anchors can reflect kinase conformations types, kinase functions and kinase inhibitor selectivity. We believe that KIDFamMap provides biological insights into kinase inhibitor selectivity and binding mechanisms. PMID:23193279

  11. Comprehensive kinase profile of pacritinib, a nonmyelosuppressive Janus kinase 2 inhibitor

    PubMed Central

    Singer, Jack W; Al-Fayoumi, Suliman; Ma, Haiching; Komrokji, Rami S; Mesa, Ruben; Verstovsek, Srdan

    2016-01-01

    Pacritinib, potent inhibitor of Janus kinase 2 (JAK2), JAK2V617F, and fms-like receptor tyrosine kinase 3, is in Phase III development in myelofibrosis. Among type 1 inhibitors, pacritinib shows a lack of myelosuppression at doses that both inhibit JAK2/signal transducer and activator of transcription 3 pathway and demonstrate clinical efficacy. To elucidate these mechanisms and identify other disease targets, a kinome analysis screened 439 recombinant kinases at 100 nM pacritinib concentration. For kinases with >50% inhibition, pacritinib was titrated from 1 to 100 nM. JAK2, JAK2V617F, FLT3, colony-stimulating factor 1 receptor, and interleukin-1 receptor-associated kinase 1 achieved half-maximal inhibitory concentrations <50 nM. Pacritinib did not inhibit JAK1 (82% control at 100 nM). Lack of myelosuppression may stem from inhibiting JAK2 without affecting JAK1 and reducing hematopoietic inhibitory cytokines by suppressing interleukin-1 receptor-associated kinase 1 or colony-stimulating factor 1 receptor. The pacritinib kinome suggests therapeutic utility in acute myeloid leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, solid tumors, and inflammatory conditions. PMID:27574472

  12. Comprehensive kinase profile of pacritinib, a nonmyelosuppressive Janus kinase 2 inhibitor.

    PubMed

    Singer, Jack W; Al-Fayoumi, Suliman; Ma, Haiching; Komrokji, Rami S; Mesa, Ruben; Verstovsek, Srdan

    2016-01-01

    Pacritinib, potent inhibitor of Janus kinase 2 (JAK2), JAK2V617F, and fms-like receptor tyrosine kinase 3, is in Phase III development in myelofibrosis. Among type 1 inhibitors, pacritinib shows a lack of myelosuppression at doses that both inhibit JAK2/signal transducer and activator of transcription 3 pathway and demonstrate clinical efficacy. To elucidate these mechanisms and identify other disease targets, a kinome analysis screened 439 recombinant kinases at 100 nM pacritinib concentration. For kinases with >50% inhibition, pacritinib was titrated from 1 to 100 nM. JAK2, JAK2V617F, FLT3, colony-stimulating factor 1 receptor, and interleukin-1 receptor-associated kinase 1 achieved half-maximal inhibitory concentrations <50 nM. Pacritinib did not inhibit JAK1 (82% control at 100 nM). Lack of myelosuppression may stem from inhibiting JAK2 without affecting JAK1 and reducing hematopoietic inhibitory cytokines by suppressing interleukin-1 receptor-associated kinase 1 or colony-stimulating factor 1 receptor. The pacritinib kinome suggests therapeutic utility in acute myeloid leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, solid tumors, and inflammatory conditions. PMID:27574472

  13. High-Throughput Kinase Profiling: A More Efficient Approach towards the Discovery of New Kinase Inhibitors

    PubMed Central

    Miduturu, Chandrasekhar V.; Deng, Xianming; Kwiatkowski, Nicholas; Yang, Wannian; Brault, Laurent; Filippakopoulos, Panagis; Chung, Eunah; Yang, Qingkai; Schwaller, Juerg; Knapp, Stefan; King, Randall W.; Lee, Jiing-Dwan; Herrgard, Sanna; Zarrinkar, Patrick; Gray, Nathanael S.

    2011-01-01

    SUMMARY Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that “high-throughput kinase profiling” is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1/PLK1–3 and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2 and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors. PMID:21802008

  14. Janus kinase inhibitors for rheumatoid arthritis.

    PubMed

    Yamaoka, Kunihiro

    2016-06-01

    Treatment of autoimmune diseases, such as rheumatoid arthritis (RA), has advanced substantially over the past decade with the development of biologics targeting inflammatory cytokines. Recent progress in treating RA has been achieved with janus kinase (JAK) inhibitors (Jakinibs), an orally available disease-modifying anti-rheumatic drug targeting the intracellular kinase JAK and with similar efficacy to biologics. The first Jakinib approved for RA was tofacitinib, which exerted superiority to methotrexate and non-inferiority to tumor necrosis factor (TNF) inhibitors. In recent years, the Jakinib baricitinib has demonstrated superiority to both methotrexate and a TNF inhibitor, adalimumab. Given these promising findings, Jakinibs are expected to represent the next generation compounds for treating RA, and a number of Jakinibs are currently in clinical trials. Jakinibs can differ substantially in their selectivity against JAKs; tofacitinib and baricitinib target multiple JAKs, whereas the most recently developed Jakinibs target only a single JAK. The influence of Jakinib selectivity on efficacy and side effects is of great interest, requiring further careful observation. PMID:26994322

  15. The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors

    PubMed Central

    Nellore, Anoma; Liu, Bianling; Patsoukis, Nikolaos; Boussiotis, Vassiliki A.; Li, Lequn

    2014-01-01

    Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms’ tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity. PMID:24631965

  16. The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors.

    PubMed

    Nellore, Anoma; Liu, Bianling; Patsoukis, Nikolaos; Boussiotis, Vassiliki A; Li, Lequn

    2014-01-01

    Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms' tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity. PMID:24631965

  17. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase

    PubMed Central

    Deacon, Sean W.; Beeser, Alexander; Fukui, Jami A.; Rennefahrt, Ulrike E. E.; Myers, Cynthia; Chernoff, Jonathan; Peterson, Jeffrey R.

    2015-01-01

    SUMMARY Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation and identified the inhibitor IPA-3. Remarkably, pre-activated Pak1 is resistant to IPA-3. IPA-3 also inhibits activation of related Pak isoforms regulated by autoinhibition, but not more distantly related Paks, nor >200 other kinases tested. Pak1 inhibition by IPA-3 in live cells supports a critical role for Pak in PDGF-stimulated Erk activation. These studies illustrate a novel strategy for kinase inhibition and introduce a highly selective, cell-permeable chemical inhibitor of Pak. PMID:18420139

  18. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

    SciTech Connect

    Jin, Hyeon-Ok; Hong, Sung-Eun; Kim, Chang Soon; Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora; Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun; Park, In-Chul; Lee, Jin Kyung

    2015-08-15

    Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.

  19. Structural investigation of protein kinase C inhibitors

    NASA Technical Reports Server (NTRS)

    Barak, D.; Shibata, M.; Rein, R.

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  20. Tyrosine kinase inhibitors and the thyroid.

    PubMed

    Sherman, Steven I

    2009-12-01

    Protein tyrosine kinase inhibitors (TKIs) have emerged as significant targets for novel cancer therapies. For patients with differentiated or medullary carcinomas unresponsive to conventional treatments, multiple novel therapies primarily targeting angiogenesis have entered clinical trials. Partial response rates up to 30% have been reported in single-agent studies, but prolonged disease stabilisation is more commonly seen. The most successful agents target the vascular endothelial growth factor receptors. Sorafenib and sunitinib have had promising preliminary results reported and are being used selectively for patients who do not qualify for clinical trials. Treatment for patients with metastatic or advanced thyroid carcinoma now emphasises clinical trial opportunities for novel agents with considerable promise. Adverse effects on thyroid function and thyroid hormone metabolism have also been seen with several TKIs, necessitating prospective thyroid function testing for all patients starting therapy. PMID:19942148

  1. Discovery of a Potent And Selective Aurora Kinase Inhibitor

    SciTech Connect

    Oslob, J.D.; Romanowski, M.J.; Allen, D.A.; Baskaran, S.; Bui, M.; Elling, R.A.; Flanagan, W.M.; Fung, A.D.; Hanan, E.J.; Harris, S.; Heumann, S.A.; Hoch, U.; Jacobs, J.W.; Lam, J.; Lawrence, C.E.; McDowell, R.S.; Nannini, M.A.; Shen, W.; Silverman, J.A.; Sopko, M.M.; Tangonan, B.T.

    2009-05-21

    This communication describes the discovery of a novel series of Aurora kinase inhibitors. Key SAR and critical binding elements are discussed. Some of the more advanced analogues potently inhibit cellular proliferation and induce phenotypes consistent with Aurora kinase inhibition. In particular, compound 21 (SNS-314) is a potent and selective Aurora kinase inhibitor that exhibits significant activity in pre-clinical in vivo tumor models.

  2. Virtual Target Screening: Validation Using Kinase Inhibitors

    PubMed Central

    Santiago, Daniel N.; Pevzner, Yuri; Durand, Ashley A.; Tran, MinhPhuong; Scheerer, Rachel R.; Daniel, Kenyon; Sung, Shen-Shu; Woodcock, H. Lee; Guida, Wayne C.; Brooks, Wesley H.

    2012-01-01

    Computational methods involving virtual screening could potentially be employed to discover new biomolecular targets for an individual molecule of interest (MOI). However, existing scoring functions may not accurately differentiate proteins to which the MOI binds from a larger set of macromolecules in a protein structural database. An MOI will most likely have varying degrees of predicted binding affinities to many protein targets. However, correctly interpreting a docking score as a hit for the MOI docked to any individual protein can be problematic. In our method, which we term “Virtual Target Screening (VTS)”, a set of small drug-like molecules are docked against each structure in the protein library to produce benchmark statistics. This calibration provides a reference for each protein so that hits can be identified for an MOI. VTS can then be used as tool for: drug repositioning (repurposing), specificity and toxicity testing, identifying potential metabolites, probing protein structures for allosteric sites, and testing focused libraries (collection of MOIs with similar chemotypes) for selectivity. To validate our VTS method, twenty kinase inhibitors were docked to a collection of calibrated protein structures. Here we report our results where VTS predicted protein kinases as hits in preference to other proteins in our database. Concurrently, a graphical interface for VTS was developed. PMID:22747098

  3. The selectivity of protein kinase inhibitors: a further update

    PubMed Central

    Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C. James; Mclauchlan, Hilary; Klevernic, Iva; Arthur, J. Simon C.; Alessi, Dario R.; Cohen, Philip

    2007-01-01

    The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70–80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes. PMID:17850214

  4. Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

    PubMed Central

    Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.

    2016-01-01

    Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524

  5. In Vitro Characterization of Derrone as an Aurora Kinase Inhibitor.

    PubMed

    Hoang, Nhung Thi My; Phuong, Thuong Thien; Nguyen, Trang Thi Nhu; Tran, Yen Thi Hai; Nguyen, Anh Thi Ngoc; Nguyen, Thanh Lai; Bui, Khanh Thi Van

    2016-06-01

    Among mitotic kinases, Aurora kinases are the most widely studied, since their expression is restricted to mitosis. They play a key role in chromosome segregation and cell polyploidy. Aurora kinases are important therapeutic targets, and several research groups have directed their efforts toward the identification of kinase inhibitors. The aim of this study is to screen and characterize Aurora kinase inhibitors from natural substances extracted from plants that are used in the Vietnamese pharmacopoeia. We have characterized in vitro Derrone, extracted from Erythrina orientalis L. MURR, as a novel Aurora kinase inhibitor. This compound exhibited an ability to inhibit the phosphorylation of histone H3 at ser10 both in kinase assay and at the cellular level. The compound was more effective against Aurora kinase B, with a lower IC50 value as compared to Aurora A. Moreover, it impaired the mitotic spindle checkpoint and led to endoreduplication in cancer cells, a phenomenon caused by an Aurora B inhibitor. Interestingly, using the xCelligence system and real-time cell analysis (RTCA) software, we set up a comparison of cell proliferation profiles between cancer cells treated with Derrone and VX680-a well-known Aurora kinase inhibitor-and we found that these profiles exhibited considerable similarity in cell morphology, growth, and death. Additionally, Derrone significantly inhibited the formation and growth of MCF7 tumor spheroids. PMID:26983907

  6. Identification of beta-escin as a novel inhibitor of signal transducer and activator of transcription 3/Janus-activated kinase 2 signaling pathway that suppresses proliferation and induces apoptosis in human hepatocellular carcinoma cells.

    PubMed

    Tan, Sandra Min-Li; Li, Feng; Rajendran, Peramaiyan; Kumar, Alan Prem; Hui, Kam M; Sethi, Gautam

    2010-07-01

    The activation of signal transducer and activator of transcription 3 (STAT3) has been linked with the proliferation, survival, invasion, and angiogenesis of a variety of human cancer cells, including hepatocellular carcinoma (HCC). Agents that can suppress STAT3 activation have potential for the prevention and treatment of HCC. In this study, we tested an agent, beta-escin, for its ability to suppress STAT3 activation. We found that beta-escin, a pentacyclic triterpenoid, inhibited both constitutive and interleukin-6-inducible STAT3 activation in a dose- and time-dependent manner in HCC cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src, Janus-activated kinase 1, and Janus-activated kinase 2. Vanadate treatment reversed the beta-escin-induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that beta-escin induced the expression of tyrosine phosphatase Src homology phosphatase 1 that correlated with the down-regulation of constitutive STAT3 activation. beta-Escin also down-regulated the expression of STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1, and vascular endothelial growth factor. Finally, beta-escin inhibited proliferation and also substantially potentiated the apoptotic effects of paclitaxel and doxorubicin in HCC cells. Overall, these results suggest that beta-escin is a novel blocker of STAT3 activation that may have potential in the suppression of proliferation and chemosensitization in HCC. PMID:20378717

  7. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells.

    PubMed

    Jin, Hyeon-Ok; Hong, Sung-Eun; Kim, Chang Soon; Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora; Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun; Park, In-Chul; Lee, Jin Kyung

    2015-08-15

    Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H+ ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. PMID:25981168

  8. Therapeutic drug monitoring and tyrosine kinase inhibitors

    PubMed Central

    Herviou, Pauline; Thivat, Emilie; Richard, Damien; Roche, Lucie; Dohou, Joyce; Pouget, Mélanie; Eschalier, Alain; Durando, Xavier; Authier, Nicolas

    2016-01-01

    The therapeutic activity of drugs can be optimized by establishing an individualized dosage, based on the measurement of the drug concentration in the serum, particularly if the drugs are characterized by an inter-individual variation in pharmacokinetics that results in an under- or overexposure to treatment. In recent years, several tyrosine kinase inhibitors (TKIs) have been developed to block intracellular signaling pathways in tumor cells. These oral drugs are candidates for therapeutic drug monitoring (TDM) due to their high inter-individual variability for therapeutic and toxic effects. Following a literature search on PubMed, studies on TKIs and their pharmacokinetic characteristics, plasma quantification and inter-individual variability was studied. TDM is commonly used in various medical fields, including cardiology and psychiatry, but is not often applied in oncology. Plasma concentration monitoring has been thoroughly studied for imatinib, in order to evaluate the usefulness of TDM. The measurement of plasma concentration can be performed by various analytical techniques, with liquid chromatography-mass spectrometry being the reference method. This method is currently used to monitor the efficacy and tolerability of imatinib treatments. Although TDM is already being used for imatinib, additional studies are required in order to improve this practice with the inclusion of other TKIs. PMID:27446421

  9. Protein kinase inhibitors against malignant lymphoma

    PubMed Central

    D’Cruz, Osmond J; Uckun, Fatih M

    2013-01-01

    Introduction Tyrosine kinases (TKs) are intimately involved in multiple signal transduction pathways regulating survival, activation, proliferation and differentiation of lymphoid cells. Deregulation or overexpression of specific oncogenic TKs is implicated in maintaining the malignant phenotype in B-lineage lymphoid malignancies. Several novel targeted TK inhibitors (TKIs) have recently emerged as active in the treatment of relapsed or refractory B-cell lymphomas that inhibit critical signaling pathways, promote apoptotic mechanisms or modulate the tumor microenvironment. Areas covered In this review, the authors summarize the clinical outcomes of newer TKIs in various B-cell lymphomas from published and ongoing clinical studies and abstracts from major cancer and hematology conferences. Expert opinion Multiple clinical trials have demonstrated that robust antitumor activity can be obtained with TKIs directed toward specific oncogenic TKs that are genetically deregulated in various subtypes of B-cell lymphomas. Clinical success of targeting TKIs is dependent upon on identifying reliable molecular and clinical markers associated with select cohorts of patients. Further understanding of the signaling pathways should stimulate the identification of novel molecular targets and expand the development of new therapeutic options and individualized therapies. PMID:23496343

  10. Kinases inhibitors in lung cancer: From benchside to bedside.

    PubMed

    Singh, Pankaj Kumar; Singh, Harpreet; Silakari, Om

    2016-08-01

    Lung cancer still remains one of the major causes of cancer related mortality around the globe. Various different molecular targets have been discovered till date for targeting lung cancer. But not every new molecular target has a successfully designed inhibitor; moreover conventional chemotherapeutics have their own limitations such as toxicity and lack of selectivity. Thus, kinases still remain the most effective molecular target in lung cancer therapy. Also, once-shunned kinase inhibitors have recently acquired renewed interest after the development and approval of irreversible kinase inhibitors (such as afatinib) that form covalent bonds with cysteine (or other nucleophilic residues) in the ATP-binding pocket of the kinases. Irreversible kinase inhibitors have a number of potential advantages over conventional reversible kinase inhibitors including prolonged pharmacodynamics, suitability for rational design, high potency etc. This review reveals the current knowledge of all the chemical scaffolds, approved and/or investigational, utilized as inhibitors in lung cancer. It also explains the rationale of designing these along with possible interactions with their targets, biological data and possible problems associated with these inhibitors. PMID:27393082

  11. A new “angle” on kinase inhibitor design: Prioritizing amphosteric activity above kinase inhibition

    PubMed Central

    Meyerowitz, Justin G; Weiss, William A; Gustafson, W Clay

    2015-01-01

    The MYCN oncoprotein has remained an elusive target for decades. We recently reported a new class of kinase inhibitors designed to disrupt the conformation of Aurora kinase A enough to block its kinase-independent interaction with MYCN, resulting in potent degradation of MYCN. These studies provide proof-of-principle for a new method of targeting enzyme activity-independent functions of kinases and other enzymes. PMID:27308435

  12. In silico design of protein kinase inhibitors: successes and failures.

    PubMed

    Dubinina, Galina G; Chupryna, Oleksandr O; Platonov, Maxim O; Borisko, Petro O; Ostrovska, Galina V; Tolmachov, Andriy O; Shtil, Alexander A

    2007-03-01

    Protein kinases are among the most exploited targets in modern drug discovery due to key roles these enzymes play in human diseases including cancer. The in silico approach, an important part of rational design of protein kinase inhibitors, is founded on vast information about 3D structures of these enzymes. This review summarizes general structural features of the kinase inhibitors and the studies applied toward a large scale chemical database for virtual screening. Analyzed are the ways of validating the modern docking tools and their combinations with different scoring functions. In particular, we discuss the kinase flexibility as a reason for failures of the docking procedure. Finally, evidence is provided for the main patterns of kinase-inhibitor interactions and creation of the hinge-region-directed 2D filters. PMID:17348826

  13. [Side effect management of tyrosine kinase inhibitors in urology : Hypertension].

    PubMed

    Sikic, D; Meidenbauer, N; Lieb, V; Keck, B

    2016-07-01

    Tyrosine kinase inhibitors like sunitinib, sorafenib, pazopanib or axintinib are regarded the standard of care in the systemic therapy of metastatic renal cell carcinoma. However, the many side effects associated with this therapy pose challenges for the treating physician and the patient. This review offers an overview of the classification and the treatment of hypertension, which is one of the major side effects induced by all tyrosine kinase inhibitors, in order to improve treatment efficacy and patient compliance. PMID:27146871

  14. The Potential Role of Aurora Kinase Inhibitors in Haematological Malignancies

    PubMed Central

    Farag, Sherif S.

    2011-01-01

    Summary Aurora kinases play an important role in the control of the cell cycle and have been implicated in tumourigenesis in a number of cancers. Among the haematological malignancies, overexpression of Aurora kinases has been reported in acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, multiple myeloma, aggressive non-Hodgkin lymphoma and Hodgkin lymphoma. A large number of Aurora kinase inhibitors are currently in different stages of clinical development. In addition to varying in their selectivity for the different Aurora kinases, some also have activity directed at other cellular kinases involved in important molecular pathways in cancer cells. This review summarizes the biology of Aurora kinases and discusses why they may be good therapeutic targets in different haematological cancers. We describe preclinical data that has served as the rationale for investigating Aurora kinase inhibitors in different haematological malignancies, and summarize published results from early phase clinical trials. While the anti-tumour effects of Aurora kinase inhibitors appear promising, we highlight important issues for future clinical research and suggest that the optimal use of these inhibitors is likely to be in combination with cytotoxic agents already in use for the treatment of various haematological cancers. PMID:21980926

  15. Suppression of Coronavirus Replication by Cyclophilin Inhibitors

    PubMed Central

    Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

    2013-01-01

    Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS), feline infectious peritonitis (FIP), mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA), could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication. PMID:23698397

  16. Suppression of coronavirus replication by cyclophilin inhibitors.

    PubMed

    Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

    2013-05-01

    Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS), feline infectious peritonitis (FIP), mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA), could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication. PMID:23698397

  17. VEGF receptor kinase inhibitors: phthalazines, anthranilamides and related structures.

    PubMed

    Dumas, Jacques; Dixon, Julie A

    2005-06-01

    Inhibition of vascular endothelial growth factor receptor (VEGFR) signalling, using either antibodies or small molecule inhibitors of the VEGFR kinase domain, has become a major area of research in oncology. The phthalazine PTK787/ZK222584, first published in the literature in 1998, is one of the most advanced VEGFR inhibitors in the clinic. This paper provides an update on the patenting activity related to the phthalazine class. In addition, newer kinase inhibitor pharmacophores derived from this class (e.g., anthranilamides) will be reviewed. PMID:20141503

  18. The Aurora kinase inhibitors in cancer research and therapy.

    PubMed

    Cicenas, Jonas

    2016-09-01

    Compounds that affect enzymatic function of kinases are valuable for the understanding of the complex biochemical processes in cells. Aurora kinases (AURKs) play a key role in the control of the mitosis. These kinases are frequently deregulated in different human cancers: overexpression, amplifications, translocations and deletions were reported in many cancer cell lines as well as patient tissues. These findings steered a rigorous hunt for small-molecule AURK inhibitors not only for research purposes as well as for therapeutic uses. In this review, we describe a number of AURK inhibitors and their use in cancer research and/or therapy. We hope to assist researchers and clinicians in deciding which inhibitor is most appropriate for their specific purpose. The review will also provide a broad overview of the clinical studies performed with some of these inhibitors (if such studies have been performed). PMID:26932147

  19. Mini-review: bmx kinase inhibitors for cancer therapy.

    PubMed

    Jarboe, John S; Dutta, Shilpa; Velu, Sadanandan E; Willey, Christopher D

    2013-09-01

    Kinase inhibitors are among the fastest growing class of anti-cancer therapies. One family of kinases that has recently gained attention as a target for treating malignant disorders is the Tec kinase family. Evidence has been published that one member of this family; the Bmx kinase, may play a role in the pathogenesis of glioblastoma, prostate, breast and lung cancer. Bmx has also shown potential as an anti-vascular therapy in combination with radiation or as a sensitizer to chemotherapeutic agents. Therefore, several companies such as Pharmacyclics, Avila Therapeutics, Merck and Co., Metaproteomics, IRM, and Moerae Matrix have developed compounds or peptides that function as Bmx kinase inhibitors. These companies have subsequently been issued patents for these inhibitors. Additionally, it has been shown that current clinical stage EGFR inhibitors can irreversibly inhibit Bmx, suggesting these compounds might be rapidly moved to clinical trials for other malignancies. This review will discuss current patents issued since 2009 that contain data specifically on inhibition of the Bmx kinase, and will also discuss the scientific literature that suggests their potential application as therapeutics in the treatment of the aforementioned malignancies. PMID:23198769

  20. Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten

    2015-05-01

    REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.

  1. Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

    PubMed Central

    Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten

    2015-01-01

    REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ. PMID:25944708

  2. Allosteric Small-Molecule Inhibitors of the AKT Kinase

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    This research addresses computational design of small druglike molecules for possible anticancer applications. AKT and SGK are kinases that control important cellular functions. They are highly homologous, having similar activators and targets. Cancers with increased SGK activity may develop resistance to AKT-specific inhibitors. Our goal was to design new molecules that would bind both AKT and SGK, thus preventing the development of drug resistance. Most kinase inhibitors target the kinase ATP-binding site. However, the high similarity in this site among kinases makes it difficult to target specifically. Furthermore, mutations in this site can cause resistance to ATP-competitive kinase inhibitors. We used existing AKT inhibitors as initial templates to design molecules that could potentially bind the allosteric sites of both AKT and SGK. Molecules with no implicit toxicities and optimal drug-like properties were used for docking studies. Binding energies of the stable complexes that the designed molecules formed with AKT and SGK were calculated. Possible applications of the designed putative inhibitors against cancers with overexpressed AKT/SGK is discussed.

  3. LRRK2 and ubiquitination: implications for kinase inhibitor therapy

    PubMed Central

    Melrose, Heather L.

    2015-01-01

    Pathogenic mutations and risk variants in LRRK2 (leucine-rich repeat kinase 2) represent the most common genetic cause of familial and sporadic PD (Parkinson's disease). LRRK2 protein is widely expressed throughout the brain and the periphery. Structurally, LRRK2 contains several functional domains, including a dual enzymatic core consisting of a kinase and GTPase domain. Disease-linked variants are found in both these enzymatic domains as well as in the COR [C-terminal of ROC (Ras of complex proteins)] and WD40 protein–protein binding domain. The kinase domain is widely believed to be linked to toxicity, and thus the thrust of pharmaceutical effort has focused on developing LRRK2 kinase inhibitors. However, recent data have suggested that inhibition of LRRK2 activity results in reduced LRRK2 levels and peripheral side effects, which are similar to those observed in homozygous LRRK2-knockout and LRRK2 kinase-dead rodent models. In a recent issue of the Biochemical Journal, a study led by Nichols reveals that dephosphorylation of LRRK2 cellular phosphorylation sites (Ser910/Ser935/Ser955/Ser973) triggers its ubiquitination and subsequent degradation and thus may account for the loss of function phenotypes observed in peripheral tissues in LRRK2-knockout/kinase-dead or inhibitor-treated rodents and primates. Albeit negative from a kinase inhibitor standpoint, the data open new avenues for LRRK2 biology and therapeutic approaches to counteract LRRK2 toxicity. PMID:26341487

  4. Recent advances in the development of sphingosine kinase inhibitors.

    PubMed

    Pitman, Melissa R; Costabile, Maurizio; Pitson, Stuart M

    2016-09-01

    Sphingosine kinase (SK) 1 and 2 are lipid kinases that catalyse the formation of sphingosine 1-phosphate (S1P), a potent signalling molecule with a wide array of cellular effects. SK1 and 2 have been shown to be up-regulated in tumours and their genetic ablation or inhibition has been shown to slow tumour growth as well as sensitise cancer cells to chemotherapeutics. The SKs have been extensively studied, with a plethora of inhibitors developed that target the sphingosine-binding pocket of the enzyme, some with nanomolar affinities. Recently, inhibitors targeting the ATP pocket of SK have also been described. Here we discuss the development of these new small molecule SK inhibitors, summarise the recent discovery of off-targets effects of many current SK inhibitors, and provide an overview of the usefulness of these inhibitors as in vitro tools and therapeutic agents. PMID:27297359

  5. Targeting the TGF-β receptor with kinase inhibitors for scleroderma therapy.

    PubMed

    Cong, Lin; Xia, Zhi-Kuan; Yang, Rong-Ya

    2014-09-01

    Scleroderma (systemic sclerosis) is a connective tissue disease that affects various organ systems; the treatment of scleroderma is still difficult and remains a challenge to the clinician. Recently, kinase inhibitors have shown great potential against fibrotic diseases and, specifically, the transforming growth factor-β receptor (TGF-βR) was found as a new and promising target for scleroderma therapy. In the current study, we propose that the large pool of existing kinase inhibitors could be exploited for inhibiting the TGF-βR to suppress scleroderma. In this respect, we developed a modeling protocol to systematically profile the inhibitory activities of 169 commercially available kinase inhibitors against the TGF-βR, from which five promising candidates were selected and tested using a standard kinase assay protocol. Consequently, two molecular entities, namely the PKB inhibitor MK-2206 and the mTOR C1/C2 inhibitor AZD8055, showed high potency when bound to the TGF-βR, with IC50 values of 97 and 86 nM, respectively, which are close to those of the recently developed TGF-βR selective inhibitors SB525334 and LY2157299 (IC50 = 14.3 and 56 nM, respectively). We also performed atomistic molecular dynamics simulations and post-molecular mechanics/Poisson-Boltzmann surface area analyses to dissect the structural basis and energetic properties of intermolecular interactions between the TGF-βR kinase domain and these potent compounds, highlighting intensive nonbonded networks across the tightly packed interface of non-cognate TGF-βR-inhibitor complexes. PMID:24917246

  6. Aminofurazans as potent inhibitors of AKT kinase

    SciTech Connect

    Rouse, Meagan B.; Seefeld, Mark A.; Leber, Jack D.; McNulty, Kenneth C.; Sun, Lihui; Miller, William H.; Zhang, ShuYun; Minthorn, Elisabeth A.; Concha, Nestor O.; Choudhry, Anthony E.; Schaber, Michael D.; Heerding, Dirk A.

    2009-06-24

    AKT inhibitors containing an imidazopyridine aminofurazan scaffold have been optimized. We have previously disclosed identification of the AKT inhibitor GSK690693, which has been evaluated in clinical trials in cancer patients. Herein we describe recent efforts focusing on investigating a distinct region of this scaffold that have afforded compounds (30 and 32) with comparable activity profiles to that of GSK690693.

  7. Fragment-based design of kinase inhibitors: a practical guide.

    PubMed

    Erickson, Jon A

    2015-01-01

    Fragment-based drug design has become an important strategy for drug design and development over the last decade. It has been used with particular success in the development of kinase inhibitors, which are one of the most widely explored classes of drug targets today. The application of fragment-based methods to discovering and optimizing kinase inhibitors can be a complicated and daunting task; however, a general process has emerged that has been highly fruitful. Here a practical outline of the fragment process used in kinase inhibitor design and development is laid out with specific examples. A guide to the overall process from initial discovery through fragment screening, including the difficulties in detection, to the computational methods available for use in optimization of the discovered fragments is reported. PMID:25709040

  8. Phospho-kinase profile of colorectal tumors guides in the selection of multi-kinase inhibitors

    PubMed Central

    Montero, Juan Carlos; Corrales-Sanchez, Verónica; Morales, Jorge Carlos; Núñez, Luz-Elena; Morís, Francisco; Pandiella, Atanasio; Ocaña, Alberto

    2015-01-01

    Protein kinases play a central role in the oncogenesis of colorectal tumors and are attractive druggable targets. Detection of activated kinases within a tumor could open avenues for drug selection and optimization of new kinase inhibitors. By using a phosphokinase arrays with human colorectal tumors we identified activated kinases, including the Epidermal Growth Factor Receptor (EGFR), components of the PI3K/mTOR pathway (AKT and S6), and STAT, among others. A pharmacological screening with kinase inhibitors against these proteins helped us to identify a new kinase inhibitor, termed EC-70124 that showed the highest anti-proliferative activity in cell lines. EC-70124 also inhibited cell migration and biochemical experiments demonstrated its effect targeting the PI3K/mTOR pathway. This drug also arrested cells at G2/M and induced apoptosis. Experiments in combination with standard chemotherapy used in the clinical setting indicated a synergistic effect. EC-70124 also reduced tumor growth in vivo and inhibited pS6 in the implanted tumors. In conclusion, by studying the kinase profile of colorectal tumors, we identified relevant activated pathways, and a new multi-kinase compound with significant antitumor properties. PMID:26418718

  9. Phospho-kinase profile of colorectal tumors guides in the selection of multi-kinase inhibitors.

    PubMed

    Serrano-Heras, Gemma; Cuenca-López, María Dolores; Montero, Juan Carlos; Corrales-Sanchez, Verónica; Morales, Jorge Carlos; Núñez, Luz-Elena; Morís, Francisco; Pandiella, Atanasio; Ocaña, Alberto

    2015-10-13

    Protein kinases play a central role in the oncogenesis of colorectal tumors and are attractive druggable targets. Detection of activated kinases within a tumor could open avenues for drug selection and optimization of new kinase inhibitors. By using a phosphokinase arrays with human colorectal tumors we identified activated kinases, including the Epidermal Growth Factor Receptor (EGFR), components of the PI3K/mTOR pathway (AKT and S6), and STAT, among others. A pharmacological screening with kinase inhibitors against these proteins helped us to identify a new kinase inhibitor, termed EC-70124 that showed the highest anti-proliferative activity in cell lines. EC-70124 also inhibited cell migration and biochemical experiments demonstrated its effect targeting the PI3K/mTOR pathway. This drug also arrested cells at G2/M and induced apoptosis. Experiments in combination with standard chemotherapy used in the clinical setting indicated a synergistic effect. EC-70124 also reduced tumor growth in vivo and inhibited pS6 in the implanted tumors. In conclusion, by studying the kinase profile of colorectal tumors, we identified relevant activated pathways, and a new multi-kinase compound with significant antitumor properties. PMID:26418718

  10. Seeding collaborations to advance kinase science with the GSK Published Kinase Inhibitor Set (PKIS).

    PubMed

    Drewry, David H; Willson, Timothy M; Zuercher, William J

    2014-01-01

    To catalyze research on historically untargeted protein kinases, we created the PKIS, an annotated set of 367 small molecule kinase inhibitors. The set has been widely distributed to academic collaborators as an open access tool. It has been used to identify chemical starting points for development of chemical probes for orphan kinases and to investigate kinase signaling in high content phenotypic assays. Access to the set comes with few restrictions other than the requirement that assay results be released into the public domain for the benefit of the entire research community. Examples from the efforts of several collaborators are summarized. PMID:24283969

  11. Redundant kinase activation and resistance of EGFR-tyrosine kinase inhibitors

    PubMed Central

    Luo, Min; Fu, Li-Wu

    2014-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic effects against that tumors harboring EGFR activating mutations in the EGFR intracytoplasmic tyrosine kinase domain and resulted in cell apoptosis. Unfortunately, a number of patients ultimately developed resistance by multiple mechanisms. Thus, elucidation of the mechanism of resistance to EGFR-TKIs can provide strategies for blocking or reversing the situation. Recent studies suggested that redundant kinase activation plays pivotal roles in escaping from the effects of EGFR-TKIs. Herein, we aimed to characterize several molecular events involved in the resistance to EGFR-TKIs mediated by redundant kinase activation. PMID:25520855

  12. Cyclin dependent kinase (CDK) inhibitors as anticancer drugs.

    PubMed

    Sánchez-Martínez, Concepción; Gelbert, Lawrence M; Lallena, María José; de Dios, Alfonso

    2015-09-01

    Sustained proliferative capacity is a hallmark of cancer. In mammalian cells proliferation is controlled by the cell cycle, where cyclin-dependent kinases (CDKs) regulate critical checkpoints. CDK4 and CDK6 are considered highly validated anticancer drug targets due to their essential role regulating cell cycle progression at the G1 restriction point. This review provides an overview of recent advances on cyclin dependent kinase inhibitors in general with special emphasis on CDK4 and CDK6 inhibitors and compounds under clinical evaluation. Chemical structures, structure activity relationships, and relevant preclinical properties will be described. PMID:26115571

  13. The azaindole framework in the design of kinase inhibitors.

    PubMed

    Mérour, Jean-Yves; Buron, Frédéric; Plé, Karen; Bonnet, Pascal; Routier, Sylvain

    2014-01-01

    This review article illustrates the growing use of azaindole derivatives as kinase inhibitors and their contribution to drug discovery and innovation. The different protein kinases which have served as targets and the known molecules which have emerged from medicinal chemistry and Fragment-Based Drug Discovery (FBDD) programs are presented. The various synthetic routes used to access these compounds and the chemical pathways leading to their synthesis are also discussed. An analysis of their mode of binding based on X-ray crystallography data gives structural insights for the design of more potent and selective inhibitors. PMID:25460315

  14. Combination of PIM and JAK2 inhibitors synergistically suppresses cell proliferation and overcomes drug resistance of myeloproliferative neoplasms

    PubMed Central

    Greco, Rita; Li, Zhifang; Sun, Fangxian; Barberis, Claude; Tabart, Michel; Patel, Vinod; Schio, Laurent; Hurley, Raelene; Chen, Bo; Cheng, Hong; Lengauer, Christoph; Pollard, Jack; Watters, James; Garcia-Echeverria, Carlos; Wiederschain, Dmitri; Adrian, Francisco; Zhang, JingXin

    2014-01-01

    Inhibitors of JAK2 kinase are emerging as an important treatment modality for myeloproliferative neoplasms (MPN). However, similar to other kinase inhibitors, resistance to JAK2 inhibitors may eventually emerge through a variety of mechanisms. Effective drug combination is one way to enhance therapeutic efficacy and combat resistance against JAK2 inhibitors. To identify potential combination partners for JAK2 compounds in MPN cell lines, we performed pooled shRNA screen targeting 5,000 genes in the presence or absence of JAK2 blockade. One of the top hits identified was MYC, an oncogenic transcription factor that is difficult to inhibit directly, but could be targeted by modulation of upstream regulatory elements such as kinases. We demonstrate herein that PIM kinase inhibitors efficiently suppress MYC protein levels in MPN cell lines. Overexpression of MYC restores the viability of PIM inhibitor-treated cells, revealing causal relationship between MYC down-regulation and cell growth inhibition by PIM compounds. Combination of various PIM inhibitors with a JAK2 inhibitor results in significant synergistic growth inhibition of multiple MPN cancer cell lines and induction of apoptosis. Mechanistic studies revealed strong downregulation of phosphorylated forms of S6 and 4EBP1 by JAK2/PIM inhibitor combination treatment. Finally, such combination was effective in eradicating in vitro JAK2 inhibitor-resistant MPN clones, where MYC is consistently up-regulated. These findings demonstrate that simultaneous suppression of JAK2 and PIM kinase activity by small molecule inhibitors is more effective than either agent alone in suppressing MPN cell growth. Our data suggest that JAK2 and PIM combination might warrant further investigation for the treatment of JAK2-driven hematologic malignancies. PMID:24830942

  15. Cellular impedance assays for predictive preclinical drug screening of kinase inhibitor cardiovascular toxicity.

    PubMed

    Lamore, Sarah D; Kamendi, Harriet W; Scott, Clay W; Dragan, Yvonne P; Peters, Matthew F

    2013-10-01

    Cardiovascular (CV) toxicity is a leading contributor to drug attrition. Implementing earlier testing has successfully reduced human Ether-à-go-go-Related Gene-related arrhythmias. How- ever, analogous assays targeting functional CV effects remain elusive. Demand to address this gap is particularly acute for kinase inhibitors (KIs) that suffer frequent CV toxicity. The drug class also presents some particularly challenging requirements for assessing functional CV toxicity. Specifically, an assay must sense a downstream response that integrates diverse kinase signaling pathways. In addition, sufficient throughput is essential for handling inherent KI nonselectivity. A new opportunity has emerged with cellular impedance technology, which detects spontaneous beating cardiomyocytes. Impedance assays sense morphology changes downstream of cardiomyocyte contraction. To evaluate cardiomyocyte impedance assays for KI screening, we investigated two distinct KI classes where CV toxicity was discovered late and target risks remain unresolved. Microtubule-associated protein/microtubule affinity regulating kinase (MARK) inhibitors decrease blood pressure in dogs, whereas checkpoint kinase (Chk) inhibitors (AZD7762, SCH900776) exhibit dose-limiting CV toxicities in clinical trials. These in vivo effects manifested in vitro as cardiomyocyte beat cessation. MARK effects were deemed mechanism associated because beat inhibition potencies correlated with kinase inhibition, and gene knockdown and microtubule-targeting agents suppressed beating. MARK inhibitor impedance and kinase potencies aligned with rat blood pressure effects. Chk inhibitor effects were judged off-target because Chk and beat inhibition potencies did not correlate and knockdowns did not alter beating. Taken together, the data demonstrate that cardiomyocyte impedance assays can address three unmet needs-detecting KI functional cardiotoxicity in vitro, determining mechanism of action, and supporting safety structure

  16. Adenosine kinase inhibitors attenuate opiate withdrawal via adenosine receptor activation.

    PubMed

    Kaplan, G B; Coyle, T S

    1998-11-27

    Previous studies have demonstrated a role for adenosine in mediating opiate effects. This study examines the effects of indirect activation of adenosine receptors, via treatment with adenosine kinase inhibitors, on the expression of opiate withdrawal in mice. Mice receive chronic morphine treatment via implantation of subcutaneous morphine pellets (75 mg) for 72 h. Mice then receive parenteral treatment with adenosine kinase inhibitors, either 5'-amino-5'-deoxyadenosine (2, 5, 20, 40 mg/kg, intraperitoneal or i.p.) or iodotubericidin (1, 2, 5 mg/kg, i.p.), followed by naloxone injection and opiate withdrawal signs are measured over 20 min. Both adenosine kinase inhibitors significantly reduce the following opiate withdrawal signs in a dose-dependent manner compared to vehicle: withdrawal jumps, teeth chattering, forepaw tremors, and forepaw treads. Additionally, 5'-amino-5'-deoxyadenosine significantly reduces withdrawal-induced diarrhea and weight loss. Effects of 5'-amino-5'-deoxyadenosine (40 mg/kg) on opiate withdrawal signs appear to be mediated via adenosine receptor activation as they are reversed by pretreatment by adenosine receptor antagonist caffeine (20 mg, i.p.) but not by selective phosphodiesterase inhibitor Ro 20-1724 (10 mg/kg, i.p.). Adenosine receptor activation via adenosine kinase inhibitor treatment attenuates opiate withdrawal and these agents may be generally useful in the treatment of drug withdrawal syndromes. PMID:9865523

  17. Novel cinnoline-based inhibitors of LRRK2 kinase activity.

    PubMed

    Garofalo, Albert W; Adler, Marc; Aubele, Danielle L; Bowers, Simeon; Franzini, Maurizio; Goldbach, Erich; Lorentzen, Colin; Neitz, R Jeffrey; Probst, Gary D; Quinn, Kevin P; Santiago, Pam; Sham, Hing L; Tam, Danny; Truong, Anh P; Ye, Xiaocong M; Ren, Zhao

    2013-01-01

    Leucine rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of cinnoline-3-carboxamides that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays. These compounds are also shown to be potent inhibitors in a cellular assay and to have good to excellent CNS penetration. PMID:23219325

  18. Polo-like kinase inhibitors in hematologic malignancies.

    PubMed

    Talati, Chetasi; Griffiths, Elizabeth A; Wetzler, Meir; Wang, Eunice S

    2016-02-01

    Polo-like kinases (Plk) are key regulators of the cell cycle and multiple aspects of mitosis. Two agents that inhibit the Plk signaling pathway have shown promising activity in patients with hematologic malignancies and are currently in phase III trials. Volasertib is a Plk inhibitor under evaluation combined with low-dose cytarabine in older patients with acute myeloid leukemia (AML) ineligible for intensive induction therapy. Rigosertib, a dual inhibitor of the Plk and phosphatidylinositol 3-kinase pathways, is under investigation in patients with myelodysplastic syndrome (MDS) who have failed azacitidine or decitabine treatment. The prognosis for patients with AML, who are ineligible for intensive induction therapy, and for those with MDS refractory/relapsed after a hypomethylating agent, remains poor. Novel approaches, such as Plk inhibitors, are urgently needed for these patients. Here, we provide a comprehensive overview of the current state of development of Plk inhibitors for the treatment of hematologic malignancies. PMID:26597019

  19. Old Tyrosine Kinase Inhibitors and Newcomers in Gastrointestinal Cancer Treatment.

    PubMed

    Erika, Giordani; Federica, Zoratto; Martina, Strudel; Anselmo, Papa; Luigi, Rossi; Marina, Minozzi; Davide, Caruso; Eleonora, Zaccarelli; Monica, Verrico; Silverio, Tomao

    2016-01-01

    Gastrointestinal cancer treatment is based more on molecular biology that has provided increasing knowledge about cancer pathogenesis on which targeted therapy is being developed. Precisely, targeted therapy is defined as a "type of treatment that uses drugs, such as monoclonal antibodies or tyrosine kinase inhibitors, to identify and attack specific cancer cells". Nowadays, the United States Food and Drug Administration has approved many targeted therapies for gastrointestinal cancer treatment, as many are in various phases of development as well. In a previous review we discussed the main monoclonal antibodies used and studied in gastrointestinal cancer. In addition to monoclonal antibodies, tyrosine kinase inhibitors represent another class of targeted therapy and following the approval of imatinib for gastrointestinal stromal tumours, other tyrosine kinase inhibitors have been approved for gastrointestinal cancers treatment such as sunitinib, regoragenib, sorafenib and erlotinib. Moving forward, the purpose of this review is to focus on the efficacy data of main tyrosine kinase inhibitors commonly used in the personalized treatment of each gastrointestinal tumour and to provide a comprehensive overview about experimental targeted therapies ongoing in this setting. PMID:26278713

  20. Discovery and Optimization of Quinazolinone-pyrrolopyrrolones as Potent and Orally Bioavailable Pan-Pim Kinase Inhibitors.

    PubMed

    Pettus, Liping H; Andrews, Kristin L; Booker, Shon K; Chen, Jie; Cee, Victor J; Chavez, Frank; Chen, Yuping; Eastwood, Heather; Guerrero, Nadia; Herberich, Bradley; Hickman, Dean; Lanman, Brian A; Laszlo, Jimmy; Lee, Matthew R; Lipford, J Russell; Mattson, Bethany; Mohr, Christopher; Nguyen, Yen; Norman, Mark H; Powers, David; Reed, Anthony B; Rex, Karen; Sastri, Christine; Tamayo, Nuria; Wang, Paul; Winston, Jeffrey T; Wu, Bin; Wu, Tian; Wurz, Ryan P; Xu, Yang; Zhou, Yihong; Tasker, Andrew S; Wang, Hui-Ling

    2016-07-14

    The high expression of proviral insertion site of Moloney murine leukemia virus kinases (Pim-1, -2, and -3) in cancers, particularly the hematopoietic malignancies, is believed to play a role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms of Pim protein appear largely redundant in their oncogenic functions. Thus, a pan-Pim kinase inhibitor is highly desirable. However, cell active pan-Pim inhibitors have proven difficult to develop because Pim-2 has a low Km for ATP and therefore requires a very potent inhibitor to effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors. In particular, compound 17 is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing. PMID:27285051

  1. Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials

    PubMed Central

    Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

    2015-01-01

    Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

  2. Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials.

    PubMed

    Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

    2015-01-01

    Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

  3. Prolonged and tunable residence time using reversible covalent kinase inhibitors

    PubMed Central

    Bradshaw, J. Michael; McFarland, Jesse M.; Paavilainen, Ville O.; Bisconte, Angelina; Tam, Danny; Phan, Vernon T.; Romanov, Sergei; Finkle, David; Shu, Jin; Patel, Vaishali; Ton, Tony; Li, Xiaoyan; Loughhead, David G.; Nunn, Philip A.; Karr, Dane E.; Gerritsen, Mary E.; Funk, Jens Oliver; Owens, Timothy D.; Verner, Erik; Brameld, Ken A.; Hill, Ronald J.; Goldstein, David M.; Taunton, Jack

    2015-01-01

    Drugs with prolonged, on-target residence time often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here, we demonstrate progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Utilizing an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrate biochemical residence times spanning from minutes to 7 days. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK more than 18 hours after clearance from the circulation. The inverted cyanoacrylamide strategy was further utilized to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating generalizability of the approach. Targeting noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates “residence time by design”, the ability to modulate and improve the duration of target engagement in vivo. PMID:26006010

  4. Design and synthesis of novel selective anaplastic lymphoma kinase inhibitors.

    PubMed

    Michellys, Pierre-Yves; Chen, Bei; Jiang, Tao; Jin, Yunho; Lu, Wenshuo; Marsilje, Thomas H; Pei, Wei; Uno, Tetsuo; Zhu, Xuefeng; Wu, Baogen; Nguyen, Truc Ngoc; Bursulaya, Badry; Lee, Christian; Li, Nanxin; Kim, Sungjoon; Tuntland, Tove; Liu, Bo; Sun, Frank; Steffy, Auzon; Hood, Tami

    2016-02-01

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Expression of ALK in normal human tissues is only found in a subset of neural cells, however it is involved in the genesis of several cancers through genetic aberrations involving translocation of the kinase domain with multiple fusion partners (e.g., NPM-ALK in anaplastic large cell lymphoma ALCL or EML4-ALK in non-small cell lung cancer) or activating mutations in the full-length receptor resulting in ligand-independent constitutive activation (e.g., neuroblastoma). Here we are reporting the discovery of novel and selective anaplastic lymphoma kinase inhibitors from specific modifications of the 2,4-diaminopyridine core present in TAE684 and LDK378. Synthesis, structure activity relationships (SAR), absorption, distribution, metabolism, and excretion (ADME) profile, and in vivo efficacy in a mouse xenograft model of anaplastic large cell lymphoma are described. PMID:26750252

  5. Clinical experience with aurora kinase inhibitors: a review.

    PubMed

    Boss, David S; Beijnen, Jos H; Schellens, Jan H M

    2009-08-01

    The aurora kinase family of serine/threonine kinases comprises three members, designated auroras A, B, and C. Auroras A and B are essential components of the mitotic pathway, ensuring proper chromosome assembly, formation of the mitotic spindle, and cytokinesis. The role of aurora C is less clear. Overexpression of aurora A and B has been observed in several tumor types, and has been linked with a poor prognosis of cancer patients. Several small molecules targeting aurora kinases A and B or both have been evaluated preclinically and in early phase I trials. In this review we aim to summarize the most recent advances in the development of aurora kinase inhibitors, with a focus on the clinical data. PMID:19684075

  6. Predictive Models for Fast and Effective Profiling of Kinase Inhibitors.

    PubMed

    Bora, Alina; Avram, Sorin; Ciucanu, Ionel; Raica, Marius; Avram, Stefana

    2016-05-23

    In this study we developed two-dimensional pharmacophore-based random forest models for the effective profiling of kinase inhibitors. One hundred seven prediction models were developed to address distinct kinases spanning over all kinase groups. Rigorous external validation demonstrates excellent virtual screening and classification potential of the predictors and, more importantly, the capacity to prioritize novel chemical scaffolds in large chemical libraries. The models built upon more diverse and more potent compounds tend to exert the highest predictive power. The analysis of ColBioS-FlavRC (Collection of Bioselective Flavonoids and Related Compounds) highlighted several potentially promiscuous derivatives with undesirable selectivity against kinases. The prediction models can be downloaded from www.chembioinf.ro . PMID:27064988

  7. Bosutinib: a novel second-generation tyrosine kinase inhibitor.

    PubMed

    Isfort, Susanne; Keller-v Amsberg, Gunhild; Schafhausen, Philippe; Koschmieder, Steffen; Brümmendorf, Tim H

    2014-01-01

    Bosutinib (SKI-606) is a 4-anilino-3-quinoline carbonitrile, which acts as a dual inhibitor of Src and ABL kinases. In addition, the BCR-ABL fusion gene product, a constitutively activated tyrosine kinase which is crucial for the development of chronic myeloid leukemia (CML), is highly sensitive to bosutinib. Interestingly, distinctly lower concentrations of bosutinib are required to ablate BCR-ABL phosphorylation when compared to the first-generation tyrosine kinase inhibitor imatinib (IM). Bosutinib is a potent inhibitor of CML cell proliferation in vitro and has demonstrated promising activity in CML patients resistant or intolerant to IM as well as in newly diagnosed patients with chronic phase CML (CML-CP). Remarkably, bosutinib has been found to be capable of overcoming the majority of IM-resistant BCR-ABL mutations. Bosutinib has the potency to induce deep and fast responses in second- and third-/fourth-line treatment, and as a consequence, the drug has recently been licensed for patients previously treated with one or more tyrosine kinase inhibitor(s) and for whom imatinib, nilotinib, and dasatinib are not considered appropriate treatment options. Due to its potency and differing toxicity profile, it promises to be a good therapeutic option for a defined cohort of patients. The most common side effects are gastrointestinal with most of the patients suffering from nausea, vomiting, or diarrhea. For the most part, these gastrointestinal symptoms occur early after treatment initiation, are manageable, and often self-limiting. Continuous monitoring of liver enzymes upon treatment initiation is necessary during bosutinib treatment. In addition to CML treatment, bosutinib has shown some efficacy in selected patients suffering from advanced-stage solid tumors. In conclusion, bosutinib is a promising novel small molecule inhibitor approved now for targeted therapy of CML and in clinical development for other malignancies. PMID:24756786

  8. Inhibitors of cyclooxygenase-2 (COX-2) suppressed the proliferation and differentiation of human leukaemia cell lines.

    PubMed

    Nakanishi, Y; Kamijo, R; Takizawa, K; Hatori, M; Nagumo, M

    2001-08-01

    Prostaglandins (PG) are known to play important roles in the proliferation and differentiation of leukaemia cells. The effect of the inhibitors of cyclooxygenase-2 (COX-2), a rate-limiting enzyme for the synthesis of PG, on the proliferation and differentiation of leukaemia cell lines was investigated. COX-2 inhibitors, NS-398 and nabumetone, suppressed the proliferation of U-937 and ML-1 cells by inducing a G0/G1 cell-cycle arrest. Cell-cycle arrest induced by these COX-2 inhibitors was not associated with an upregulation of the cyclin-dependent kinase inhibitors. COX-2 inhibitors also inhibited the differentiation of these cells induced by interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and retinoic acid (RA). Treatment with NS-398 did not suppress the levels of PGs produced by these cells. Although COX-2 antisense oligonucleotide showed a similar inhibitory effect on these cells, its inhibitory effect was smaller than that of NS-398. These results suggest that COX-2 inhibitors may suppress the proliferation and differentiation of leukaemia cells both via COX-2-dependent and -independent pathways. PMID:11506967

  9. The novel combination of dual mTOR inhibitor AZD2014 and pan-PIM inhibitor AZD1208 inhibits growth in acute myeloid leukemia via HSF pathway suppression

    PubMed Central

    Yamamoto, Shinichi; Kaur, Surinder; Arslan, Dirim; Ramirez, Santiago; Jacamo, Rodrigo; Platanias, Leonidas; Matsushita, Hiromichi; Fujimura, Tsutomu; Kazuno, Saiko; Kojima, Kensuke; Tabe, Yoko; Konopleva, Marina

    2015-01-01

    Mammalian target of rapamycin (mTOR) signaling is a critical pathway in the biology of acute myeloid leukemia (AML). Proviral integration site for moloney murine leukemia virus (PIM) serine/threonine kinase signaling takes part in various pathways exerting tumorigenic properties. We hypothesized that the combination of a PIM kinase inhibitor with an mTOR inhibitor might have complementary growth-inhibitory effects against AML. The simultaneous inhibition of the PIM kinase by pan-PIM inhibitor AZD1208 and of mTOR by selective mTORC1/2 dual inhibitor AZD2014 exerted anticancer properties in AML cell lines and in cells derived from primary AML samples with or without supportive stromal cell co-culture, leading to suppressed proliferation and increased apoptosis. The combination of AZD1208 and AZD2014 rapidly activated AMPKα, a negative regulator of translation machinery through mTORC1/2 signaling in AML cells; profoundly inhibited AKT and 4EBP1 activation; and suppressed polysome formation. Inhibition of both mTOR and PIM counteracted induction of heat-shock family proteins, uncovering the master negative regulation of heat shock factor 1 (HSF1), the dominant transcription factor controlling cellular stress responses. The novel combination of the dual mTOR inhibitor and pan-PIM inhibitor synergistically inhibited AML growth by effectively reducing protein synthesis through heat shock factor pathway suppression. PMID:26473447

  10. Luteolin Suppresses Cancer Cell Proliferation by Targeting Vaccinia-Related Kinase 1

    PubMed Central

    Shin, Joon; Harikishore, Amaravadhi; Lim, Jong-Kwan; Jung, Youngseob; Lyu, Ha-Na; Baek, Nam-In; Choi, Kwan Yong; Yoon, Ho Sup; Kim, Kyong-Tai

    2014-01-01

    Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1) is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF), histone H3, and the cAMP response element (CRE)-binding protein (CREB). In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy. PMID:25310002

  11. A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions.

    PubMed

    Szyniarowski, Piotr; Corcelle-Termeau, Elisabeth; Farkas, Thomas; Høyer-Hansen, Maria; Nylandsted, Jesper; Kallunki, Tuula; Jäättelä, Marja

    2011-08-01

    Macroautophagy is a catabolic process that maintains cellular homeostasis and protects cells against various external stresses including starvation. Except for the identification of the Akt-mTORC1 pathway as a major negative regulator, little is known about signaling networks that control macroautophagy under optimal growth conditions. Therefore, we screened a human kinome siRNA library for siRNAs that increase the number of autophagosomes in normally growing MCF-7 human breast carcinoma cells, and identified 10 kinases as regulators of constitutive macroautophagy. Further analysis of these kinases with respect to the autophagic flux, kinase signaling and endolysosomal function identified WNK2 as a positive regulator of autophagosome maturation and nine others as macroautophagy inhibitors. The depletion of MK2, PACSIN1, DAPK2, CDKL3 and SCYL1 functioned upstream of Akt-mTORC1 pathway, whereas CSNK1A1, BUB1, PKLR and NEK4 suppressed autophagosome formation downstream or independent of mTORC1. Importantly, all identified kinases except for BUB1 regulated macroautophagy also in immortalized MCF-10A breast epithelial cells. The kinases identified here shed light to the complex regulation of macroautophagy and open new possibilities for its pharmacological manipulation. PMID:21508686

  12. Indolinones as promising scaffold as kinase inhibitors: a review.

    PubMed

    Prakash, C R; Raja, S

    2012-02-01

    Kinases are probably the most important signaling enzymes, which represent about 20% of the druggable genome. Currently, more than 150 kinases are known. So, kinase inhibition therapy has become a very important area of drug research since most of our diseases are related to intra or intercellular signaling by kinases. Indole alkaloids are extensively studied for their biological activities in several pharmaceutical areas, including, for example, antitumor. Among this chemical family, indolinone displays very promising antitumor properties by inhibiting various kinase families. These small molecules have a low molecular weight and most of them bind to protein kinases competing with ATP for the ATP-binding site. This review focuses on the indolinone based drugs approved for the treatment of cancer, drugs under clinical trial and then chemical diversity of various synthetic analogues of indolinone and their metabolites as various kinase inhibitors. This review also focused on structural activity relationship (SAR), mechanisms of action and biological targets through which indolinone and its derivatives display their antitumor activity. PMID:22372601

  13. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2006-05-30

    Compounds of formula 1: ##STR00001## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0 3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  14. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2000-01-01

    Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  15. Combinations of Kinase Inhibitors Protecting Myoblasts against Hypoxia

    PubMed Central

    Kang, Yunyi; Tierney, Matthew; Ong, Edison; Zhang, Linda; Piermarocchi, Carlo; Sacco, Alessandra; Paternostro, Giovanni

    2015-01-01

    Cell-based therapies to treat skeletal muscle disease are limited by the poor survival of donor myoblasts, due in part to acute hypoxic stress. After confirming that the microenvironment of transplanted myoblasts is hypoxic, we screened a kinase inhibitor library in vitro and identified five kinase inhibitors that protected myoblasts from cell death or growth arrest in hypoxic conditions. A systematic, combinatorial study of these compounds further improved myoblast viability, showing both synergistic and additive effects. Pathway and target analysis revealed CDK5, CDK2, CDC2, WEE1, and GSK3β as the main target kinases. In particular, CDK5 was the center of the target kinase network. Using our recently developed statistical method based on elastic net regression we computationally validated the key role of CDK5 in cell protection against hypoxia. This method provided a list of potential kinase targets with a quantitative measure of their optimal amount of relative inhibition. A modified version of the method was also able to predict the effect of combinations using single-drug response data. This work is the first step towards a broadly applicable system-level strategy for the pharmacology of hypoxic damage. PMID:26042811

  16. (-)-β-hydrastine suppresses the proliferation and invasion of human lung adenocarcinoma cells by inhibiting PAK4 kinase activity.

    PubMed

    Guo, Bingyu; Li, Xiaodong; Song, Shuai; Chen, Meng; Cheng, Maosheng; Zhao, Dongmei; Li, Feng

    2016-04-01

    (-)-β-hydrastine is one of the main active components of the medicinal plant, Hydrastis canadensis, which is used in many dietary supplements intended to enhance the immune system. However, whether (-)-β-hydrastine affects the tumor signaling pathway remains unexplored. In the present study, we found that (-)-β-hydrastine inhibited the kinase activity of p21-activated kinase 4 (PAK4), which is involved in the regulation of cytoskeletal reorganization, cell proliferation, gene transcription, oncogenic transformation and cell invasion. In the present study, (-)-β-hydrastine suppressed lung adenocarcinoma cell proliferation by inhibiting expression of cyclin D1/D3 and CDK2/4/6, leading to cell cycle arrest at the G1 phase, in a PAK4 kinase-dependent manner. Moreover, inhibition of PAK4 kinase activity by (-)-β-hydrastine also promoted the early apoptosis of lung adenocarcinoma cells through the mitochondrial apoptosis pathway. In addition, (-)-β-hydrastine significantly suppressed the migration and invasion of human lung adenocarcinoma cells in conjunction with concomitant blockage of the PAK4/LIMK1/cofilin, PAK4/SCG10 and PAK4/MMP2 pathways. All of these data indicate that (-)-β-hydrastine, as a novel PAK4 inhibitor, suppresses the proliferation and invasion of lung adenocarcinoma cells. Taken together, these results provide novel insight into the development of a PAK4 kinase inhibitor and a potential therapeutic strategy for lung cancer. PMID:26821251

  17. Quercetin: a pleiotropic kinase inhibitor against cancer.

    PubMed

    Russo, Gian Luigi; Russo, Maria; Spagnuolo, Carmela; Tedesco, Idolo; Bilotto, Stefania; Iannitti, Roberta; Palumbo, Rosanna

    2014-01-01

    Increased consumption of fruits and vegetables can represent an easy strategy to significantly reduce the incidence of cancer. From this observation, derived mostly from epidemiological data, the new field of chemoprevention has emerged in the primary and secondary prevention of cancer. Chemoprevention is defined as the use of natural or synthetic compounds able to stop, reverse, or delay the process of tumorigenesis in its early stages. A large number of phytochemicals are potentially capable of simultaneously inhibiting and modulating several key factors regulating cell proliferation in cancer cells. Quercetin is a flavonoid possessing potential chemopreventive properties. It is a functionally pleiotropic molecule, possessing multiple intracellular targets, affecting different cell signaling processes usually altered in cancer cells, with limited toxicity on normal cells. Simultaneously targeting multiple pathways may help to kill malignant cells and slow down the onset of drug resistance. Among the different substrates triggered by quercetin, we have reviewed the ability of the molecule to inhibit protein kinases involved in deregulated cell growth in cancer cells. PMID:24114481

  18. Rho-associated kinase inhibitors: a novel glaucoma therapy.

    PubMed

    Inoue, Toshihiro; Tanihara, Hidenobu

    2013-11-01

    The rho-associated kinase (ROCK) signaling pathway is activated via secreted bioactive molecules or via integrin activation after extracellular matrix binding. These lead to polymerization of actin stress fibers and formation of focal adhesions. Accumulating evidence suggests that actin cytoskeleton-modulating signals are involved in aqueous outflow regulation. Aqueous humor contains various biologically active factors, some of which are elevated in glaucomatous eyes. These factors affect aqueous outflow, in part, through ROCK signaling modulation. Various drugs acting on the cytoskeleton have also been shown to increase aqueous outflow by acting directly on outflow tissue. In vivo animal studies have shown that the trabecular meshwork (TM) actin cytoskeleton in glaucomatous eyes is more disorganized and more randomly oriented than in non-glaucomatous control eyes. In a previous study, we introduced ROCK inhibitors as a potential glaucoma therapy by showing that a selective ROCK inhibitor significantly lowered rabbit IOP. Rho-associated kinase inhibitors directly affect the TM and Schlemm's canal (SC), differing from the target sight of other glaucoma drugs. The TM is affected earlier and more strongly than ciliary muscle cells by ROCK inhibitors, largely because of pharmacological affinity differences stemming from regulatory mechanisms. Additionally, ROCK inhibitors disrupt tight junctions, result in F-actin depolymerization, and modulate intracellular calcium level, effectively increasing SC-cell monolayer permeability. Perfusion of an enucleated eye with a ROCK inhibitor resulted in wider empty spaces in the juxtacanalicular (JCT) area and more giant vacuoles in the endothelial cells of SC, while the endothelial lining of SC was intact. Interestingly, ROCK inhibitors also increase retinal blood flow by relaxing vascular smooth muscle cells, directly protecting neurons against various stresses, while promoting wound healing. These additional effects may help

  19. The STAT5 inhibitor pimozide decreases survival of chronic myelogenous leukemia cells resistant to kinase inhibitors

    PubMed Central

    Nelson, Erik A.; Walker, Sarah R.; Weisberg, Ellen; Bar-Natan, Michal; Barrett, Rosemary; Gashin, Laurie B.; Terrell, Shariya; Klitgaard, Josephine L.; Santo, Loredana; Addorio, Martha R.; Ebert, Benjamin L.; Griffin, James D.

    2011-01-01

    The transcription factor STAT5 is an essential mediator of the pathogenesis of chronic myelogenous leukemia (CML). In CML, the BCR/ABL fusion kinase causes the constitutive activation of STAT5, thereby driving the expression of genes promoting survival. BCR/ABL kinase inhibitors have become the mainstay of therapy for CML, although CML cells can develop resistance through mutations in BCR/ABL. To overcome this problem, we used a cell-based screen to identify drugs that inhibit STAT-dependent gene expression. Using this approach, we identified the psychotropic drug pimozide as a STAT5 inhibitor. Pimozide decreases STAT5 tyrosine phosphorylation, although it does not inhibit BCR/ABL or other tyrosine kinases. Furthermore, pimozide decreases the expression of STAT5 target genes and induces cell cycle arrest and apoptosis in CML cell lines. Pimozide also selectively inhibits colony formation of CD34+ bone marrow cells from CML patients. Importantly, pimozide induces similar effects in the presence of the T315I BCR/ABL mutation that renders the kinase resistant to presently available inhibitors. Simultaneously inhibiting STAT5 with pimozide and the kinase inhibitors imatinib or nilotinib shows enhanced effects in inhibiting STAT5 phosphorylation and in inducing apoptosis. Thus, targeting STAT5 may be an effective strategy for the treatment of CML and other myeloproliferative diseases. PMID:21233313

  20. Targeting Angiogenesis in Colorectal Cancer: Tyrosine Kinase Inhibitors.

    PubMed

    Kircher, Sheetal Mehta; Nimeiri, Halla S; Benson, Al B

    2016-01-01

    Colorectal cancer is commonly diagnosed throughout the world, and treatment options have greatly expanded over the last 2 decades. Targeting angiogenesis has been a major focus of study in a variety of malignancy types. Targeting angiogenesis has been achieved by several mechanisms in colorectal cancer, including use of antiangiogenic small molecule tyrosine kinase inhibitors (TKIs). There have been many attempts and failures to prove efficacy of TKIs in the treatment of colorectal cancer including sorafenib, sunitinib, vatalanib, and tivozanib. Regorafenib was the first TKI to demonstrate efficacy and is an orally active inhibitor of angiogenic (including the vascular endothelial growth factor receptors 1, 2, and 3), stromal, and oncogenic receptor tyrosine kinases. There are ongoing investigations of both regorafenib and ninetanib; however, there remains a critical need to better understand novel combinations with TKIs that could prove more efficacious than available options. PMID:27341596

  1. Endocrine side effects of broad-acting kinase inhibitors

    PubMed Central

    Lodish, Maya B.; Stratakis, Constantine A.

    2011-01-01

    Targeted therapy in oncology consists of drugs that specifically interfere with abnormal signaling pathways that are dysregulated in cancer cells. Tyrosine kinase inhibitors (TKIs) take advantage of unique oncogenes that are activated in certain types of cancer, and also target common mechanisms of growth, invasion, metastasis, and angiogenesis. However, many kinase inhibitors for cancer therapy are somewhat nonselective, and most have additional mechanisms of action at the cellular level which are not completely understood. The use of these agents has increased our knowledge of important side effects, of which the practicing clinician must be aware. Recently proposed endocrine-related side effects of these agents include alterations in thyroid function, bone metabolism, linear growth, gonadal function, fetal development, and glucose metabolism, and adrenal function. This review summarizes the most recent data on the endocrine side effects of TKIs. PMID:20603395

  2. A protein kinaseinhibitor attenuates multidrug resistance of neuroblastoma cells

    PubMed Central

    Svensson, Karin; Larsson, Christer

    2003-01-01

    Background The acquisition of drug resistance is a major reason for poor outcome of neuroblastoma. Protein kinase C (PKC) has been suggested to influence drug resistance in cancer cells. The aim of this study was to elucidate whether inhibition of PKCβ isoforms influences drug-resistance of neuroblastoma cells. Methods The effect of the PKCβ inhibitor LY379196 on the growth-suppressing effects of different chemotherapeutics on neuroblastoma cells was analyzed with MTT assays. The effect of LY379196 on the accumulation of [3H]vincristine was also investigated Results The PKCβ inhibitor LY379196 suppressed the growth of three neuroblastoma cell lines. LY379196 also augmented the growth-suppressive effect of doxorubicin, etoposide, paclitaxel, and vincristine, but not of carboplatin. The effect was most marked for vincristine and for the cell-line (SK-N-BE(2)) that was least sensitive to vincristine. No effect was observed on the non-resistant IMR-32 cells. Two other PKC inhibitors, Gö6976 and GF109203X, also enhanced the vincristine effect. The PKC inhibitors caused an increased accumulation of [3H]vincristine in SK-N-BE(2) cells. Conclusions This indicates that inhibition of PKCβ could attenuate multidrug resistance in neuroblastoma cells by augmenting the levels of natural product anticancer drugs in resistant cells. PMID:12697075

  3. Tyrosine Kinase Inhibitors and Diabetes: A Novel Treatment Paradigm?

    PubMed

    Fountas, Athanasios; Diamantopoulos, Leonidas-Nikolaos; Tsatsoulis, Agathocles

    2015-11-01

    Deregulation of protein tyrosine kinase (PTK) activity is implicated in various proliferative conditions. Multi-target tyrosine kinase inhibitors (TKIs) are increasingly used for the treatment of different malignancies. Recently, several clinical cases of the reversal of both type 1 and 2 diabetes mellitus (T1DM, T2DM) during TKI administration have been reported. Experimental in vivo and in vitro studies have elucidated some of the mechanisms behind this effect. For example, inhibition of Abelson tyrosine kinase (c-Abl) results in β cell survival and enhanced insulin secretion, while platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) inhibition leads to improvement in insulin sensitivity. In addition, inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) reduces the degree of islet cell inflammation (insulitis). Therefore, targeting several PTKs may provide a novel approach for correcting the pathophysiologic disturbances of diabetes. PMID:26492832

  4. Novel Bruton’s tyrosine kinase inhibitors currently in development

    PubMed Central

    D’Cruz, Osmond J; Uckun, Fatih M

    2013-01-01

    Bruton’s tyrosine kinase (Btk) is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas. PMID:23493945

  5. Phosphatidylinositol 3-kinase inhibitors block differentiation of skeletal muscle cells.

    PubMed

    Kaliman, P; Viñals, F; Testar, X; Palacín, M; Zorzano, A

    1996-08-01

    Skeletal muscle differentiation involves myoblast alignment, elongation, and fusion into multinucleate myotubes, together with the induction of regulatory and structural muscle-specific genes. Here we show that two phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, blocked an essential step in the differentiation of two skeletal muscle cell models. Both inhibitors abolished the capacity of L6E9 myoblasts to form myotubes, without affecting myoblast proliferation, elongation, or alignment. Myogenic events like the induction of myogenin and of glucose carrier GLUT4 were also blocked and myoblasts could not exit the cell cycle, as measured by the lack of mRNA induction of p21 cyclin-dependent kinase inhibitor. Overexpresssion of MyoD in 10T1/2 cells was not sufficient to bypass the myogenic differentiation blockade by LY294002. Upon serum withdrawal, 10T1/2-MyoD cells formed myotubes and showed increased levels of myogenin and p21. In contrast, LY294002-treated cells exhibited none of these myogenic characteristics and maintained high levels of Id, a negative regulator of myogenesis. These data indicate that whereas phosphatidylinositol 3-kinase is not indispensable for cell proliferation or in the initial events of myoblast differentiation, i.e. elongation and alignment, it appears to be essential for terminal differentiation of muscle cells. PMID:8702591

  6. Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1

    PubMed Central

    2014-01-01

    Background Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous in vitro and in vivo studies have demonstrated the therapeutic effects of inhibiting DCLK1 with small interfering RNA (siRNA) as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Therefore, we assessed the effects of inhibiting DCLK1 kinase activity using the novel small molecule kinase inhibitor, LRRK2-IN-1, which demonstrates significant affinity for DCLK1. Results Here we report that LRRK2-IN-1 demonstrates potent anti-cancer activity including inhibition of cancer cell proliferation, migration, and invasion as well as induction of apoptosis and cell cycle arrest. Additionally we found that it regulates stemness, epithelial-mesenchymal transition, and oncogenic targets on the molecular level. Moreover, we show that LRRK2-IN-1 suppresses DCLK1 kinase activity and downstream DCLK1 effector c-MYC, and demonstrate that DCLK1 kinase activity is a significant factor in resistance to LRRK2-IN-1. Conclusions Given DCLK1’s tumor stem cell marker status, a strong understanding of its biological role and interactions in gastrointestinal tumors may lead to discoveries that improve patient outcomes. The results of this study suggest that small molecule inhibitors of DCLK1 kinase should be further investigated as they may hold promise as anti-tumor stem cell drugs. PMID:24885928

  7. Daurinol Enhances the Efficacy of Radiotherapy in Lung Cancer via Suppression of Aurora Kinase A/B Expression.

    PubMed

    Woo, Jong Kyu; Kang, Ju-Hee; Shin, DongYun; Park, Seong-Hyeok; Kang, Kyungsu; Nho, Chu Won; Seong, Je Kyung; Lee, Sang-Jin; Oh, Seung Hyun

    2015-07-01

    The aurora kinases constitute one family of serine/threonine kinases whose activity is essential for mitotic progression. The aurora kinases are frequently upregulated in human cancers and are associated with sensitivity to chemotherapy in certain ones. In the present study, we investigated whether aurora kinases could be a target to overcome radioresistance or enhance the radiosensitivity of lung cancer. For that purpose, we determined the therapeutic potential of daurinol, an investigational topoisomerase inhibitor, alone and in combination with radiation, by observing its effect on aurora kinases. Daurinol decreased cell viability and proliferation in human colon and lung cancer cells. Gene expression in daurinol-treated human colon cancer cells was evaluated using RNA microarray. The mRNA expression of 18 genes involved in the mitotic spindle check point, including aurora kinase A (AURKA) and aurora kinase B (AURKB), was decreased in daurinol-treated human colon cancer cells as compared with vehicle-treated cells. As expected, radiation increased expression levels of AURKA and AURKB. This increase was effectively attenuated by siRNAs against AURKA and AURKB, which suppressed cell growth and increased apoptosis under radiation. Furthermore, the expression of AURKA and AURKB was suppressed by daurinol in the presence or absence of radiation in colon and lung cancer cells. Daurinol alone or in combination with radiation decreased lung cancer growth in xenograft mouse models. Our data clearly confirm the antitumor and radiosensitizing activity of daurinol in human lung cancer cells through the inhibition of AURKA and AURKB. PMID:25882311

  8. Identification of quinones as novel PIM1 kinase inhibitors.

    PubMed

    Schroeder, Richard L; Goyal, Navneet; Bratton, Melyssa; Townley, Ian; Pham, Nancy A; Tram, Phan; Stone, Treasure; Geathers, Jasmine; Nguyen, Kathy; Sridhar, Jayalakshmi

    2016-07-01

    PIM1 is a proto-oncogene encoding the serine/threonine PIM1 kinase. PIM1 kinase plays important roles in regulating aspects of cell cycle progression, apoptosis resistance, and has been implicated in the development of such malignancies as prostate cancer and acute myeloid leukemia among others. Knockout of PIM1 kinase in mice has been shown to be non-lethal without any obvious phenotypic changes, making it an attractive therapeutic target. Our investigation of anthraquinones as kinase inhibitors revealed a series of quinone analogs showing high selectivity for inhibition of the PIM kinases. Molecular modeling studies were used to identify key interactions and binding poses of these compounds within the PIM1 binding pocket. Compounds 1, 4, 7 and 9 inhibited the growth of DU-145 prostate cancer cell lines with a potency of 8.21μM, 4.06μM, 3.21μM and 2.02μM. PMID:27173800

  9. Characterization of irreversible kinase inhibitors by directly detecting covalent bond formation: a tool for dissecting kinase drug resistance.

    PubMed

    Klüter, Sabine; Simard, Jeffrey R; Rode, Haridas B; Grütter, Christian; Pawar, Vijaykumar; Raaijmakers, Hans C A; Barf, Tjeerd A; Rabiller, Matthias; van Otterlo, Willem A L; Rauh, Daniel

    2010-12-10

    Targeting protein kinases in cancer therapy with irreversible small-molecule inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-associated drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biology and biochemical studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants. PMID:21080395

  10. Adenosine kinase inhibition and suppression of RNA silencing by geminivirus AL2 and L2 proteins.

    PubMed

    Wang, Hui; Buckley, Kenneth J; Yang, Xiaojuan; Buchmann, R Cody; Bisaro, David M

    2005-06-01

    Most plant viruses are initiators and targets of RNA silencing and encode proteins that suppress this adaptive host defense. The DNA-containing geminiviruses are no exception, and the AL2 protein (also known as AC2, C2, and transcriptional activator protein) encoded by members of the genus Begomovirus has been shown to act as a silencing suppressor. Here, a three-component, Agrobacterium-mediated transient assay is used to further examine the silencing suppression activity of AL2 from Tomato golden mosaic virus (TGMV, a begomovirus) and to determine if the related L2 protein of Beet curly top virus (BCTV, genus Curtovirus) also has suppression activity. We show that TGMV AL2, AL2(1-100) (lacking the transcriptional activation domain), and BCTV L2 can all suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene when silencing is induced by a construct expressing an inverted repeat GFP RNA (dsGFP). We previously found that these viral proteins interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and methyl cycle maintenance. Using the GFP-dsGFP system, we demonstrate here that codelivery of a construct expressing an inverted repeat ADK RNA (dsADK), or addition of an ADK inhibitor (the adenosine analogue A-134974), suppresses GFP-directed silencing in a manner similar to the geminivirus proteins. In addition, AL2/L2 suppression phenotypes and nucleic acid binding properties are shown to be different from those of the RNA virus suppressors HC-Pro and p19. These findings provide strong evidence that ADK activity is required to support RNA silencing, and indicate that the geminivirus proteins suppress silencing by a novel mechanism that involves ADK inhibition. Further, since AL2(1-100) is as effective a suppressor as the full-length AL2 protein, activation and silencing suppression appear to be independent activities. PMID:15919897

  11. Antitumoral activity of allosteric inhibitors of protein kinase CK2

    PubMed Central

    Sautel, Céline F.; Teillet, Florence; Barette, Caroline; Lafanechere, Laurence; Receveur-Brechot, Veronique; Cochet, Claude

    2011-01-01

    Introduction Due to its physiological role into promoting cell survival and its dysregulation in most cancer cells, protein kinase CK2 is a relevant physiopathological target for development of chemical inhibitors. We report the discovery of azonaphthalene derivatives, as a new family of highly specific CK2 inhibitors. First, we demonstrated that CK2 inhibition (IC50= 0.4 μM) was highly specific, reversible and non ATP-competitive. Small Angle X-ray Scattering experiments showed that this inhibition was due to large conformational change of CK2α upon binding of these inhibitors. We showed that several compounds of the family were cell-potent CK2 inhibitors promoting cell cycle arrest of human glioblastoma U373 cells. Finally, in vitro and in vivo assays showed that these compounds could decrease U373 cell tumor mass by 83% emphasizing their efficacy against these apoptosis-resistant tumors. In contrast, Azonaphthalene derivatives inactive on CK2 activity showed no effect in colony formation and tumor regression assays. These findings illustrate the emergence of nonclassical CK2 inhibitors and provide exciting opportunities for the development of novel allosteric CK2 inhibitors. Background CK2 is an emerging therapeutic target and ATP-competitive inhibitors have been identified. CK2 is endowed with specific structural features providing alternative strategies for inhibition. Results Azonaphthalene compounds are allosteric CK2 inhibitors showing antitumor activity. Conclusion CK2 may be targeted allosterically. Significance These inhibitors provide a foundation for a new paradigm for specific CK2 inhibition. PMID:22184283

  12. Pharmacological cyclin dependent kinase inhibitors: Implications for colorectal cancer

    PubMed Central

    Balakrishnan, Archana; Vyas, Arpita; Deshpande, Kaivalya; Vyas, Dinesh

    2016-01-01

    Colorectal cancer accounts for a significant proportion of cancer deaths worldwide. The need to develop more chemotherapeutic agents to combat this disease is critical. Cyclin dependent kinases (CDKs), along with its binding partner cyclins, serve to control the growth of cells through the cell cycle. A new class of drugs, termed CDK inhibitors, has been studied in preclinical and now clinical trials. These inhibitors are believed to act as an anti-cancer drug by blocking CDKs to block the uncontrolled cellular proliferation that is hallmark of cancers like colorectal cancer. CDK article provides overview of the emerging drug class of CDK inhibitors and provides a list of ones that are currently in clinical trials. PMID:26900281

  13. Design and synthesis of constrained analogs of LCRF-0004 as potent RON tyrosine kinase inhibitors.

    PubMed

    Raeppel, Stéphane L; Therrien, Eric; Raeppel, Franck

    2015-09-01

    New fused bicyclic lactam head groups as rigidified analogs of thieno[3,2-b]pyridine-based kinase inhibitor LCRF-0004 were designed and synthesized. Depending on the functionalities and the size of these bicyclic head groups, potent inhibitors of RON tyrosine kinase with various level of selectivity against c-Met tyrosine kinase were obtained. PMID:26112445

  14. ROS1 Kinase Inhibitors for Molecular-Targeted Therapies.

    PubMed

    Al-Sanea, M M; Abdelazem, A Z; Park, B S; Yoo, K H; Sim, T; Kwon, Y J; Lee, S H

    2016-01-01

    ROS1 is a pivotal transmembrane receptor protein tyrosine kinase which regulates several cellular processes like apoptosis, survival, differentiation, proliferation, cell migration, and transformation. There is increasing evidence supporting that ROS1 plays an important role in different malignancies including glioblastoma, colorectal cancer, gastric adenocarcinoma, inflammatory myofibroblastic tumor, ovarian cancer, angiosarcoma, and non small cell lung cancer; thus, ROS1 has become a potential drug discovery target. ROS1 shares about 49% sequence homology with ALK primary structure; therefore, wide range of ALK kinase inhibitors have shown in vitro inhibitory activity against ROS1 kinase. After Crizotinib approval by FDA for the management of ALK-rearranged lung cancer, ROS1-positive tumors have been focused. Although significant advancements have been achieved in understanding ROS1 function and its signaling pathways plus recent discovery of small molecules modulating ROS1 protein, a vital need of medicinal chemistry efforts is still required to produce selective and potent ROS1 inhibitors as an important therapeutic strategy for different human malignancies. This review focuses on the current knowledge about different scaffolds targeting ROS1 rearrangements, methods to synthesis, and some biological data about the most potent compounds that have delivered various scaffold structures. PMID:26438251

  15. Discovery of a Highly Selective STK16 Kinase Inhibitor.

    PubMed

    Liu, Feiyang; Wang, Jinhua; Yang, Xingxing; Li, Binhua; Wu, Hong; Qi, Shuang; Chen, Cheng; Liu, Xiaochuan; Yu, Kailin; Wang, Wenchao; Zhao, Zheng; Wang, Aoli; Chen, Yongfei; Wang, Li; Gray, Nathanael S; Liu, Jing; Zhang, Xin; Liu, Qingsong

    2016-06-17

    STK16, a serine/threonine protein kinase, is ubiquitously expressed and is conserved among all eukaryotes. STK16 has been implicated to function in a variety of cellular processes such as VEGF and cargo secretion, but the pathways through which these effects are mediated remain to be elucidated. Through screening of our focused library of kinase inhibitors, we discovered a highly selective ATP competitive inhibitor, STK16-IN-1, which exhibits potent inhibitory activity against STK16 kinase (IC50: 0.295 μM) with excellent selectivity across the kinome as assessed using the KinomeScan profiling assay (S score (1) = 0.0). In MCF-7 cells, treatment with STK16-IN-1 results in a reduction in cell number and accumulation of binucleated cells, which can be recapitulated by RNAi knockdown of STK16. Co-treatment of STK16-IN-1 with chemotherapeutics such as cisplatin, doxorubicin, colchicine, and paclitaxel results in a slight potentiation of the antiproliferative effects of the chemotherapeutics. STK16-IN-1 provides a useful tool compound for further elucidating the biological functions of STK16. PMID:27082499

  16. Some implications of receptor kinase signaling pathway for development of multitargeted kinase inhibitors.

    PubMed

    Mitrasinovic, Petar M

    2013-03-01

    Epidermal growth factor receptors (EGFRs) belong to the ErbB family of receptor tyrosine kinases (TKs). Based on the role of EGFR signaling pathway in malignant progression of various types of tumors, a growing interest in the use of EGFR-TK inhibitors as probes for molecular imaging of EGFR-overexpressing tumors via positron emission tomography (PET) and single photon emission computed tomography (SPECT) is being notable. On one side, such noninvasive and repetitive monitoring of the activity of EGFR at the kinase level is intended to provide a direct measure of EGFR occupancy and inhibition by EGFR-targeting drugs. On the other side, all oncologic imaging tracers are molecularly targeted radiopharmaceuticals, which are strongly dependent on the tumor biochemistry including increased metabolism, hyperproliferation, angiogenesis, hypoxia, apoptosis, and specific tumor biomarkers (tumor specific antigens and tumor-specific receptors). The present article is an attempt to reconcile these two vital standpoints influencing the choice of appropriate radiolabeled agents for PET and SPECT imaging aimed to support the development of a new generation of multi-targeted kinase inhibitors in the time ahead, because the routine accomplishment of drug selectivity for particular protein kinases is a substantial challenge. PMID:23278847

  17. Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

    PubMed

    Nutt, Joyce E; O'Toole, Kieran; Gonzalez, David; Lunec, John

    2009-06-01

    Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma. PMID:19318229

  18. Receptor tyrosine kinase inhibitors: Are they real tumor killers?

    PubMed

    Gaumann, Andreas K A; Kiefer, Friedemann; Alfer, Joachim; Lang, Sven A; Geissler, Edward K; Breier, Georg

    2016-02-01

    Inhibiting tumor growth by targeting the tumor vasculature was first proposed by Judah Folkman almost 40 years ago. Since then, different approaches and numerous drugs and agents have been developed to achieve this goal, either with the aim of inhibiting tumor neoangiogenesis or normalizing the tumor vasculature. Among the most promising therapeutic targets are receptor tyrosine kinases (RTKs), some of which are predominantly expressed on tumor endothelial cells, although they are sometimes also present on tumor cells. The majority of RTK inhibitors investigated over the past two decades competes with ATP at the active site of the kinase and therefore block the phosphorylation of intracellular targets. Some of these drugs have been approved for therapy, whereas others are still in clinical trials. Here, we discuss the scientific basis, current status, problems and future prospects of RTK inhibition in anti-tumor therapy. PMID:25716346

  19. Tailoring Tyrosine Kinase Inhibitors to Fit the Lung Cancer Genome

    PubMed Central

    Looyenga, Brendan D; Cherni, Irene; MacKeigan, Jeffrey P; Weiss, Glen J

    2011-01-01

    Tyrosine kinase inhibitors (TKIs) have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies with defined genetic lesions has proven to be remarkably effective. Recent efforts to characterize the spectrum of genetic lesions found in non-small cell lung carcinoma (NSCLC) have provided important insights into the molecular basis of this disease and have also revealed a wide array of tyrosine kinases that might be effectively targeted for rationally designed therapies. The findings of these studies, however, also provide a cautionary tale about the limitations of single-agent therapies, which fail to account for the genetic heterogeneity and pathway redundancy that characterize advanced NSCLC. Emergence of drug resistance mechanisms to specific TKIs, such as gefitinib and erlotinib, suggests that more sophisticated chemotherapeutic paradigms that target multiple pathways at the same time will be required to effectively treat this disease. PMID:21461169

  20. A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors.

    PubMed

    Lanning, Bryan R; Whitby, Landon R; Dix, Melissa M; Douhan, John; Gilbert, Adam M; Hett, Erik C; Johnson, Theodore O; Joslyn, Chris; Kath, John C; Niessen, Sherry; Roberts, Lee R; Schnute, Mark E; Wang, Chu; Hulce, Jonathan J; Wei, Baoxian; Whiteley, Laurence O; Hayward, Matthew M; Cravatt, Benjamin F

    2014-09-01

    Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors. PMID:25038787

  1. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization

    PubMed Central

    Lavoie, Hugo; Thevakumaran, Neroshan; Gavory, Gwenaëlle; Li, John; Padeganeh, Abbas; Guiral, Sébastien; Duchaine, Jean; Mao, Daniel Y. L.; Bouvier, Michel; Sicheri, Frank; Therrien, Marc

    2016-01-01

    RAF kinases play a prominent role in cancer. Their mode of activation is complex, but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and as a result undesirably stimulate RAS/ERK-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe BRET-based biosensors for the extended RAF family enabling the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization as well as for probing structural determinants of RAF dimerization in vivo. Our findings, which appear generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain. PMID:23685672

  2. A chrysin derivative suppresses skin cancer growth by inhibiting cyclin-dependent kinases.

    PubMed

    Liu, Haidan; Liu, Kangdong; Huang, Zunnan; Park, Chan-Mi; Thimmegowda, N R; Jang, Jae-Hyuk; Ryoo, In-Ja; He, Long; Kim, Sun-Ok; Oi, Naomi; Lee, Ki Won; Soung, Nak-Kyun; Bode, Ann M; Yang, Yifeng; Zhou, Xinmin; Erikson, Raymond L; Ahn, Jong-Seog; Hwang, Joonsung; Kim, Kyoon Eon; Dong, Zigang; Kim, Bo-Yeon

    2013-09-01

    Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency. PMID:23888052

  3. Role of glycogen synthase kinase-3β inhibitor AZD1080 in ovarian cancer

    PubMed Central

    Chen, Shuo; Sun, Kai-Xuan; Feng, Miao-Xiao; Sang, Xiu-Bo; Liu, Bo-Liang; Zhao, Yang

    2016-01-01

    Background Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays an important role in cancer tumorigenesis and progression. We investigated the role of the GSK-3β inhibitor AZD1080 in ovarian cancer cell lines. Methods A2780 and OVCAR3 ovarian cancer cell lines were exposed to AZD1080, after which cell proliferation, cell cycle, invasion, and migration assays were performed. Phalloidin staining was used to observe lamellipodia formation. Reverse transcription polymerase chain reaction and Western blot were used to assess the respective mRNA and protein expression levels of GSK-3β, CDK2, CDK1, cyclin D1, matrix metalloproteinase-9 (MMP9), and Bcl-xL. Results AZD1080 exposure suppressed ovarian cancer cell proliferation, invasion, migration, and lamellipodia formation, and induced G1 arrest, which was concentration dependent. AZD1080 also significantly downregulated GSK-3β, CDK2, CDK1, cyclin D1, MMP9, and Bcl-xL expression at both mRNA and protein levels. Conclusion Taken together, our results demonstrate that the GSK-3β inhibitor AZD1080 suppresses ovarian cancer development and therefore may indicate a new direction for ovarian cancer treatment. PMID:27051274

  4. Linking phenotype to kinase: identification of a novel benzoxaborole hinge-binding motif for kinase inhibition and development of high-potency rho kinase inhibitors.

    PubMed

    Akama, Tsutomu; Dong, Chen; Virtucio, Charlotte; Sullivan, David; Zhou, Yasheen; Zhang, Yong-Kang; Rock, Fernando; Freund, Yvonne; Liu, Liang; Bu, Wei; Wu, Anne; Fan, Xiao-Qing; Jarnagin, Kurt

    2013-12-01

    Benzoxaboroles are a novel class of drug-like compounds that have been rich sources of novel inhibitors for various enzymes and of new drugs. While examining benzoxaborole activity in phenotypic screens, our attention was attracted by the (aminomethylphenoxy)benzoxaborole family, which potently inhibited Toll-like receptor-stimulated cytokine secretion from leukocytes. After considering their structure-activity relationships and the central role of kinases in leukocyte biology, we performed a kinome-wide screen to investigate the members of the (aminomethylphenoxy)benzoxaborole family. This technique identified Rho-activated kinase (ROCK) as a target. We showed competitive behavior, with respect to ATP, and then determined the ROCK2-drug cocrystal structure. The drug occupies the ATP site in which the oxaborole moiety provides hydrogen bond donors and acceptors to the hinge, and the aminomethyl group interacts with the magnesium/ATP-interacting aspartic acid common to protein kinases. The series exhibits excellent selectivity against most of the kinome, with greater than 15-fold selectivity against the next best member of the AGC protein kinase subfamily. Medicinal chemistry efforts with structure-based design resulted in a compound with a Ki of 170 nM. Cellular studies revealed strong enzyme inhibition rank correlation with suppression of intracellular phosphorylation of a ROCK substrate. The biochemical potencies of these compounds also translated to functional activity, causing smooth muscle relaxation in rat aorta and guinea pig trachea. The series exhibited oral availability and one member reduced rat blood pressure, consistent with ROCK's role in smooth muscle contraction. Thus, the benzoxaborole moiety represents a novel hinge-binding kinase scaffold that may have potential for therapeutic use. PMID:24049062

  5. Optogenetically controlled RAF to characterize BRAF and CRAF protein kinase inhibitors

    PubMed Central

    Chatelle, Claire V.; Hövermann, Désirée; Müller, Anne; Wagner, Hanna J.; Weber, Wilfried; Radziwill, Gerald

    2016-01-01

    Here, we applied optoRAF, an optogenetic tool for light-controlled clustering and activation of RAF proteins that mimics the natural occurring RAS-mediated dimerization. This versatile tool allows studying the effect on BRAF and CRAF homodimer- as well as heterodimer-induced RAF signaling. Vemurafenib and dabrafenib are two clinically approved inhibitors for BRAF that efficiently suppress the kinase activity of oncogenic BRAF (V600E). However in wild-type BRAF expressing cells, BRAF inhibitors can exert paradoxical activation of wild-type CRAF. Using optoRAF, vemurafenib was identified as paradoxical activator of BRAF and CRAF homo- and heterodimers. Dabrafenib enhanced activity of light-stimulated CRAF at low dose and inhibited CRAF signaling at high dose. Moreover, dabrafenib increased the protein level of CRAF proteins but not of BRAF proteins. Increased CRAF levels correlate with elevated RAF signaling in a dabrafenib-dependent manner, independent of light activation. PMID:27025703

  6. Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases

    PubMed Central

    Schonbrunn, Ernst; Betzi, Stephane; Alam, Riazul; Martin, Mathew P.; Becker, Andreas; Han, Huijong; Francis, Rawle; Chakrasali, Ramappa; Jakkaraj, Sudhakar; Kazi, Aslamuzzaman; Sebti, Said M.; Cubitt, Christopher L.; Gebhard, Anthony W.; Hazlehurst, Lori A.; Tash, Joseph S.; Georg, Gunda I.

    2013-01-01

    Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009 – 0.0015 µM) from a single hit compound with weak inhibitory activity (IC50 = 15 µM), discovered by high-throughput screening. Structure-based design was performed using 35 co-crystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4 and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 µM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics. PMID:23600925

  7. Design, synthesis and structure-activity relationships of novel biarylamine-based Met kinase inhibitors

    SciTech Connect

    Williams, David K; Chen, Xiao-Tao; Tarby, Christine; Kaltenbach, Robert; Cai, Zhen-Wei; Tokarski, John S; An, Yongmi; Sack, John S; Wautlet, Barri; Gullo-Brown, Johnni; Henley, Benjamin J; Jeyaseelan, Robert; Kellar, Kristen; Manne, Veeraswamy; Trainor, George L; Lombardo, Louis J; Fargnoli, Joseph; Borzilleri, Robert M

    2010-09-03

    Biarylamine-based inhibitors of Met kinase have been identified. Lead compounds demonstrate nanomolar potency in Met kinase biochemical assays and significant activity in the Met-driven GTL-16 human gastric carcinoma cell line. X-ray crystallography revealed that these compounds adopt a bioactive conformation, in the kinase domain, consistent with that previously seen with 2-pyridone-based Met kinase inhibitors. Compound 9b demonstrated potent in vivo antitumor activity in the GTL-16 human tumor xenograft model.

  8. Novel Aurora/vascular endothelial growth factor receptor dual kinase inhibitor as treatment for hepatocellular carcinoma.

    PubMed

    Nakao, Keisuke; Tanaka, Shinji; Miura, Tomoya; Sato, Kota; Matsumura, Satoshi; Aihara, Arihiro; Mitsunori, Yusuke; Ban, Daisuke; Ochiai, Takanori; Kudo, Atsushi; Arii, Shigeki; Tanabe, Minoru

    2015-08-01

    We previously identified Aurora B kinase as the only independent factor predictive of the aggressive recurrence of hepatocellular carcinoma (HCC). In this preclinical study, JNJ-28841072, a novel Aurora/vascular endothelial growth factor receptor dual kinase inhibitor, was evaluated for treatment of HCC. In vitro and in vivo effects of JNJ-28841072 were analyzed using human HCC cell cultures and xenograft models. An orthotopic liver xenograft model was used for the pharmacobiological effects on Aurora kinase and vascularization in hepatic tumors. JNJ-28841072 suppressed in vitro phosphorylation of histone H3 with induction of cell polyploidy and death in a dose-dependent manner (IC50  = 0.8-1.2 μM). In s.c. human HCC xenografts, remarkable inhibition of tumor growth was observed after JNJ-28841072 treatment (P = 0.0005). In orthotopic liver xenografts, the treatment with JNJ-28841072 significantly suppressed in vivo phosphorylation of histone H3 (P = 0.0008), vessel formation (P = 0.018), normoxic area (P = 0.0001), and hepatoma growth (P = 0.038). Our preclinical studies indicate that JNJ-28841072 is a promising novel therapeutic approach for the treatment of HCC. It might be worthy of evaluation in further studies. PMID:26011703

  9. Purvalanol A, a CDK inhibitor, effectively suppresses Src-mediated transformation by inhibiting both CDKs and c-Src.

    PubMed

    Hikita, Tomoya; Oneyama, Chitose; Okada, Masato

    2010-10-01

    The nonreceptor tyrosine kinase c-Src is frequently over-expressed or hyperactivated in various human cancers and contributes to cancer progression in cooperation with up-regulated growth factor receptors. However, Src-selective anticancer drugs are still in clinical trials. To identify more effective inhibitors of c-Src-mediated cancer progression, we developed a new screening platform using Csk-deficient cells that can be transformed by c-Src. We found that purvalanol A, developed as a CDK inhibitor, potently suppressed the anchorage-independent growth of c-Src-transformed cells, indicating that the activation of CDKs contributes to the c-Src transformation. We also found that purvalanol A suppressed the c-Src activity as effectively as the Src-selective inhibitor PP2, and that it reverted the transformed morphology to a nearly normal shape with less cytotoxicity than PP2. Purvalanol A induced a strong G2-M arrest, whereas PP2 weakly acted on the G1-S transition. Furthermore, when compared with PP2, purvalanol A more effectively suppressed the growth of human colon cancer HT29 and SW480 cells, in which Src family kinases and CDKs are activated. These findings demonstrate that the coordinated inhibition of cell cycle progression and tyrosine kinase signaling by the multi-selective purvalanol A is effective in suppressing cancer progression associated with c-Src up-regulation. PMID:20825494

  10. Glycogen synthase kinase-3 inhibitors: Rescuers of cognitive impairments

    PubMed Central

    King, Margaret K.; Pardo, Marta; Cheng, Yuyan; Downey, Kimberlee; Jope, Richard S.; Beurel, Eléonore

    2013-01-01

    Impairment of cognitive processes is a devastating outcome of many diseases, injuries, and drugs affecting the central nervous system (CNS). Most often, very little can be done by available therapeutic interventions to improve cognitive functions. Here we review evidence that inhibition of glycogen synthase kinase-3 (GSK3) ameliorates cognitive deficits in a wide variety of animal models of CNS diseases, including Alzheimer's disease, Fragile X syndrome, Down syndrome, Parkinson's disease, spinocerebellar ataxia type 1, traumatic brain injury, and others. GSK3 inhibitors also improve cognition following impairments caused by therapeutic interventions, such as cranial irradiation for brain tumors. These findings demonstrate that GSK3 inhibitors are able to ameliorate cognitive impairments caused by a diverse array of diseases, injury, and treatments. The improvements in impaired cognition instilled by administration of GSK3 inhibitors appear to involve a variety of different mechanisms, such as supporting long-term potentiation and diminishing long-term depression, promotion of neurogenesis, reduction of inflammation, and increasing a number of neuroprotective mechanisms. The potential for GSK3 inhibitors to repair cognitive deficits associated with many conditions warrants further investigation of their potential for therapeutic interventions, particularly considering the current dearth of treatments available to reduce loss of cognitive functions. PMID:23916593

  11. Structural Determinants of CX-4945 Derivatives as Protein Kinase CK2 Inhibitors: A Computational Study

    PubMed Central

    Liu, Hongbo; Wang, Xia; Wang, Jian; Wang, Jinghui; Li, Yan; Yang, Ling; Li, Guohui

    2011-01-01

    Protein kinase CK2, also known as casein kinase-2, is involved in a broad range of physiological events including cell growth, proliferation and suppression of apoptosis which are related to human cancers. A series of compounds were identified as CK2 inhibitors and their inhibitory activities varied depending on their structures. In order to explore the structure-activity correlation of CX-4945 derivatives as inhibitors of CK2, in the present study, a set of ligand- and receptor-based 3D-QSAR models were developed employing Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Index Analysis (CoMSIA). The optimum CoMFA (Rcv2 = 0.618, Rpred2 = 0.892) and CoMSIA (Rcv2 = 0.681, Rpred2 = 0.843) models exhibited reasonable statistical characteristics for CX-4945 derivatives. The results indicated that electrostatic effects contributed the most to both CoMFA and CoMSIA models. The combination of docking analysis and molecular dynamics (MD) simulation showed that Leu45, Lys68, Glu81, Val116, Asp175 and Trp176 of CK2 which formed several direct or water-bridged H-bonds with CX-4945 are crucial for CX-4945 derivatives recognition to CK2. These results can offer useful theoretical references for designing more potent CK2 inhibitors. PMID:22072932

  12. Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance

    PubMed Central

    Yoda, Akinori; Adelmant, Guillaume; Tamburini, Jerome; Chapuy, Bjoern; Shindoh, Nobuaki; Yoda, Yuka; Weigert, Oliver; Kopp, Nadja; Wu, Shuo-Chieh; Kim, Sunhee S.; Liu, Huiyun; Tivey, Trevor; Christie, Amanda L.; Elpek, Kutlu G.; Card, Joseph; Gritsman, Kira; Gotlib, Jason; Deininger, Michael W.; Makishima, Hideki; Turley, Shannon J.; Javidi-Sharifi, Nathalie; Maciejewski, Jaroslaw P.; Jaiswal, Siddhartha; Ebert, Benjamin L.; Rodig, Scott J.; Tyner, Jeffrey W.; Marto, Jarrod A.; Weinstock, David M.; Lane, Andrew A.

    2014-01-01

    Activating mutations of G protein alpha subunits (Gα) occur in 4–5% of all human cancers1 but oncogenic alterations in beta subunits (Gβ) have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors, and disrupt Gα-Gβγ interactions. Different mutations in Gβ proteins clustered to some extent based on lineage; for example, all eleven GNB1 K57 mutations were in myeloid neoplasms while 7 of 8 GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 alleles in Cdkn2a-deficient bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K/mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, GNB1 mutations co-occurred with oncogenic kinase alterations, including BCR/ABL, JAK2 V617F and BRAF V600K. Co-expression of patient-derived GNB1 alleles with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 mutations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling. PMID:25485910

  13. Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways

    PubMed Central

    Zwang, N. A.; Zhang, R.; Germana, S.; Fan, M. Y.; Hastings, W. D.; Cao, A.; Turka, L. A.

    2016-01-01

    Phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated (MEK) signaling are central to the survival and proliferation of many cell types. Multiple lines of investigation in murine models have shown that control of the PI3K pathway is particularly important for regulatory T cell (Treg) stability and function. PI3K and MEK inhibitors are being introduced into the clinic, and we hypothesized that pharmacologic inhibition of PI3K, and possibly MEK, in mixed cultures of human mononuclear cells would preferentially affect CD4+ and CD8+ lymphocytes compared with Tregs. We tested this hypothesis using four readouts: proliferation, activation, functional suppression, and signaling. Results showed that Tregs were less susceptible to inhibition by both δ and α isoform–specific PI3K inhibitors and by an MEK inhibitor compared with their conventional CD4+ and CD8+ counterparts. These studies suggest less functional reliance on PI3K and MEK signaling in Tregs compared with conventional CD4+ and CD8+ lymphocytes. Therefore, the PI3K and MEK pathways are attractive pharmacologic targets for transplantation and treatment of autoimmunity. PMID:27017850

  14. Toward the rational design of protein kinase casein kinase-2 inhibitors.

    PubMed

    Sarno, Stefania; Moro, Stefano; Meggio, Flavio; Zagotto, Giuseppe; Dal Ben, Diego; Ghisellini, Paola; Battistutta, Roberto; Zanotti, Giuseppe; Pinna, Lorenzo A

    2002-01-01

    Casein kinase-2 (CK2) probably is the most pleiotropic member of the protein kinase family, with more than 200 substrates known to date. Unlike the great majority of protein kinases, which are tightly regulated enzymes, CK2 is endowed with high constitutive activity, a feature that is suspected to underlie its oncogenic potential and possible implication in viral infections. This makes CK2 an attractive target for anti-neoplastic and antiviral drugs. Here, we present an overview of our present knowledge about CK2 inhibitors, with special reference to the information drawn from two recently solved crystal structures of CK2alpha in complex with emodin and with 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), this latter being the most specific CK2 inhibitor known to date. A comparison with a series of anthraquinone and xanthenone derivatives highlights the crucial relevance of the hydroxyl group at position 3 for inhibition by emodin, and discloses the possibility of increasing the inhibitory potency by placing an electron withdrawing group at position 5. We also present mutational data corroborating the relevance of two hydrophobic residues unique to CK2, Val66 and Ile174, for the interactions with emodin and TBB, but not with the flavonoid inhibitors quercetin and fisetin. In particular, the CK2alpha mutant V66A displays 27- and 11-fold higher IC(50) values with emodin and TBB, respectively, as compared with the wild-type, while the IC(50) value with quercetin is unchanged. The data presented pave the road toward the rational design of more potent and selective inhibitors of CK2 and the generation of CK2 mutants refractory to inhibition, useful to probe the implication of CK2 in specific cellular functions. PMID:12191608

  15. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors.

    PubMed

    Tan, Li; Wang, Jun; Tanizaki, Junko; Huang, Zhifeng; Aref, Amir R; Rusan, Maria; Zhu, Su-Jie; Zhang, Yiyun; Ercan, Dalia; Liao, Rachel G; Capelletti, Marzia; Zhou, Wenjun; Hur, Wooyoung; Kim, NamDoo; Sim, Taebo; Gaudet, Suzanne; Barbie, David A; Yeh, Jing-Ruey Joanna; Yun, Cai-Hong; Hammerman, Peter S; Mohammadi, Moosa; Jänne, Pasi A; Gray, Nathanael S

    2014-11-11

    The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a "DFG-out" covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket. PMID:25349422

  16. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors

    PubMed Central

    Tan, Li; Wang, Jun; Tanizaki, Junko; Huang, Zhifeng; Aref, Amir R.; Rusan, Maria; Zhu, Su-Jie; Zhang, Yiyun; Ercan, Dalia; Liao, Rachel G.; Capelletti, Marzia; Zhou, Wenjun; Hur, Wooyoung; Kim, NamDoo; Sim, Taebo; Gaudet, Suzanne; Barbie, David A.; Yeh, Jing-Ruey Joanna; Yun, Cai-Hong; Hammerman, Peter S.; Mohammadi, Moosa; Jänne, Pasi A.; Gray, Nathanael S.

    2014-01-01

    The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a “DFG-out” covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket. PMID:25349422

  17. Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies

    PubMed Central

    Winiarska, Magdalena; Bojarczuk, Kamil; Pyrzynska, Beata; Bil, Jacek; Siernicka, Marta; Dwojak, Michal; Bobrowicz, Malgorzata; Miazek, Nina; Zapala, Piotr; Zagozdzon, Agnieszka; Krol, Magdalena; Syta, Aleksandra; Podszywalow-Bartnicka, Paulina; Pilch, Zofia; Dabrowska-Iwanicka, Anna; Juszczynski, Przemyslaw; Efremov, Dimitar G; Slabicki, Mikolaj; Zenz, Thorsten; Roy, Aude Le; Olive, Daniel; Rygiel, Tomasz P; Leusen, Jeanette HW; Golab, Jakub

    2014-01-01

    Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells. PMID:25517315

  18. Tyrosine kinase inhibitors: New class of antimalarials on the horizon?

    PubMed

    Pathak, Vrushali; Colah, Roshan; Ghosh, Kanjaksha

    2015-08-01

    Development of the antimalarial drug resistant strains has currently become a major public health challenge. There is an urgent need to develop new antimalarial drugs. Tyrosine kinase inhibitors (TKIs) are receiving increasing attention as anticancer therapy. It has revolutionarised the management of CML to say the least. TKIs are also increasingly being implicated in complicated but vital life cycle of malaria parasite. Hence we tested two commonly used but different classes of TKIs (imatinib and sorafenib) in-vitro for their antimalarial activity and possible synergistic activity with existing antimalarial drug. Antimalarial activity was tested with the help of modified WHO microtest technique in-vitro for five different Plasmodium falciparum laboratory strains (3D7, Dd2, 7G8, MRC2, PKL9). Imatinib and sorafenib showed a promising antimalarial activity with all the strains. These compounds caused dose dependent inhibition of parasite maturation. The isobologram analysis of the interactions of these TKIs with standard antimalarial drug, artesunate revealed distinct patterns of synergism, additivity and antagonism at different ratios. Imatinib showed worthwhile synergism with artesunate indicating imatinib and other tyrosine kinase inhibitors may have significant antimalarial activity and can be used in combination therapy. PMID:26142327

  19. Crystal structure of inhibitor of ;#954;B kinase [beta

    SciTech Connect

    Xu, Guozhou; Lo, Yu-Chih; Li, Qiubai; Napolitano, Gennaro; Wu, Xuefeng; Jiang, Xuliang; Dreano, Michel; Karin, Michael; Wu, Hao

    2011-07-26

    Inhibitor of {kappa}B (I{kappa}B) kinase (IKK) phosphorylates I{kappa}B proteins, leading to their degradation and the liberation of nuclear factor {kappa}B for gene transcription. Here we report the crystal structure of IKK{beta} in complex with an inhibitor, at a resolution of 3.6 {angstrom}. The structure reveals a trimodular architecture comprising the kinase domain, a ubiquitin-like domain (ULD) and an elongated, {alpha}-helical scaffold/dimerization domain (SDD). Unexpectedly, the predicted leucine zipper and helix-loop-helix motifs do not form these structures but are part of the SDD. The ULD and SDD mediate a critical interaction with I{kappa}B{alpha} that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKK{beta} dimerization, but dimerization per se is not important for maintaining IKK{beta} activity and instead is required for IKK{beta} activation. Other IKK family members, IKK{alpha}, TBK1 and IKK-i, may have a similar trimodular architecture and function.

  20. Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

    PubMed Central

    Moccia, Marialuisa; Liu, Qingsong; Guida, Teresa; Federico, Giorgia; Brescia, Annalisa; Zhao, Zheng; Choi, Hwan Geun; Deng, Xianming; Tan, Li; Wang, Jinhua; Billaud, Marc; Gray, Nathanael S.

    2015-01-01

    Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET. PMID:26046350

  1. An evaluation of indirubin analogues as phosphorylase kinase inhibitors.

    PubMed

    Begum, Jaida; Skamnaki, Vassiliki T; Moffatt, Colin; Bischler, Nicolas; Sarrou, Josephine; Skaltsounis, Alexios-Leandros; Leonidas, Demetres D; Oikonomakos, Nikos G; Hayes, Joseph M

    2015-09-01

    Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360μM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8μM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work. PMID:26364215

  2. Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor That Attenuates Disease Progression in Alzheimer’s Disease Mouse Models

    PubMed Central

    2015-01-01

    The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150’s exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior. PMID:25676389

  3. Acetylcholinesterase inhibitors used in treatment of Alzheimer's disease prevent glutamate neurotoxicity via nicotinic acetylcholine receptors and phosphatidylinositol 3-kinase cascade.

    PubMed

    Takada-Takatori, Yuki; Kume, Toshiaki; Sugimoto, Mitsuhiro; Katsuki, Hiroshi; Sugimoto, Hachiro; Akaike, Akinori

    2006-09-01

    We show here that donepezil, galanathamine and tacrine, therapeutic acetylcholinesterase inhibitors currently being used for treatment of Alzheimer's disease, protect neuronal cells in a time- and concentration-dependent manner from glutamate neurotoxicity that involves apoptosis. The neuroprotective effects were antagonized by mecamylamine, an inhibitor of nicotinic acetylcholine receptors (nAChRs). Dihydro-beta-erythroidine and methyllycaconitine, antagonists for alpha4-nAChR and alpha7-nAChR, respectively, antagonized the protective effect of donepezil and galanthamine, but not that of tacrine. Previous reports suggest the involvement of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway in the nicotine-induced neuroprotection. Inhibitors for a non-receptor type tyrosine kinase, Fyn, and janus-activated kinase 2, suppressed the neuroprotective effect of donepezil and galanthamine, but not that of tacrine. Furthermore, LY294002, a PI3K inhibitor, also suppressed the neuroprotective effect of donepezil and galanthamine, but not that of tacrine. The phosphorylation of Akt, an effector of PI3K, and the expression level of Bcl-2, an anti-apoptotic protein, increased with donepezil and galanthamine treatment, but not with tacrine treatment. These results suggest that donepezil and galanthamine prevent glutamate neurotoxicity through alpha4- and alpha7-nAChRs, followed by the PI3K-Akt pathway, and that tacrine protects neuronal cells through a different pathway. PMID:16762377

  4. HPLC-DAD protein kinase inhibitor analysis in human serum.

    PubMed

    Dziadosz, Marek; Lessig, Rüdiger; Bartels, Heidemarie

    2012-04-15

    We here describe an HPLC-DAD method to analyse different protein kinase inhibitors. Potential applications of this method are pharmacokinetic studies and therapeutic drug monitoring. Optimised chromatography conditions resulted in a very good separation of seven inhibitors (vatalanib, bosutinib, canertinib, tandutinib, pazopanib, dasatinib - internal standard and erlotinib). The good sensitivity makes this method competitive with LC/MS/MS. The separation was performed with a Lichrospher 100-5 RP8, 250 mm × 4 mm column maintained at 30 ± 1 °C, and with a mobile phase of 0.05 M H(3)PO(4)/KH(2)PO(4) (pH=2.3)-acetonitrile (7:3, v/v) at a flow rate of 0.7 mL/min. A simple and fast sample preparation sequence with liquid-liquid extraction led to good recoveries (73-90%) of all analytes. The recovery hardly reached 50% only for pazopanib. This method can also be used for targeted protein kinase inhibitor quantification. A perfect linearity in the validated range (20-10,000 ng/mL) and an LOQ of 20 ng/mL were achieved. The relative standard deviations and accuracies of all examined drug concentrations gave values much lower than 15% both for between- and within-batch calculations. All analysed PKIs were stable for 6 months in a 1mg/mL dimethyl sulfoxide stock solution. Vatalanib, bosutinib and erlotinib were also stable in human serum in the whole examined concentration range. PMID:22425385

  5. Protein kinase inhibitors in the treatment of inflammatory and autoimmune diseases

    PubMed Central

    Patterson, H; Nibbs, R; McInnes, I; Siebert, S

    2014-01-01

    Protein kinases mediate protein phosphorylation, which is a fundamental component of cell signalling, with crucial roles in most signal transduction cascades: from controlling cell growth and proliferation to the initiation and regulation of immunological responses. Aberrant kinase activity is implicated in an increasing number of diseases, with more than 400 human diseases now linked either directly or indirectly to protein kinases. Protein kinases are therefore regarded as highly important drug targets, and are the subject of intensive research activity. The success of small molecule kinase inhibitors in the treatment of cancer, coupled with a greater understanding of inflammatory signalling cascades, has led to kinase inhibitors taking centre stage in the pursuit for new anti-inflammatory agents for the treatment of immune-mediated diseases. Herein we discuss the main classes of kinase inhibitors; namely Janus kinase (JAK), mitogen-activated protein kinase (MAPK) and spleen tyrosine kinase (Syk) inhibitors. We provide a mechanistic insight into how these inhibitors interfere with kinase signalling pathways and discuss the clinical successes and failures in the implementation of kinase-directed therapeutics in the context of inflammatory and autoimmune disorders. PMID:24313320

  6. Small-molecule inhibitors of IκB kinase (IKK) and IKK-related kinases.

    PubMed

    Llona-Minguez, Sabin; Baiget, Jessica; Mackay, Simon P

    2013-07-01

    The transcription factors NF-κB and IFN control important signaling cascades and mediate the expression of a number of important pro-inflammatory cytokines, adhesion molecules, growth factors and anti-apoptotic survival proteins. IκB kinase (IKK) and IKK-related kinases (IKKε and TBK1) are key regulators of these biological pathways and, as such, modulators of these enzymes may be useful in the treatment of inflammatory diseases and cancer. We have reviewed the most recent IKK patent literature (2008-2012), added publications of interest overlooked in previous patent reviews and identified all the players involved in small-molecule inhibitors of the IKKs. This will provide the reader with a decisive summary of the IKK arena, a field that has reached maturity over a decade of research. PMID:24237125

  7. Cheminfomatic-based Drug Discovery of Human Tyrosine Kinase Inhibitors.

    PubMed

    Reid, Terry-Elinor; Fortunak, Joseph M; Wutoh, Anthony; Simon Wang, Xiang

    2016-01-01

    Receptor Tyrosine Kinases (RTKs) are essential components for regulating cell-cell signaling and communication events in cell growth, proliferation, differentiation, survival and metabolism. Deregulation of RTKs and their associated signaling pathways can lead to a wide variety of human diseases such as immunodeficiency, diabetes, arterosclerosis, psoriasis and cancer. Thus RTKs have become one of the most important drug targets families in recent decade. Pharmaceutical companies have dedicated their research efforts towards the discovery of small-molecule inhibitors of RTKs, many of which had been approved by the U.S. Food and Drug Administration (US FDA) or are currently in clinical trials. The great successes in the development of small-molecule inhibitors of RTKs are largely attributed to the use of modern cheminformatic approaches to identifying lead scaffolds. Those include the quantitative structure-activity relationship (QSAR) modeling, as well as the structure-, and ligand-based pharmacophore modeling techniques in this case. Herein we inspected the literature thoroughly in an effort to conduct a comparative analysis of major findings regarding the essential structure-activity relationships (SARs)/pharmacophore features of known active RTK inhibitors, most of which were collected from cheminformatic modeling approaches. PMID:26369823

  8. Bruton's tyrosine kinase inhibitors in chronic lymphocytic leukemia and lymphoma.

    PubMed

    Varma, Gaurav; Johnson, Tyler P; Advani, Ranjana H

    2016-07-01

    The development of Bruton's tyrosine kinase (BTK) inhibitors and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL) and other B-cell lymphomas. Although ibrutinib is the only BTK inhibitor that has been approved by the US Food and Drug Administration, several others are under investigation. Ibrutinib is currently approved for use in relapsed/refractory CLL, CLL with 17p deletion (del[17p]), relapsed or refractory mantle cell lymphoma, and Waldenström macroglobulinemia. Although it is clear that ibrutinib has altered treatment paradigms and outcomes in these diseases, several questions remain regarding (1) its role in frontline vs salvage therapy; (2) its use as a single agent vs in combination with biologic agents, other small molecules, or traditional chemoimmunotherapy; (3) the optimal duration of treatment; and (4) the treatment of patients who cannot tolerate or have disease resistant to ibrutinib. Because sparse clinical data are available on other BTK inhibitors, it is unclear at present whether their clinical efficacy and toxicity will differ from those of ibrutinib. PMID:27379948

  9. Identification of “Preferred” Human Kinase Inhibitors for Sleeping Sickness Lead Discovery. Are Some Kinases Better than Others for Inhibitor Repurposing?

    PubMed Central

    2016-01-01

    A kinase-targeting cell-based high-throughput screen (HTS) against Trypanosoma brucei was recently reported, and this screening set included the Published Kinase Inhibitor Set (PKIS). From the PKIS was identified 53 compounds with pEC50 ≥ 6. Utilizing the published data available for the PKIS, a statistical analysis of these active antiparasitic compounds was performed, allowing identification of a set of human kinases having inhibitors that show a high likelihood for blocking T. brucei cellular proliferation in vitro. This observation was confirmed by testing other established inhibitors of these human kinases and by mining past screening campaigns at GlaxoSmithKline. Overall, although the parasite targets of action are not known, inhibitors of this set of human kinases displayed an enhanced hit rate relative to a random kinase-targeting HTS campaign, suggesting that repurposing efforts should focus primarily on inhibitors of these specific human kinases. We therefore term this statistical analysis-driven approach “preferred lead repurposing”. PMID:26998514

  10. The Next Wave of EGFR Tyrosine Kinase Inhibitors Enter the Clinic.

    PubMed

    Politi, Katerina; Ayeni, Deborah; Lynch, Thomas

    2015-06-01

    The T790M mutation in EGFR accounts for approximately half of all lung cancer cases with acquired resistance to the current clinical EGFR tyrosine kinase inhibitors. In tyrosine kinase inhibitor-resistant lung tumors, rociletinib and AZD9291 are highly active when T790M is present and modestly active when T790M is absent. PMID:26058074

  11. Discovery of CX-6258. A Potent, Selective, and Orally Efficacious pan-Pim Kinases Inhibitor.

    PubMed

    Haddach, Mustapha; Michaux, Jerome; Schwaebe, Michael K; Pierre, Fabrice; O'Brien, Sean E; Borsan, Cosmin; Tran, Joe; Raffaele, Nicholas; Ravula, Suchitra; Drygin, Denis; Siddiqui-Jain, Adam; Darjania, Levan; Stansfield, Ryan; Proffitt, Chris; Macalino, Diwata; Streiner, Nicole; Bliesath, Joshua; Omori, May; Whitten, Jeffrey P; Anderes, Kenna; Rice, William G; Ryckman, David M

    2012-02-01

    Structure-activity relationship analysis in a series of 3-(5-((2-oxoindolin-3-ylidene)methyl)furan-2-yl)amides identified compound 13, a pan-Pim kinases inhibitor with excellent biochemical potency and kinase selectivity. Compound 13 exhibited in vitro synergy with chemotherapeutics and robust in vivo efficacy in two Pim kinases driven tumor models. PMID:24900437

  12. Mechanisms of resistance to EGFR tyrosine kinase inhibitors

    PubMed Central

    Huang, Lihua; Fu, Liwu

    2015-01-01

    Since the discovery that non-small cell lung cancer (NSCLC) is driven by epidermal growth factor receptor (EGFR) mutations, the EGFR tyrosine kinase inhibitors (EGFR-TKIs, e.g., gefitinib and elrotinib) have been effectively used for clinical treatment. However, patients eventually develop drug resistance. Resistance to EGFR-TKIs is inevitable due to various mechanisms, such as the secondary mutation (T790M), activation of alternative pathways (c-Met, HGF, AXL), aberrance of the downstream pathways (K-RAS mutations, loss of PTEN), impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism), histologic transformation, ATP binding cassette (ABC) transporter effusion, etc. Here we review and summarize the known resistant mechanisms to EGFR-TKIs and provide potential targets for development of new therapeutic strategies. PMID:26579470

  13. Discovery of a Selective Aurora A Kinase Inhibitor by Virtual Screening.

    PubMed

    Kilchmann, Falco; Marcaida, Maria J; Kotak, Sachin; Schick, Thomas; Boss, Silvan D; Awale, Mahendra; Gönczy, Pierre; Reymond, Jean-Louis

    2016-08-11

    Here we report the discovery of a selective inhibitor of Aurora A, a key regulator of cell division and potential anticancer target. We used the atom category extended ligand overlap score (xLOS), a 3D ligand-based virtual screening method recently developed in our group, to select 437 shape and pharmacophore analogs of reference kinase inhibitors. Biochemical screening uncovered two inhibitor series with scaffolds unprecedented among kinase inhibitors. One of them was successfully optimized by structure-based design to a potent Aurora A inhibitor (IC50 = 2 nM) with very high kinome selectivity for Aurora kinases. This inhibitor locks Aurora A in an inactive conformation and disrupts binding to its activator protein TPX2, which impairs Aurora A localization at the mitotic spindle and induces cell division defects. This phenotype can be rescued by inhibitor-resistant Aurora A mutants. The inhibitor furthermore does not induce Aurora B specific effects in cells. PMID:27391133

  14. Small Molecule Substrate Phosphorylation Site Inhibitors of Protein Kinases: Approaches and Challenges

    PubMed Central

    2015-01-01

    Protein kinases are important mediators of cellular communication and attractive drug targets for many diseases. Although success has been achieved with developing ATP-competitive kinase inhibitors, the disadvantages of ATP-competitive inhibitors have led to increased interest in targeting sites outside of the ATP binding pocket. Kinase inhibitors with substrate-competitive, ATP-noncompetitive binding modes are promising due to the possibility of increased selectivity and better agreement between biochemical and in vitro potency. However, the difficulty of identifying these types of inhibitors has resulted in significantly fewer small molecule substrate phosphorylation site inhibitors being reported compared to ATP-competitive inhibitors. This review surveys reported substrate phosphorylation site inhibitors and methods that can be applied to the discovery of such inhibitors, including a discussion of the challenges inherent to these screening methods. PMID:25494294

  15. Bumped kinase inhibitor prohibits egression in Babesia bovis.

    PubMed

    Pedroni, Monica J; Vidadala, Rama Subba Rao; Choi, Ryan; Keyloun, Katelyn R; Reid, Molly C; Murphy, Ryan C; Barrett, Lynn K; Van Voorhis, Wesley C; Maly, Dustin J; Ojo, Kayode K; Lau, Audrey O T

    2016-01-15

    Babesiosis is a global zoonotic disease acquired by the bite of a Babesia-infected Ixodes tick or through blood transfusion with clinical relevance affecting humans and animals. In this study, we evaluated a series of small molecule compounds that have previously been shown to target specific apicomplexan enzymes in Plasmodium, Toxoplasma and Cryptosporidium. The compounds, bumped kinase inhibitors (BKIs), have strong therapeutic potential targeting apicomplexa-specific calcium dependent protein kinases (CDPKs). We investigated if BKIs also show inhibitory activities against piroplasms such as Babesia. Using a subset of BKIs that have promising inhibitory activities to Plasmodium and Toxoplasma, we determined that their actions ranged from 100% and no inhibition against Babesia bovis blood stages. One specific BKI, RM-1-152, showed complete inhibition against B. bovis within 48h and was the only BKI that showed noticeable phenotypic changes to the parasites. Focusing our study on this BKI, we further demonstrated that RM-1-152 has Babesia-static activity and involves the prohibition of merozoite egress while replication and re-invasion of host cells are unaffected. The distinct, abnormal phenotype induced by RM-1-152 suggests that this BKI can be used to investigate less studied cellular processes such as egression in piroplasm. PMID:26790733

  16. Mitochondrial-Targeting MET Kinase Inhibitor Kills Erlotinib-Resistant Lung Cancer Cells.

    PubMed

    Yang, Tianming; Ng, Wai Har; Chen, Huan; Chomchopbun, Kamon; Huynh, The Hung; Go, Mei Lin; Kon, Oi Lian

    2016-08-11

    Lung cancer cells harboring activating EGFR mutations acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) by activating several bypass mechanisms, including MET amplification and overexpression. We show that a significant proportion of activated MET protein in EGFR TKI-resistant HCC827 lung cancer cells resides within the mitochondria. Targeting the total complement of MET in the plasma membrane and mitochondria should render these cells more susceptible to cell death and hence provide a means of circumventing drug resistance. Herein, the mitochondrial targeting triphenylphosphonium (TPP) moiety was introduced to the selective MET kinase inhibitor PHA665752. The resulting TPP analogue rapidly localized to the mitochondria of MET-overexpressing erlotinib-resistant HCC827 cells, partially suppressed the phosphorylation (Y1234/Y1235) of MET in the mitochondrial inner membrane and was as cytotoxic and apoptogenic as the parent compound. These findings provide support for the targeting of mitochondrial MET with a TPP-TKI conjugate as a means of restoring responsiveness to chemotherapy. PMID:27563407

  17. The protein kinase 2 inhibitor tetrabromobenzotriazole protects against renal ischemia reperfusion injury

    PubMed Central

    Ka, Sun-O; Hwang, Hong Pil; Jang, Jong-Hwa; Hyuk Bang, In; Bae, Ui-Jin; Yu, Hee Chul; Cho, Baik Hwan; Park, Byung-Hyun

    2015-01-01

    Protein kinase 2 (CK2) activation was reported to enhance reactive oxygen species production and activate the nuclear factor κB (NF-κB) pathway. Because oxidative stress and inflammation are critical events for tissue destruction during ischemia reperfusion (I/R), we sought to determine whether CK2 was important in the renal response to I/R. Mice underwent 25 min of renal ischemia and were then reperfused. We confirmed an increased expression of CK2α during the reperfusion period, while expression of CK2β remained consistent. We administered tetrabromobenzotriazole (TBBt), a selective CK2α inhibitor before inducing I/R injury. Mice subjected to I/R injury showed typical patterns of acute kidney injury; blood urea nitrogen and serum creatinine levels, tubular necrosis and apoptosis, inflammatory cell infiltration and proinflammatory cytokine production, and oxidative stress were markedly increased when compared to sham mice. However, pretreatment with TBBt abolished these changes and improved renal function and architecture. Similar renoprotective effects of CK2α inhibition were observed for emodin. Renoprotective effects of CK2α inhibition were associated with suppression of NF-κB and mitogen activated protein kinase (MAPK) pathways. Taken together, these results suggest that CK2α mediates proapoptotic and proinflammatory signaling, thus the CK2α inhibitor may be used to prevent renal I/R injuries observed in clinical settings. PMID:26423352

  18. Norathyriol Suppresses Skin Cancers Induced by Solar Ultraviolet Radiation by Targeting ERK Kinases

    SciTech Connect

    Li, Jixia; Malakhova, Margarita; Mottamal, Madhusoodanan; Reddy, Kanamata; Kurinov, Igor; Carper, Andria; Langfald, Alyssa; Oi, Naomi; Kim, Myoung Ok; Zhu, Feng; Sosa, Carlos P.; Zhou, Keyuan; Bode, Ann M.; Dong, Zigang

    2012-06-27

    Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, prompting great interest in chemopreventive agents for this disease. In this study, we report the discovery of norathyriol, a plant-derived chemopreventive compound identified through an in silico virtual screening of the Chinese Medicine Library. Norathyriol is a metabolite of mangiferin found in mango, Hypericum elegans, and Tripterospermum lanceolatum and is known to have anticancer activity. Mechanistic investigations determined that norathyriol acted as an inhibitor of extracellular signal-regulated kinase (ERK)1/2 activity to attenuate UVB-induced phosphorylation in mitogen-activated protein kinases signaling cascades. We confirmed the direct and specific binding of norathyriol with ERK2 through a cocrystal structural analysis. The xanthone moiety in norathyriol acted as an adenine mimetic to anchor the compound by hydrogen bonds to the hinge region of the protein ATP-binding site on ERK2. Norathyriol inhibited in vitro cell growth in mouse skin epidermal JB6 P+ cells at the level of G{sub 2}-M phase arrest. In mouse skin tumorigenesis assays, norathyriol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis indicated that norathyriol mediates its chemopreventive activity by inhibiting the ERK-dependent activity of transcriptional factors AP-1 and NF-{kappa}B during UV-induced skin carcinogenesis. Taken together, our results identify norathyriol as a safe new chemopreventive agent that is highly effective against development of UV-induced skin cancer.

  19. Aurora Kinases and Potential Medical Applications of Aurora Kinase Inhibitors: A Review

    PubMed Central

    Gavriilidis, Paschalis; Giakoustidis, Alexandros; Giakoustidis, Dimitrios

    2015-01-01

    Aurora kinases (AKs) represent a novel group of serine/threonine kinases. They were originally described in 1995 by David Glover in the course of studies of mutant alleles characterized with unusual spindle pole configuration in Drosophila melanogaster. Thus far, three AKs A, B, and C have been discovered in human healthy and neoplastic cells. Each one locates in different subcellular locations and they are all nuclear proteins. AKs are playing an essential role in mitotic events such as monitoring of the mitotic checkpoint, creation of bipolar mitotic spindle and alignment of centrosomes on it, also regulating centrosome separation, bio-orientation of chromosomes and cytokinesis. Any inactivation of them can have catastrophic consequences on mitotic events of spindle formation, alignment of centrosomes and cytokinesis, resulting in apoptosis. Overexpression of AKs has been detected in diverse solid and hematological cancers and has been linked with a dismal prognosis. After discovery and identification of the first aurora kinase inhibitor (AKI) ZM447439 as a potential drug for targeted therapy in cancer treatment, approximately 30 AKIs have been introduced in cancer treatment. PMID:26345296

  20. 4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives as New Axl Kinase Inhibitors.

    PubMed

    Tan, Li; Zhang, Zhang; Gao, Donglin; Luo, Jinfeng; Tu, Zheng-Chao; Li, Zhengqiu; Peng, Lijie; Ren, Xiaomei; Ding, Ke

    2016-07-28

    Axl is a new potential target for anticancer drug discovery. A series of 4-oxo-1,4-dihydroquinoline-3-carboxamides were designed and synthesized as highly potent Axl kinase inhibitors. One of the most promising compounds, 9im, tightly bound with Axl protein and potently inhibited its kinase function with a Kd value of 2.7 nM and an IC50 value of 4.0 nM, respectively, while was obviously less potent against most of the 403 wild-type kinases evaluated at a relatively high concentration. The compound dose-dependently inhibited the TGF-β1-induced epithelial-mesenchymal transition (EMT) and suppressed the migration and invasion of MDA-MB-231 breast cancer cells. In addition, 9im also demonstrated reasonable pharmacokinetics properties in rats and exhibited in vivo therapeutic effect on hepatic metastasis in a xenograft model of highly metastatic 4T1 murine breast cancer cells. Compound 9im may serve as a lead compound for new anticancer drug discovery and a valuable research probe for further biological investigation on Axl. PMID:27379978

  1. A mitogen-activated protein kinase kinase inhibitor induced compound skin toxicity with oedema in metastatic malignant melanoma.

    PubMed

    Thomas, C L; Mortimer, P S; Larkin, J M; Basu, T N; Gore, M E; Fearfield, L

    2016-04-01

    We report three cases of skin toxicity associated with oral mitogen-activated protein kinase kinase (MEK) inhibitor treatment for metastatic malignant melanoma (MM). All three patients developed oedema, and a single patient experienced eyelash trichomegaly. This is the first known report of eyelash trichomegaly secondary to MEK inhibitor use. We also discuss possible mechanisms for MEK inhibitor-associated oedema development. This series supports the role of the dermatologist in the screening and management of patients in the rapidly developing oncology setting, as new targeted agents can give rise to marked skin toxicity. PMID:26411345

  2. Can kinomics and proteomics bridge the gap between pediatric cancers and newly designed kinase inhibitors?

    PubMed

    van der Sligte, Naomi E; Kampen, Kim R; de Bont, Eveline S J M

    2015-10-01

    The introduction of kinase inhibitors in cancer medicine has transformed chronic myeloid leukemia from a fatal disease into a leukemia subtype with a favorable prognosis by interfering with the constitutively active kinase BCR-ABL. This success story has resulted in the development of multiple kinase inhibitors. We are currently facing significant limitations in implementing these kinase inhibitors into the clinic for the treatment of pediatric malignancies. As many hallmarks of cancer are known to be regulated by intracellular protein signaling networks, we suggest focusing on these networks to improve the implementation of kinase inhibitors. This viewpoint will provide a short overview of currently used strategies for the implementation of kinase inhibitors as well as reasons why kinase inhibitors have unfortunately not yet been widely used for the treatment of pediatric cancers. We argue that by using a future personalized medicine strategy combining kinomics, proteomics, and drug screen approaches, the gap between pediatric cancers and the use of kinase inhibitors may be bridged. PMID:26321002

  3. Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts

    PubMed Central

    Shugg, Ryan P. P.; Thomson, Ashley; Tanabe, Natsuko; Kashishian, Adam; Steiner, Bart H.; Puri, Kamal D.; Pereverzev, Alexey; Lannutti, Brian J.; Jirik, Frank R.; Dixon, S. Jeffrey; Sims, Stephen M.

    2013-01-01

    Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics. PMID:24133210

  4. AMP-activated protein kinase suppresses matrix metalloproteinase-9 expression in mouse embryonic fibroblasts.

    PubMed

    Morizane, Yuki; Thanos, Aristomenis; Takeuchi, Kimio; Murakami, Yusuke; Kayama, Maki; Trichonas, George; Miller, Joan; Foretz, Marc; Viollet, Benoit; Vavvas, Demetrios G

    2011-05-01

    Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPKα deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPKα1 and AMPKα2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. The AMPK activators 5-amino-4-imidazole carboxamide riboside and A769662 suppressed MMP-9 expression in WT MEFs, and AMPK inhibition by the overexpression of dominant negative (DN) AMPKα elevated MMP-9 expression. However, in AMPKα(-/-) MEFs transduced with DN AMPKα, MMP-9 expression was suppressed. AMPKα(-/-) MEFs showed increased phosphorylation of IκBα, expression of IκBα mRNA, nuclear localization of nuclear factor-κB (NF-κB), and DNA-binding activity of NF-κB compared with WT. Consistently, selective NF-κB inhibitors BMS345541 and SM7368 decreased MMP-9 expression in AMPKα(-/-) MEFs. Overall, our results suggest that both AMPKα isoforms suppress MMP-9 expression and that both the activity and presence of AMPKα contribute to its function as a regulator of MMP-9 expression by inhibiting the NF-κB pathway. PMID:21402702

  5. Comparative analysis of the human and zebrafish kinomes: focus on the development of kinase inhibitors

    PubMed Central

    Wlodarchak, Nathan; Tariq, Rehan; Striker, Rob

    2016-01-01

    Targeting kinases with semi-selective kinase inhibitors is one of the most successful drug development strategies of the 21st century. Zebrafish have become an increasingly useful model for pharmaceutical development. Water-soluble compounds can be screened for zebrafish phenotypes in a high throughput format against a living vertebrate, and cell-signaling events can be imaged in transparent living fish. Despite zebrafish being a more relevant model than more distantly related systems such as the well-annotated kinome of yeast and drosophila, there is no comparative analysis of the human and zebrafish kinome. Furthermore most approved kinase inhibitors, often called ‘DFG in’ ATP competitive inhibitors, act on conserved active site residues in the kinase. Since the active site residues can be identified by examining the primary sequence, primary sequence identity can be a rough guide as to whether a particular inhibitor will have activity against another kinase. There is a need to evaluate the utility of zebrafish as a drug development model for active site inhibitors of kinases. Here we offer a systematic comparison of the catalytic domains of classical human kinases with the catalytic domains of all annotated zebrafish kinases. We found a high degree of identity between the catalytic domains of most human kinases and their zebrafish homologs, and we ranked 504 human kinase catalytic domains by order of similarity. We found only 23 human kinases with no easily recognizable homologous zebrafish catalytic domain. On the other hand we found 78 zebrafish kinase catalytic domains with no close human counterpart. These ‘additional kinase active sites’ could represent potential mediators of zebrafish toxicity that may not be relevant to human kinase inhibitors. We used two clinically approved human kinase inhibitors, one targeting a highly homologous target and one targeting a lesser homologous target, and we compared the known human kinase target structures with

  6. Troglitazone inhibits endothelial cell proliferation through suppression of casein kinase 2 activity

    SciTech Connect

    Lee, Kuy-Sook; Park, Jin-Hee; Lee, Seahyoung; Lim, Hyun-Joung; Jang, Yangsoo; Park, Hyun-Young . E-mail: hypark65@nih.go.kr

    2006-07-21

    Troglitazone, an agonist of peroxisome proliferator activated receptor{gamma} (PPAR{gamma}), has been reported to inhibit endothelial cell proliferation by suppressing Akt activation. Recently, it has been also proposed that phosphatase and tensin homolog deleted from chromosome 10 (PTEN) plays an important role in such effect of troglitazone. However, the mechanism of how troglitazone regulates PTEN remains to be elucidated. We therefore investigated the effects of troglitazone on casein kinase 2 (CK2), which is known to negatively regulate PTEN activity. Troglitazone significantly inhibited serum-induced proliferation of HUVEC in a concentration dependent manner. Serum-induced Akt and its downstream signaling pathway activation was attenuated by troglitazone (10 {mu}M) pretreatment. The phosphorylation of PTEN, which was directly related to Akt activation, was decreased with troglitazone pretreatment and was inversely proportional to CK2 activity. DRB, a CK2 inhibitor, also showed effects similar to that of troglitazone on Akt and its downstream signaling molecules. In conclusion, our results suggest that troglitazone inhibits proliferation of HUVECs through suppression of CK2 activity rendering PTEN to remain activated, and this effect of troglitazone in HUVECs seems to be PPAR{gamma} independent.

  7. The Protein Kinase 2 Inhibitor CX-4945 Regulates Osteoclast and Osteoblast Differentiation In Vitro

    PubMed Central

    Son, You Hwa; Moon, Seong Hee; Kim, Jiyeon

    2013-01-01

    Drug repositioning can identify new therapeutic applications for existing drugs, thus mitigating high R&D costs. The Protein kinase 2 (CK2) inhibitor CX-4945 regulates human cancer cell survival and angiogenesis. Here we found that CX-4945 significantly inhibited the RANKL-induced osteoclast differentiation, but enhanced the BMP2-induced osteoblast differentiation in a cell culture model. CX-4945 inhibited the RANKL-induced activation of TRAP and NFATc1 expression accompanied with suppression of Akt phosphorylation, but, in contrast, it enhanced the BMP2-mediated ALP induction and MAPK ERK1/2 phosphorylation. CX-4945 is thus a novel drug candidate for bone-related disorders such as osteoporosis. PMID:24293011

  8. A chemoproteomic method for identifying cellular targets of covalent kinase inhibitors

    PubMed Central

    Chen, Ying-Chu; Zhang, Chao

    2016-01-01

    Protein kinases are attractive drug targets for numerous human diseases including cancers, diabetes and neurodegeneration. A number of kinase inhibitors that covalently target a cysteine residue in their target kinases have recently entered use in the cancer clinic. Despite the advantages of covalent kinases inhibitors, their inherent reactivity can lead to non-specific binding to other cellular proteins and cause off- target effects in cells. It is thus essential to determine the identity of these off targets in order to fully account for the phenotype and to improve the selectivity and efficacy of covalent inhibitors. Herein we present a detailed protocol for a chemoproteomic method to enrich and identify cellular targets of covalent kinase inhibitors. PMID:27551330

  9. Protein kinase small molecule inhibitors for rheumatoid arthritis: Medicinal chemistry/clinical perspectives

    PubMed Central

    Malemud, Charles J; Blumenthal, David E

    2014-01-01

    Medicinal chemistry strategies have contributed to the development, experimental study of and clinical trials assessment of the first type of protein kinase small molecule inhibitor to target the Janus kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling pathway. The orally administered small molecule inhibitor, tofacitinib, is the first drug to target the JAK/STAT pathway for entry into the armamentarium of the medical therapy of rheumatoid arthritis. The introduction of tofacitinib into general rheumatologic practice coupled with increasing understanding that additional cellular signal transduction pathways including the mitogen-activated protein kinase and phosphatidylinositide-3-kinase/Akt/mammalian target of rapamycin pathways as well as spleen tyrosine kinase also contribute to immune-mediated inflammatory in rheumatoid arthritis makes it likely that further development of orally administered protein kinase small molecule inhibitors for rheumatoid arthritis will occur in the near future. PMID:25232525

  10. Benzothiophene inhibitors of MK2. Part 2: Improvements in kinase selectivity and cell potency

    SciTech Connect

    Anderson, David R.; Meyers, Marvin J.; Kurumbail, Ravi G.; Caspers, Nicole; Poda, Gennadiy I.; Long, Scott A.; Pierce, Betsy S.; Mahoney, Matthew W.; Mourey, Robert J.; Parikh, Mihir D.; Pfizer

    2010-10-01

    Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.

  11. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

    SciTech Connect

    Zhou, Wenjun; Ercan, Dalia; Chen, Liang; Yun, Cai-Hong; Li, Danan; Capelletti, Marzia; Cortot, Alexis B.; Chirieac, Lucian; Iacob, Roxana E.; Padera, Robert; Engen, John R.; Wong, Kwok-Kin; Eck, Michael J.; Gray, Nathanael S.; Jänne, Pasi A.

    2010-01-12

    The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.

  12. Targeting Protein Kinase CK2 Suppresses Pro-survival Signaling Pathways and Growth of Glioblastoma

    PubMed Central

    Zheng, Ying; McFarland, Braden C.; Drygin, Denis; Yu, Hao; Bellis, Susan L.; Kim, Hyunsoo; Bredel, Markus; Benveniste, Etty N.

    2014-01-01

    Purpose Gliomas are the most frequently occurring primary malignancies in the brain, and glioblastoma (GBM) is the most aggressive of these tumors. Protein kinase CK2 is composed of two catalytic subunits (α and/or α’) and two β regulatory subunits. CK2 suppresses apoptosis, promotes neo-angiogenesis, and enhances activation of the JAK/STAT, NF-κB, PI3K/AKT, Hsp90, Wnt and Hedgehog pathways. Aberrant activation of the NF-κB, PI3K/AKT and JAK/STAT-3 pathways is implicated in GBM progression. Since CK2 is involved in their activation, the expression and function of CK2 in GBM was evaluated. Experimental Design and Results Analysis of 537 GBMs from The Cancer Genome Atlas Project demonstrates the CSNK2A1 gene, encoding CK2α, has gene dosage gains in GBM (33.7%), and is significantly associated with the classical GBM subtype. Inhibition of CK2 activity by CX-4945, a selective CK2 inhibitor, or CK2 knockdown by siRNA suppresses activation of the JAK/STAT, NF-κB and AKT pathways and downstream gene expression in human GBM xenografts. On a functional level, CX-4945 treatment decreases the adhesion and migration of GBM cells, in part through inhibition of integrin β1 and α4 expression. In vivo, CX-4945 inhibits activation of STAT-3, NF-κB p65 and AKT, and promotes survival of mice with intracranial human GBM xenografts. Conclusions CK2 inhibitors may be considered for treatment of patients with GBM. PMID:24036851

  13. Pharmaceutical inhibition of glycogen synthetase kinase 3 beta suppresses wear debris-induced osteolysis.

    PubMed

    Geng, Dechun; Wu, Jian; Shao, Hongguo; Zhu, Shijun; Wang, Yijun; Zhang, Wen; Ping, Zichuan; Hu, Xuanyang; Zhu, Xuesong; Xu, Yaozeng; Yang, Huilin

    2015-11-01

    Aseptic loosening is associated with the development of wear debris-induced peri-implant osteolytic bone disease caused by an increased osteoclastic bone resorption and decreased osteoblastic bone formation. However, no effective measures for the prevention and treatment of peri-implant osteolysis currently exist. The aim of this study was to determine whether lithium chloride (LiCl), a selective inhibitor of glycogen synthetase kinase 3 beta (GSK-3β), mitigates wear debris-induced osteolysis in a murine calvarial model of osteolysis. GSK-3β is activated by titanium (Ti) particles, and implantation of Ti particles on the calvarial surface in C57BL/6 mice resulted in osteolysis caused by an increase in the number of osteoclasts and a decrease in the number of osteoblasts. Mice implanted with Ti particles were gavage-fed LiCl (50 or 200 mg kg(-1)d(-1)), 6 days per week for 2 weeks. The LiCl treatment significantly inhibited GSK-3β activity and increased β-catenin and axin-2 expression in a dose-dependent manner, dramatically mitigating the Ti particle-induced suppression of osteoblast numbers and the expression of bone formation markers. Finally, we demonstrated that inhibition of GSK-3β suppresses osteoclast differentiation and reduces the severity of Ti particle-induced osteolysis. The results of this study indicate that Ti particle-induced osteolysis is partly dependent on GSK-3β and, therefore, the canonical Wnt signaling pathway. This suggests that selective inhibitors of GSK-3β such as LiCl may help prevent and treat wear debris-induced osteolysis. PMID:26275858

  14. Targeting polo-like kinase 1 suppresses essential functions of alloreactive T cells.

    PubMed

    Berges, Carsten; Chatterjee, Manik; Topp, Max S; Einsele, Hermann

    2016-06-01

    Acute graft-versus-host disease (aGvHD) is still a major cause of transplant-related mortality after allogeneic stem cell transplantation (ASCT). It requires immunosuppressive treatments that broadly abrogate T cell responses including beneficial ones directed against tumor cells or infective pathogens. Polo-like kinase 1 (PLK1) is overexpressed in many cancer types including leukemia, and clinical studies demonstrated that targeting PLK1 using selective PLK1 inhibitors resulted in inhibition of proliferation and induction of apoptosis predominantly in tumor cells, supporting the feasibility of PLK1 as target for anticancer therapy. Here, we show that activation of alloreactive T cells (Tallo) up-regulate expression of PLK1, suggesting that PLK1 is a potential new candidate for dual therapy of aGvHD and leukemia after ASCT. Inhibition of PLK1, using PLK1-specific inhibitor GSK461364A selectively depletes Tallo by preventing activation and by inducing apoptosis in already activated Tallo, while memory T cells are preserved. Activated Tallo cells which survive exposure to PLK1 undergo inhibition of proliferation by induction of G2/M cell cycle arrest, which is accompanied by accumulation of cell cycle regulator proteins p21(WAF/CIP1), p27(Kip1), p53 and cyclin B1, whereas abundance of CDK4 decreased. We also show that suppressive effects of PLK1 inhibition on Tallo were synergistically enhanced by concomitant inhibition of molecular chaperone Hsp90. Taken together, our data suggest that PLK1 inhibition represents a reasonable dual strategy to suppress residual tumor growth and efficiently deplete Tallo, and thus provide a rationale to selectively prevent and treat aGvHD. PMID:26724940

  15. Combined HSP90 and kinase inhibitor therapy: Insights from The Cancer Genome Atlas.

    PubMed

    Schwartz, Harvey; Scroggins, Brad; Zuehlke, Abbey; Kijima, Toshiki; Beebe, Kristin; Mishra, Alok; Neckers, Len; Prince, Thomas

    2015-09-01

    The merging of knowledge from genomics, cellular signal transduction and molecular evolution is producing new paradigms of cancer analysis. Protein kinases have long been understood to initiate and promote malignant cell growth and targeting kinases to fight cancer has been a major strategy within the pharmaceutical industry for over two decades. Despite the initial success of kinase inhibitors (KIs), the ability of cancer to evolve resistance and reprogram oncogenic signaling networks has reduced the efficacy of kinase targeting. The molecular chaperone HSP90 physically supports global kinase function while also acting as an evolutionary capacitor. The Cancer Genome Atlas (TCGA) has compiled a trove of data indicating that a large percentage of tumors overexpress or possess mutant kinases that depend on the HSP90 molecular chaperone complex. Moreover, the overexpression or mutation of parallel activators of kinase activity (PAKA) increases the number of components that promote malignancy and indirectly associate with HSP90. Therefore, targeting HSP90 is predicted to complement kinase inhibitors by inhibiting oncogenic reprogramming and cancer evolution. Based on this hypothesis, consideration should be given by both the research and clinical communities towards combining kinase inhibitors and HSP90 inhibitors (H90Ins) in combating cancer. The purpose of this perspective is to reflect on the current understanding of HSP90 and kinase biology as well as promote the exploration of potential synergistic molecular therapy combinations through the utilization of The Cancer Genome Atlas. PMID:26070366

  16. Kinase Pathway Dependence in Primary Human Leukemias Determined by Rapid Inhibitor Screening

    PubMed Central

    Tyner, Jeffrey W.; Yang, Wayne F.; Bankhead, Armand; Fan, Guang; Fletcher, Luke B.; Bryant, Jade; Glover, Jason M.; Chang, Bill H.; Spurgeon, Stephen E.; Fleming, William H.; Kovacsovics, Tibor; Gotlib, Jason R.; Oh, Stephen T.; Deininger, Michael W.; Zwaan, C. Michel; Den Boer, Monique L.; van den Heuvel-Eibrink, Marry M.; O’Hare, Thomas; Druker, Brian J.; Loriaux, Marc M.

    2012-01-01

    Kinases are dysregulated in most cancer but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway dysregulation, ultimately enabling a correlation of patient genetic profiles to clinically effective kinase inhibitors. Our methodology assessed the sensitivity of primary leukemia patient samples to a panel of 66 small-molecule kinase inhibitors over 3 days. Screening of 151 leukemia patient samples revealed a wide diversity of drug sensitivities, with 70% of the clinical specimens exhibiting hypersensitivity to one or more drugs. From this data set, we developed an algorithm to predict kinase pathway dependence based on analysis of inhibitor sensitivity patterns. Applying this algorithm correctly identified pathway dependence in proof-of-principle specimens with known oncogenes, including a rare FLT3 mutation outside regions covered by standard molecular diagnostic tests. Interrogation of all 151 patient specimens with this algorithm identified a diversity of gene targets and signaling pathways that could aid prioritization of deep sequencing data sets, permitting a cumulative analysis to understand kinase pathway dependence within leukemia subsets. In a proof-of-principle case, we showed that in vitro drug sensitivity could predict both a clinical response and the development of drug resistance. Taken together, our results suggested that drug target scores derived from a comprehensive kinase inhibitor panel could predict pathway dependence in cancer cells while simultaneously identifying potential therapeutic options. PMID:23087056

  17. Activated Drosophila Ras1 is selectively suppressed by isoprenyl transferase inhibitors.

    PubMed Central

    Kauffmann, R C; Qian, Y; Vogt, A; Sebti, S M; Hamilton, A D; Carthew, R W

    1995-01-01

    Ras CAAX (C = cysteine, A = aliphatic amino acid, and X = any amino acid) peptidomimetic inhibitors of farnesyl protein transferase suppress Ras-dependent cell transformation by preventing farnesylation of the Ras oncoprotein. These compounds are potential anticancer agents for tumors associated with Ras mutations. The peptidomimetic FTI-254 was tested for Ras1-inhibiting activity in whole animals by injection of activated Ras1val12 Drosophila larvae. FTI-254 decreased the ability of Ras1val12 to form supernumerary R7 photoreceptor cells in the compound eye of transformed flies. In contrast, it had no effect on the related supernumerary R7 phenotypes of flies transformed with either the activated sevenless receptor tyrosine kinase, Raf kinase, or a chimeric Ras1val12 protein that is membrane associated through myristylation instead of isoprenylation. Therefore, FTI-254 acts as an isoprenylation inhibitor to selectively inhibit Ras1val12 signaling activity in a whole-animal model system. Images Fig. 2 PMID:7479910

  18. Discovery of Bivalent Kinase Inhibitors via Enzyme-Templated Fragment Elaboration

    PubMed Central

    2015-01-01

    We have employed novel fragment-based screening methodology to discover bivalent kinase inhibitors with improved selectivity. Starting from a low molecular weight promiscuous kinase inhibitor, we appended a thiol for subsequent reaction with a library of acrylamide electrophiles. Enzyme-templated screening was performed to identify acrylamides that assemble into bivalent inhibitors of c-Src kinase. Upon identification of acrylamide fragments that improve the binding affinity of our lead thiol, we characterized the resulting bivalent inhibitors and identified a series of kinase inhibitors with improved potency and selectivity compared to the thiol-containing precursor. Provided that protein can be prepared free of endogenous reactive cysteines, our methodology is general and could be applied to nearly any enzyme of interest. PMID:26286460

  19. Protein kinase inhibitors in plants of the myrtaceae, proteaceae, and leguminosae.

    PubMed

    Larkin, M; Brazier, J; Ternai, B; Polya, G M

    1993-12-01

    Methanolic extracts of leaves, flowers, stems, bark, and other parts of representative plants of the Myrtaceae, specifically of the EUCALYPTUS, MELALEUCA, THRYPTOMENA, CALLISTOMEN, ACMENA, AND ANGOPHORA genera, variously contain high levels of inhibitors of plant Ca (2+)-dependent protein kinase (CDPK) and of Ca (2+)-calmodulin-dependent myosin light chain kinase (MLCK). In terms of the protein kinase inhibition unit (PKIU), defined as the amount in the standard protein kinase assays causing 50% inhibition of protein kinase activity, these inhibitor levels ranged from the non-detectable to 179,000 PKIU (gram fresh weight) (-1) [(g FW) (-1)] and there was no consistent pattern of inhibitor distribution. A variety of other plants tested had low or non-detectable levels of CDPK and MLCK inhibitors. Plants of the EUCALYPTUS, MELALEUCA, ANGOPHORA, and GREVILLEA genera contained inhibitors of the catalytic subunit of the cyclic AMP-dependent protein kinase (cAK), inhibitor levels ranging from 20,000 to 9,600,000 PKIU (g FW) (-1). In general, cAK inhibitor levels found in the Myrtaceae were mostly much higher than levels of CDPK and MLCK inhibitors and reversed phase HPLC of such plant extracts revealed a multiplicity of components associated with cAK inhibitory activity. These IN VITRO screening procedures enable rapid detection and quantitation of levels of bioactive plant defence compounds with medicinal potential. PMID:17230363

  20. Mutations in G protein β subunits promote transformation and kinase inhibitor resistance.

    PubMed

    Yoda, Akinori; Adelmant, Guillaume; Tamburini, Jerome; Chapuy, Bjoern; Shindoh, Nobuaki; Yoda, Yuka; Weigert, Oliver; Kopp, Nadja; Wu, Shuo-Chieh; Kim, Sunhee S; Liu, Huiyun; Tivey, Trevor; Christie, Amanda L; Elpek, Kutlu G; Card, Joseph; Gritsman, Kira; Gotlib, Jason; Deininger, Michael W; Makishima, Hideki; Turley, Shannon J; Javidi-Sharifi, Nathalie; Maciejewski, Jaroslaw P; Jaiswal, Siddhartha; Ebert, Benjamin L; Rodig, Scott J; Tyner, Jeffrey W; Marto, Jarrod A; Weinstock, David M; Lane, Andrew A

    2015-01-01

    Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling. PMID:25485910

  1. Discovery, Biological Evaluation and Structure-Activity Relationship of Amidine-Based Sphingosine Kinase Inhibitors

    PubMed Central

    Mathews, Thomas P.; Kennedy, Andrew J.; Kharel, Yugesh; Kennedy, Perry C.; Nicoara, Oana; Sunkara, Manjula; Morris, Andrew J.; Wamhoff, Brian R.; Lynch, Kevin R.; Macdonald, Timothy L.

    2010-01-01

    Sphingosine 1-phosphate (S1P), a potent phospholipid growth and trophic factor, is synthesized in vivo by two sphingosine kinases. Thus these kinases have been proposed as important drug targets for treatment of hyper-proliferative diseases and inflammation. We report here a new class of amidine-based sphingosine analogs that are competitive inhibitors of sphingosine kinases exhibiting varying degrees of enzyme selectivity. These inhibitors display KI values in the submicromolar range for both sphingosine kinases and, in cultured vascular smooth muscle cells, decrease S1P levels and initiate growth arrest. PMID:20205392

  2. The Rho kinase inhibitor Y-27632 facilitates the differentiation of bone marrow mesenchymal stem cells.

    PubMed

    Liu, Xiao; Zhang, Zhengzheng; Yan, Xianliang; Liu, He; Zhang, Licai; Yao, Aiming; Guo, Chengcheng; Liu, Xiaoyun; Xu, Tie

    2014-12-01

    The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10 µg/l) and basic fibroblastic growth factor (bFGF, 10 µg/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10 µM). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20 ± 8.677 (positive ratio 0.2140 ± 0.0119) to 109.20 ± 8.430 (positive ratio 0.3193 ± 0.0161) per visual field in the presence of Y-27632, while GFAP-positive cell number increased from 96.30 ± 8.486 (positive ratio 0.18 ± 0.0152) to 107.50 ± 8.683 (positive ratio 0.27 ± 0.0115) (P < 0.05 for both). Both NSE and GFAP protein expression levels were enhanced significantly by Y-27632 treatment (NSE: 0.74 ± 0.05 vs. 1.03 ± 0.06; GFAP: 0.64 ± 0.08 vs. 0.97 ± 0.05, both P < 0.01) as indicated by Western blots. The Rho kinase inhibitor Y-27632 concomitant with EGF and bFGF stimulation promotes BMSC differentiation into neural cells. Control of Rho kinase activity may enhance the efficiency of stem cell-based treatments for neurodegenerative diseases. PMID:25178638

  3. Protein kinase c inhibitor attenuates cyanide toxicity in vivo

    SciTech Connect

    Maduh, E.U.; Nealley, E.W.; Song, H.; Wang, P.C.; Baskin, S.I.

    1995-12-31

    We have examined the effect of pretreatment with a potent protein kinase C (PKC) inhibitor, l-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7), against metabolic alterations induced by sodium cyanide (NaCN), 4.2 mg/kg, in brain of anesthetized male micropigs (6-10 kg). Brain high energy phosphates were analyzed using a 3/P nuclear magnetic resonance (NMR) spectroscopic surface coil in a 4.7 Tesla horizontal bore magnet. H-7, I mg/kg, was given intravenously (i.v.) 30 min before NaCN challenge (H-7 + CN). Prior to NaCN, H-7, or H-7 + CN administration, baseline 31P resonance spectra of 1-min duration were acquired for 5-10 min, and continued for an additional 60 min following i.v. NaCN injection, each animal serving as its own control. Peaks were identified as phosphomonoester (PME), inorganic phosphate (Pi), phosphodiester (PDE), phosphocreatine (PCr) and adenosine triphosphate (ATP), based on their respective chemical shifts. Without H-7 pretreatment, NaCN effects were marked by a rising Pi and a declining PCr peak 2 min after injection, with only 2/5 of the animals surviving the 60 min experiment. Through a pretreatment period of 30 min, H-7 did not affect baseline cell energy profile as reflected by the 31P-NMR spectra, but in its presence, those changes (i.e. diminishing PCr and rising Pi peaks) elicited by NaCN were markedly blunted; 4/5 of the animals in this group survived the NaCN challenge. It is proposed that H-7, a pharmacologic inhibitor of PKC, may be useful in CN antagonism, underscoring the role of PKC in cyanide intoxication.

  4. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    SciTech Connect

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  5. Checkpoint kinase inhibitors: SAR and radioprotective properties of a series of 2-arylbenzimidazoles.

    PubMed

    Arienti, Kristen L; Brunmark, Anders; Axe, Frank U; McClure, Kelly; Lee, Alice; Blevitt, Jon; Neff, Danielle K; Huang, Liming; Crawford, Shelby; Pandit, Chennagiri R; Karlsson, Lars; Breitenbucher, J Guy

    2005-03-24

    The discovery of a series of novel, potent, and highly selective inhibitors of the DNA damage control kinase chk2 is disclosed. Here we report the first SAR study around inhibitors of this kinase. High-throughput screening of purified human chk2 led to the identification of a novel series of 2-arylbenzimidazole inhibitors of the kinase. Optimization was facilitated using homology models of chk2 and docking of inhibitors, leading to the highly potent 2-arylbenzimidazole 2h (IC(50) 15 nM). Compound 2h is an ATP-competitive inhibitor of chk2 that dose dependently protects human CD4(+) and CD8(+) T-cells from apoptosis due to ionizing radiation. This work suggests that a selective small molecule inhibitor of chk2 could be a useful adjuvant to radiotherapy, increasing the therapeutic window of such treatment. PMID:15771432

  6. A roadmap to evaluate the proteome-wide selectivity of covalent kinase inhibitors

    PubMed Central

    Dix, Melissa M.; Douhan, John; Gilbert, Adam M.; Hett, Erik C.; Johnson, Theodore O.; Joslyn, Chris; Kath, John C.; Niessen, Sherry; Roberts, Lee R.; Schnute, Mark E.; Wang, Chu; Hulce, Jonathan J.; Wei, Baoxian; Whiteley, Laurence O.; Hayward, Matthew M.; Cravatt, Benjamin F.

    2014-01-01

    Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active-sites have emerged as valuable probes and approved drugs. Many protein classes, however, possess functional cysteines and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative mass spectrometry to globally map the targets, both specific and non-specific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent non-kinase proteins that, interestingly, possess conserved, active-site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental roadmap to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors. PMID:25038787

  7. Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia

    PubMed Central

    Green, Alexa S.; Maciel, Thiago T.; Hospital, Marie-Anne; Yin, Chae; Mazed, Fetta; Townsend, Elizabeth C.; Pilorge, Sylvain; Lambert, Mireille; Paubelle, Etienne; Jacquel, Arnaud; Zylbersztejn, Florence; Decroocq, Justine; Poulain, Laury; Sujobert, Pierre; Jacque, Nathalie; Adam, Kevin; So, Jason C. C.; Kosmider, Olivier; Auberger, Patrick; Hermine, Olivier; Weinstock, David M.; Lacombe, Catherine; Mayeux, Patrick; Vanasse, Gary J.; Leung, Anskar Y.; Moura, Ivan C.; Bouscary, Didier; Tamburini, Jerome

    2015-01-01

    ABSTRACT Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD–induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD+ cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy. PMID:26601252

  8. Have adjuvant tyrosine kinase inhibitors lost their shine?

    PubMed Central

    Sabari, Joshua K.

    2016-01-01

    Despite broad advances in molecularly targeted therapies, lung cancer remains the leading cause of cancer related mortality in the United States. Epidermal growth factor receptor (EGFR) mutations occur in approximately 17% of advanced non-small cell lung cancer (NSCLC) in the US population. The remarkable efficacy of small-molecule EGFR tyrosine kinase inhibitors (TKIs) in this unique subset of patients has revolutionized the therapeutic approach to lung cancer. The success of these agents in the metastatic setting leads to the logical question of what role these drugs may have in the adjuvant setting for patients with earlier stage disease. RADIANT, an international randomized, double-blind, placebo controlled phase III study in patients with completely resected stage IB to IIIA NSLC whose tumors expressed EGFR by IHC and EGFR amplification by FISH, attempted to answer the question of whether erlotinib would improve disease free survival and overall survival in the adjuvant setting. While RADIANT does not conclude for or against adjuvant use of EGFR-TKIs, all data points towards benefit in a selected population. As clinicians, we must continue to enroll to potentially practice changing therapeutic neoadjuvant and adjuvant chemotherapy studies internationally. PMID:27568486

  9. Have adjuvant tyrosine kinase inhibitors lost their shine?

    PubMed

    Sabari, Joshua K; Chaft, Jamie E

    2016-08-01

    Despite broad advances in molecularly targeted therapies, lung cancer remains the leading cause of cancer related mortality in the United States. Epidermal growth factor receptor (EGFR) mutations occur in approximately 17% of advanced non-small cell lung cancer (NSCLC) in the US population. The remarkable efficacy of small-molecule EGFR tyrosine kinase inhibitors (TKIs) in this unique subset of patients has revolutionized the therapeutic approach to lung cancer. The success of these agents in the metastatic setting leads to the logical question of what role these drugs may have in the adjuvant setting for patients with earlier stage disease. RADIANT, an international randomized, double-blind, placebo controlled phase III study in patients with completely resected stage IB to IIIA NSLC whose tumors expressed EGFR by IHC and EGFR amplification by FISH, attempted to answer the question of whether erlotinib would improve disease free survival and overall survival in the adjuvant setting. While RADIANT does not conclude for or against adjuvant use of EGFR-TKIs, all data points towards benefit in a selected population. As clinicians, we must continue to enroll to potentially practice changing therapeutic neoadjuvant and adjuvant chemotherapy studies internationally. PMID:27568486

  10. Trichomonas vaginalis thymidine kinase: purification, characterization and search for inhibitors.

    PubMed Central

    Strosselli, S; Spadari, S; Walker, R T; Basnak, I; Focher, F

    1998-01-01

    We report that a thymidine kinase (TK) activity is present in Trichomonas vaginalis and can be separated from the deoxyribonucleoside phosphotransferase. T. vaginalis TK, purified 11200-fold to apparent homogeneity, has a molecular mass of 31500 Da. It phosphorylates not only thymidine (Km 0.18 microM) but also deoxycytidine (Km 0.88 microM) and deoxyuridine (Km 0.14 microM). In contrast with T. vaginalis deoxyribonucleoside phosphotransferase, the TK activity is strongly inhibited by novel deoxyuridine analogues such as 5-methyl-4'-thio-2'-deoxyuridine (MTdU) (Ki 20 nM) and 5-iodo-4'-thio-2'-deoxyuridine (ITdU) (Ki 24 nM). MTdU and ITdU are phosphorylated by T. vaginalis TK in vitro. In vivo they inhibit [3H]thymidine incorporation in T. vaginalis cultured cells and T. vaginalis growth (IC50 7.5 and 24 microM respectively; minimal lethal dose 100 microM). Thus the TK inhibitors described here demonstrate the key role of T. vaginalis TK for protozoal growth and viability and indicate TK as a new target for the design of antitrichomonal drugs. PMID:9693096

  11. Identification of inhibitors of checkpoint kinase 1 through template screening.

    PubMed

    Matthews, Thomas P; Klair, Suki; Burns, Samantha; Boxall, Kathy; Cherry, Michael; Fisher, Martin; Westwood, Isaac M; Walton, Michael I; McHardy, Tatiana; Cheung, Kwai-Ming J; Van Montfort, Rob; Williams, David; Aherne, G Wynne; Garrett, Michelle D; Reader, John; Collins, Ian

    2009-08-13

    Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells. PMID:19572549

  12. Discovery of Clinical Candidate CEP-37440, a Selective Inhibitor of Focal Adhesion Kinase (FAK) and Anaplastic Lymphoma Kinase (ALK).

    PubMed

    Ott, Gregory R; Cheng, Mangeng; Learn, Keith S; Wagner, Jason; Gingrich, Diane E; Lisko, Joseph G; Curry, Matthew; Mesaros, Eugen F; Ghose, Arup K; Quail, Matthew R; Wan, Weihua; Lu, Lihui; Dobrzanski, Pawel; Albom, Mark S; Angeles, Thelma S; Wells-Knecht, Kevin; Huang, Zeqi; Aimone, Lisa D; Bruckheimer, Elizabeth; Anderson, Nathan; Friedman, Jay; Fernandez, Sandra V; Ator, Mark A; Ruggeri, Bruce A; Dorsey, Bruce D

    2016-08-25

    Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein. PMID:27527804

  13. Differential inhibitor sensitivity between human kinases VRK1 and VRK2.

    PubMed

    Vázquez-Cedeira, Marta; Barcia-Sanjurjo, Iria; Sanz-García, Marta; Barcia, Ramiro; Lazo, Pedro A

    2011-01-01

    Human vaccinia-related kinases (VRK1 and VRK2) are atypical active Ser-Thr kinases implicated in control of cell cycle entry, apoptosis and autophagy, and affect signalling by mitogen activated protein kinases (MAPK). The specific structural differences in VRK catalytic sites make them suitable candidates for development of specific inhibitors. In this work we have determined the sensitivity of VRK1 and VRK2 to kinase inhibitors, currently used in biological assays or in preclinical studies, in order to discriminate between the two proteins as well as with respect to the vaccinia virus B1R kinase. Both VRK proteins and vaccinia B1R are poorly inhibited by inhibitors of different types targeting Src, MEK1, B-Raf, JNK, p38, CK1, ATM, CHK1/2 and DNA-PK, and most of them have no effect even at 100 µM. Despite their low sensitivity, some of these inhibitors in the low micromolar range are able to discriminate between VRK1, VRK2 and B1R. VRK1 is more sensitive to staurosporine, RO-31-8220 and TDZD8. VRK2 is more sensitive to roscovitine, RO 31-8220, Cdk1 inhibitor, AZD7762, and IC261. Vaccinia virus B1R is more sensitive to staurosporine, KU55933, and RO 31-8220, but not to IC261. Thus, the three kinases present a different pattern of sensitivity to kinase inhibitors. This differential response to known inhibitors can provide a structural framework for VRK1 or VRK2 specific inhibitors with low or no cross-inhibition. The development of highly specific VRK1 inhibitors might be of potential clinical use in those cancers where these kinases identify a clinical subtype with a poorer prognosis, as is the case of VRK1 in breast cancer. PMID:21829721

  14. Benzobisthiazoles Represent a Novel Scaffold for Kinase Inhibitors of CLK Family Members

    PubMed Central

    2015-01-01

    Protein kinases are essential regulators of most cellular processes and are involved in the etiology and progression of multiple diseases. The cdc2-like kinases (CLKs) have been linked to various neurodegenerative disorders, metabolic regulation, and virus infection, and the kinases have been recognized as potential drug targets. Here, we have developed a screening workflow for the identification of potent CLK2 inhibitors and identified compounds with a novel chemical scaffold structure, the benzobisthiazoles, that has not been previously reported for kinase inhibitors. We propose models for binding of these compounds to CLK family proteins and key residues in CLK2 that are important for the compound interactions and the kinase activity. We identified structural elements within the benzobisthiazole that determine CLK2 and CLK3 inhibition, thus providing a rationale for selectivity assays. In summary, our results will inform structure-based design of CLK family inhibitors based on the novel benzobisthiazole scaffold. PMID:26701387

  15. A unified approach to the important protein kinase inhibitor balanol and a proposed analogue

    PubMed Central

    Saha, Tapan; Maitra, Ratnava

    2013-01-01

    Summary A common approach to the important protein kinase inhibitor (−)-balanol and an azepine-ring-modified balanol derivative has been developed using an efficient fragment coupling protocol which proceeded in good overall yield. PMID:24454570

  16. Cytoplasmic kinases downstream of GPR30 suppress gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone secretion from bovine anterior pituitary cells

    PubMed Central

    RUDOLF, Faidiban O.; KADOKAWA, Hiroya

    2015-01-01

    GPR30 is known as a membrane receptor for picomolar concentrations of estradiol. The GPR30-specific agonist G1 causes a rapid, non-genomic suppression of gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion from bovine anterior pituitary (AP) cells. A few studies have recently clarified that protein kinase A (PKA) and phosphorylated extracellular signal-regulated kinase (pERK) might be involved in cytoplasmic signaling pathways of GPR30 in other cells. Therefore, we tested the hypothesis that PKA and ERK kinase (MEK) are important cytoplasmic mediators for GPR30-associated non-genomic suppression of GnRH-induced LH secretion from bovine AP cells. Bovine AP cells (n = 8) were cultured for 3 days under steroid-free conditions. The AP cells were previously treated for 30 min with one of the following: 5000 nM of PKA inhibitor (H89), 1000 nM of MEK inhibitor (U0126), or a combination of H89 and U0126. Next, the AP cells were treated with 0.01 nM estradiol for 5 min before GnRH stimulation. Estradiol treatment without inhibitor pretreatment significantly suppressed GnRH-induced LH secretion (P < 0.01). In contrast, estradiol treatment after pretreatment with H89, U0126 or their combination had no suppressive effect on GnRH-induced LH secretion. The inhibitors also inhibited the G1 suppression of GnRH-induced LH secretion. Therefore, these data supported the hypothesis that PKA and MEK (thus, also pERK) are the intracellular mediators downstream of GPR30 that induce the non-genomic suppression of GnRH-induced LH secretion from bovine AP cells by estradiol or G1. PMID:26522383

  17. Selective interleukin-1 receptor–associated kinase 4 inhibitors for the treatment of autoimmune disorders and lymphoid malignancy

    PubMed Central

    Kelly, Priscilla N.; Romero, Donna L.; Yang, Yibin; Shaffer, Arthur L.; Chaudhary, Divya; Robinson, Shaughnessy; Miao, Wenyan; Rui, Lixin; Westlin, William F.; Kapeller, Rosana

    2015-01-01

    Pathological activation of the Toll-like receptor signaling adaptor protein MYD88 underlies many autoimmune and inflammatory disease states. In the activated B cell–like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), the oncogenic MYD88 L265P mutation occurs in 29% of cases, making it the most prevalent activating mutation in this malignancy. IRAK4 kinase accounts for almost all of the biological functions of MYD88, highlighting IRAK4 as a therapeutic target for diseases driven by aberrant MYD88 signaling. Using innovative structure-based drug design methodologies, we report the development of highly selective and bioavailable small molecule IRAK4 inhibitors, ND-2158 and ND-2110. These small molecules suppressed LPS-induced TNF production, alleviated collagen-induced arthritis, and blocked gout formation in mouse models. IRAK4 inhibition promoted killing of ABC DLBCL lines harboring MYD88 L265P, by down-modulating survival signals, including NF-κB and autocrine IL-6/IL-10 engagement of the JAK–STAT3 pathway. In ABC DLBCL xenograft models, IRAK4 inhibition suppressed tumor growth as a single agent, and in combination with the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib or the Bcl-2 inhibitor ABT-199. Our findings support pharmacological inhibition of IRAK4 as a therapeutic strategy in autoimmune disorders, in a genetically defined population of ABC DLBCL, and possibly other malignancies dependent on aberrant MYD88 signaling. PMID:26621451

  18. Selective interleukin-1 receptor-associated kinase 4 inhibitors for the treatment of autoimmune disorders and lymphoid malignancy.

    PubMed

    Kelly, Priscilla N; Romero, Donna L; Yang, Yibin; Shaffer, Arthur L; Chaudhary, Divya; Robinson, Shaughnessy; Miao, Wenyan; Rui, Lixin; Westlin, William F; Kapeller, Rosana; Staudt, Louis M

    2015-12-14

    Pathological activation of the Toll-like receptor signaling adaptor protein MYD88 underlies many autoimmune and inflammatory disease states. In the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), the oncogenic MYD88 L265P mutation occurs in 29% of cases, making it the most prevalent activating mutation in this malignancy. IRAK4 kinase accounts for almost all of the biological functions of MYD88, highlighting IRAK4 as a therapeutic target for diseases driven by aberrant MYD88 signaling. Using innovative structure-based drug design methodologies, we report the development of highly selective and bioavailable small molecule IRAK4 inhibitors, ND-2158 and ND-2110. These small molecules suppressed LPS-induced TNF production, alleviated collagen-induced arthritis, and blocked gout formation in mouse models. IRAK4 inhibition promoted killing of ABC DLBCL lines harboring MYD88 L265P, by down-modulating survival signals, including NF-κB and autocrine IL-6/IL-10 engagement of the JAK-STAT3 pathway. In ABC DLBCL xenograft models, IRAK4 inhibition suppressed tumor growth as a single agent, and in combination with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib or the Bcl-2 inhibitor ABT-199. Our findings support pharmacological inhibition of IRAK4 as a therapeutic strategy in autoimmune disorders, in a genetically defined population of ABC DLBCL, and possibly other malignancies dependent on aberrant MYD88 signaling. PMID:26621451

  19. Quinalizarin as a potent, selective and cell-permeable inhibitor of protein kinase CK2.

    PubMed

    Cozza, Giorgio; Mazzorana, Marco; Papinutto, Elena; Bain, Jenny; Elliott, Matthew; di Maira, Giovanni; Gianoncelli, Alessandra; Pagano, Mario A; Sarno, Stefania; Ruzzene, Maria; Battistutta, Roberto; Meggio, Flavio; Moro, Stefano; Zagotto, Giuseppe; Pinna, Lorenzo A

    2009-08-01

    Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone) is a moderately potent and poorly selective inhibitor of protein kinase CK2, one of the most pleiotropic serine/threonine protein kinases, implicated in neoplasia and in other global diseases. By virtual screening of the MMS (Molecular Modeling Section) database, we have now identified quinalizarin (1,2,5,8-tetrahydroxyanthraquinone) as an inhibitor of CK2 that is more potent and selective than emodin. CK2 inhibition by quinalizarin is competitive with respect to ATP, with a Ki value of approx. 50 nM. Tested at 1 microM concentration on a panel of 75 protein kinases, quinalizarin drastically inhibits only CK2, with a promiscuity score (11.1), which is the lowest ever reported so far for a CK2 inhibitor. Especially remarkable is the ability of quinalizarin to discriminate between CK2 and a number of kinases, notably DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase), PIM (provirus integration site for Moloney murine leukaemia virus) 1, 2 and 3, HIPK2 (homeodomain-interacting protein kinase-2), MNK1 [MAPK (mitogen-activated protein kinase)-interacting kinase 1], ERK8 (extracellular-signal-regulated kinase 8) and PKD1 (protein kinase D 1), which conversely tend to be inhibited as drastically as CK2 by commercially available CK2 inhibitors. The determination of the crystal structure of a complex between quinalizarin and CK2alpha subunit highlights the relevance of polar interactions in stabilizing the binding, an unusual characteristic for a CK2 inhibitor, and disclose other structural features which may account for the narrow selectivity of this compound. Tested on Jurkat cells, quinalizarin proved able to inhibit endogenous CK2 and to induce apoptosis more efficiently than the commonly used CK2 inhibitors TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole). PMID:19432557

  20. Targeting colorectal cancer cells by a novel sphingosine kinase 1 inhibitor PF-543.

    PubMed

    Ju, TongFa; Gao, DaQuan; Fang, Zheng-yu

    2016-02-12

    In this study, we showed that PF-543, a novel sphingosine kinase 1 (SphK1) inhibitor, exerted potent anti-proliferative and cytotoxic effects against a panel of established (HCT-116, HT-29 and DLD-1) and primary human colorectal cancer (CRC) cells. Its sensitivity was negatively associated with SphK1 expression level in the CRC cells. Surprisingly, PF-543 mainly induced programmed necrosis, but not apoptosis, in the CRC cells. CRC cell necrotic death was detected by lactate dehydrogenase (LDH) release, mitochondrial membrane potential (MMP) collapse and mitochondrial P53-cyclophilin-D (Cyp-D) complexation. Correspondingly, the necrosis inhibitor necrostatin-1 largely attenuated PF-543-induced cytotoxicity against CRC cells. Meanwhile, the Cyp-D inhibitors (sanglifehrin A and cyclosporin A), or shRNA-mediated knockdown of Cyp-D, remarkably alleviated PF-543-induced CRC cell necrotic death. Reversely, over-expression of wild-type Cyp-D in HCT-116 cells significantly increased PF-543's sensitivity. In vivo, PF-543 intravenous injection significantly suppressed HCT-116 xenograft growth in severe combined immunodeficient (SCID) mice, whiling remarkably improving the mice survival. The in vivo activity by PF-543 was largely attenuated when combined with the Cyp-D inhibitor cyclosporin A. Collectively, our results demonstrate that PF-543 exerts potent anti-CRC activity in vitro and in vivo. Mitochondrial programmed necrosis pathway is likely the key mechanism responsible for PF-543's actions in CRC cells. PMID:26775841

  1. General Ser/Thr Kinases Pharmacophore Approach for Selective Kinase Inhibitors Search as Exemplified by Design of Potent and Selective Aurora A Inhibitors.

    PubMed

    Vasilevich, Natalya I; Aksenova, Elena A; Kazyulkin, Denis N; Afanasyev, Ilya I

    2016-07-01

    A general pharmachophore model for various types of Ser/Thr kinases was developed. Search for the molecules fitting to this pharmacophore among ASINEX proprietary library revealed a number of compounds, which were tested and appeared to possess some activity against several Ser/Thr kinases such as Aurora A, Aurora B and Haspin. The possibility of performing the fine-tuning of the general Ser/Thr pharmacophore to desired types of kinase to get active and selective inhibitors was exemplified by Aurora A kinase. As a result, several hits in 3-5 nm range of activity against Aurora A kinase with rather good selectivity and ADME properties were obtained. PMID:26825399

  2. An inhibitor of Janus kinase 2 prevents polycythemia in mice.

    PubMed

    Mathur, Anjili; Mo, Jan-Rung; Kraus, Manfred; O'Hare, Erin; Sinclair, Peter; Young, Jonathan; Zhao, Shuxia; Wang, Yuxun; Kopinja, Johnny; Qu, Xianlu; Reilly, John; Walker, Deborah; Xu, Lin; Aleksandrowicz, Daniel; Marshall, Gary; Scott, Martin L; Kohl, Nancy E; Bachman, Eric

    2009-08-15

    Polycythemia vera (PV) is a myeloproliferative disorder characterized by increased red cell mass and splenomegaly in the absence of secondary causes [Tefferi A., Spivak J.L., Polycythemia vera: scientific advances and current practice. Semin Hematol 2005;42(4):206-20.]. Recently, several laboratories have discovered that the vast majority of patients with PV carry a single, activating mutation (V617F) in the pseudokinase domain of Janus kinase 2 (Jak2) [Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, et al., Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005;280(24):22788-92; James C, Ugo V, Le Couédic JP, Staerk J, Delhommeau F, Lacout C, et al., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythemia vera. Nature 2005;434(7037):1144-8; Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al., A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352(17):1779-90; Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al., Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005;7(4):387-97.]. This discovery has spurred interest in developing therapies for PV via inhibition of Jak2. We induced polycythemia in mice by administering high dose recombinant erythropoietin (Epo) and determined that administration recapitulates almost all of the major and minor diagnostic features of human PV. We then tested a selective, small molecule inhibitor of Jak2 (Jak2i) and showed that this treatment prevents polycythemia. This prevention of polycythemia was accompanied by lower hematocrits, reduced spleen sizes and reductions in Stat5 phosphorylation (pStat5). Surprisingly, Epo rapidly (<1h) induces mobilization of activated erythroid precursors into the blood, thus allowing drug-response relationships to guide discovery. We conclude that inhibition of Jak2

  3. Discovery of orally active pyrrolopyridine- and aminopyridine-based Met kinase inhibitors

    SciTech Connect

    Cai, Zhen-Wei; Wei, Donna; Schroeder, Gretchen M.; Cornelius, Lyndon A.M.; Kim, Kyoung; Chen, Xiao-Tao; Schmidt, Robert J.; Williams, David K.; Tokarski, John S.; An, Yongmi; Sack, John S.; Manne, Veeraswamy; Kamath, Amrita; Zhang, Yueping; Marathe, Punit; Hunt, John T.; Lombardo, Louis J.; Fargnoli, Joseph; Borzilleri, Robert M.

    2008-09-10

    A series of acylurea analogs derived from pyrrolopyridine and aminopyridine scaffolds were identified as potent inhibitors of Met kinase activity. The SAR at various positions of the two kinase scaffolds was investigated. These studies led to the discovery of compounds 3b and 20b, which demonstrated favorable pharmacokinetic properties in mice and significant antitumor activity in a human gastric carcinoma xenograft model.

  4. Novel Anthraquinone-based Derivatives as Potent Inhibitors for Receptor Tyrosine Kinases

    PubMed Central

    Stasevych, M.; Zvarych, V.; Lunin, V.; Halenova, T.; Savchuk, O.; Dudchak, O.; Vovk, M.; Novikov, V.

    2015-01-01

    The influence of new derivatives of 9,10-anthraquinone with benzoylthiourea, thiazole, triazole and amino acid fragments on the activity of membrane-associated tyrosine kinases was investigated. Inhibitors of protein tyrosine kinase activity of the membrane fraction, as promising agents to search for new potential anticancer agents among the studied compounds, were discovered. PMID:26798182

  5. MAP KINASE ERK 1/2 INHIBITORS INDUCE DYSMORPHOLOGY IN MOUSE WHOLE EMBRYO CULTURE

    EPA Science Inventory

    ROSEN, M.B. and E. S. HUNTER. Reproductive Toxicology Division, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina. MAP kinase Erk1/2 inhibitors induce dysmorphology in mouse whole embryo culture.

    MAP Kinase signal transduction is associated with a variety ...

  6. Hypothermia-induced hyperphosphorylation: a new model to study tau kinase inhibitors

    PubMed Central

    Bretteville, Alexis; Marcouiller, François; Julien, Carl; El Khoury, Noura B.; Petry, Franck R.; Poitras, Isabelle; Mouginot, Didier; Lévesque, Georges; Hébert, Sébastien S.; Planel, Emmanuel

    2012-01-01

    Tau hyperphosphorylation is one hallmark of Alzheimer's disease (AD) pathology. Pharmaceutical companies have thus developed kinase inhibitors aiming to reduce tau hyperphosphorylation. One obstacle in screening for tau kinase inhibitors is the low phosphorylation levels of AD-related phospho-epitopes in normal adult mice and cultured cells. We have shown that hypothermia induces tau hyperphosphorylation in vitro and in vivo. Here, we hypothesized that hypothermia could be used to assess tau kinase inhibitors efficacy. Hypothermia applied to models of biological gradual complexity such as neuronal-like cells, ex vivo brain slices and adult non-transgenic mice leads to tau hyperphosphorylation at multiple AD-related phospho-epitopes. We show that Glycogen Synthase Kinase-3 inhibitors LiCl and AR-A014418, as well as roscovitine, a cyclin-dependent kinase 5 inhibitor, decrease hypothermia-induced tau hyperphosphorylation, leading to different tau phosphorylation profiles. Therefore, we propose hypothermia-induced hyperphosphorylation as a reliable, fast, convenient and inexpensive tool to screen for tau kinase inhibitors. PMID:22761989

  7. Inhibitors of cyclic nucleotide phosphodiesterase isozymes type-III and type-IV suppress mitogenesis of rat mesangial cells.

    PubMed Central

    Matousovic, K; Grande, J P; Chini, C C; Chini, E N; Dousa, T P

    1995-01-01

    We studied interactions between the mitogen-activated protein kinase (MAPK) signalling pathway and cAMP-protein kinase (PKA) signaling pathway in regulation of mitogenesis of mesangial cells (MC) determined by [3H]thymidine incorporation, with or without added EGF. Forskolin or dibutyryl cAMP strongly (by 60-70%) inhibited [3H]thymidine incorporation into MC. Cilostamide, lixazinone or cilostazol selective inhibitors of cAMP-phosphodiesterase (PDE) isozyme PDE-III, inhibited mitogenesis to similar extent as forskolin and DBcAMP and activated in situ PKA, but without detectable increase in cAMP levels. Cilostamide and cilostazol were more than three times more effective at inhibiting mesangial mitogenesis than rolipram and denbufylline, inhibitors of isozyme PDE-IV, even though PDE-IV was two times more abundant in MC than was PDE-III. On the other hand, when incubated with forskolin, rolipram-enhanced cAMP accumulation was far greater (10-100x) than with cilostamide. EGF increased MAPK activity (+300%); PDE isozyme inhibitors which suppressed mitogenesis also inhibited MAPK. PDE isozyme inhibitors also suppressed PDGF-stimulated MC proliferation. We conclude that cAMP inhibits the mitogen-dependent MAPK-signaling pathway probably by decreasing the activity of Raf-1 due to PKA-catalyzed phosphorylation. Further, we surmise that minor increase in the cAMP pool metabolized by PDE-III is intimately related to regulation of mesangial proliferation. Thus, PDE isozyme inhibitors have the potential to suppress MC proliferation by a focused effect upon signaling pathways. Images PMID:7615811

  8. [MORPHOFUNCTIONAL STATE OF BLOOD CELLS AFTER CHRONIC EXPOSURE OF THE PROTEIN KINASES INHIBITOR MALEIMIDE DERIVATIVE].

    PubMed

    Byelinska, I V; Lynchak, O V; Tsyvinska, S M; Rybalchenko, V K

    2015-01-01

    The effect of the protein kinases inhibitor maleimide derivative (MI-1, 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrole-2,5-dione), inhibitor of VEGF-R1,2,3, FGF-R1, EGF-R(h), PDK1, Src(h), Syk(h), YES, ZAP70 et al. with antineoplastic activity, on blood cells parameters of rats after chronic exposure has been studied. Administration of MI-1 at doses 0.027 and 2.7 mg/kg (suppress colon carcinogenesis) for 20 and 26 weeks does not affect the morphofunctional state of red blood cells in healthy rats. This is confirmed by the lack of differences in the concentration of hemoglobin in blood, red blood cells count, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration, hematocrit and mean corpuscular volume, and the number of reticulocytes in blood after 20 and 26 weeks of exposure compared with the control group. MI-1 at indicated doses does not influence total leukocytes count and content (eosinophilic and neutrophilic granulocytes, lymphocytes, monocytes) and does not inhibit thrombocytopoiesis (platelet count remains unchanged). No negative effect of MI-1 on hematopoiesis is not limited (by the hemopoietic system) use of this compound as a potential antitumor drug PMID:26552308

  9. Preclinical Evidence That Trametinib Enhances the Response to Antiangiogenic Tyrosine Kinase Inhibitors in Renal Cell Carcinoma.

    PubMed

    Bridgeman, Victoria L; Wan, Elaine; Foo, Shane; Nathan, Mark R; Welti, Jonathan C; Frentzas, Sophia; Vermeulen, Peter B; Preece, Natasha; Springer, Caroline J; Powles, Thomas; Nathan, Paul D; Larkin, James; Gore, Martin; Vasudev, Naveen S; Reynolds, Andrew R

    2016-01-01

    Sunitinib and pazopanib are antiangiogenic tyrosine kinase inhibitors (TKI) used to treat metastatic renal cell carcinoma (RCC). However, the ability of these drugs to extend progression-free and overall survival in this patient population is limited by drug resistance. It is possible that treatment outcomes in RCC patients could be improved by rationally combining TKIs with other agents. Here, we address whether inhibition of the Ras-Raf-MEK-ERK1/2 pathway is a rational means to improve the response to TKIs in RCC. Using a xenograft model of RCC, we found that tumors that are resistant to sunitinib have a significantly increased angiogenic response compared with tumors that are sensitive to sunitinib in vivo. We also observed significantly increased levels of phosphorylated ERK1/2 in the vasculature of resistant tumors, when compared with sensitive tumors. These data suggested that the Ras-Raf-MEK-ERK1/2 pathway, an important driver of angiogenesis in endothelial cells, remains active in the vasculature of TKI-resistant tumors. Using an in vitro angiogenesis assay, we identified that the MEK inhibitor (MEKI) trametinib has potent antiangiogenic activity. We then show that, when trametinib is combined with a TKI in vivo, more effective suppression of tumor growth and tumor angiogenesis is achieved than when either drug is utilized alone. In conclusion, we provide preclinical evidence that combining a TKI, such as sunitinib or pazopanib, with a MEKI, such as trametinib, is a rational and efficacious treatment regimen for RCC. PMID:26487278

  10. Enzastaurin (LY317615), a Protein Kinase C Beta Selective Inhibitor, Enhances Antiangiogenic Effect of Radiation

    SciTech Connect

    Willey, Christopher D.; Xiao Dakai; Tu Tianxiang; Kim, Kwang Woon; Moretti, Luigi; Niermann, Kenneth J.; Tawtawy, Mohammed N.; Quarles, Chad C. Ph.D.; Lu Bo

    2010-08-01

    Purpose: Angiogenesis has generated interest in oncology because of its important role in cancer growth and progression, particularly when combined with cytotoxic therapies, such as radiotherapy. Among the numerous pathways influencing vascular growth and stability, inhibition of protein kinase B(Akt) or protein kinase C(PKC) can influence tumor blood vessels within tumor microvasculature. Therefore, we wanted to determine whether PKC inhibition could sensitize lung tumors to radiation. Methods and Materials: The combination of the selective PKC{beta} inhibitor Enzastaurin (ENZ, LY317615) and ionizing radiation were used in cell culture and a mouse model of lung cancer. Lung cancer cell lines and human umbilical vascular endothelial cells (HUVEC) were examined using immunoblotting, cytotoxic assays including cell proliferation and clonogenic assays, and Matrigel endothelial tubule formation. In vivo, H460 lung cancer xenografts were examined for tumor vasculature and proliferation using immunohistochemistry. Results: ENZ effectively radiosensitizes HUVEC within in vitro models. Furthermore, concurrent ENZ treatment of lung cancer xenografts enhanced radiation-induced destruction of tumor vasculature and proliferation by IHC. However, tumor growth delay was not enhanced with combination treatment compared with either treatment alone. Analysis of downstream effectors revealed that HUVEC and the lung cancer cell lines differed in their response to ENZ and radiation such that only HUVEC demonstrate phosphorylated S6 suppression, which is downstream of mTOR. When ENZ was combined with the mTOR inhibitor, rapamycin, in H460 lung cancer cells, radiosensitization was observed. Conclusion: PKC appears to be crucial for angiogenesis, and its inhibition by ENZ has potential to enhance radiotherapy in vivo.

  11. Effects of tyrosine kinase inhibitors on the contractility of rat mesenteric resistance arteries.

    PubMed Central

    Toma, C; Jensen, P E; Prieto, D; Hughes, A; Mulvany, M J; Aalkjaer, C

    1995-01-01

    1. A pharmacological characterization of tyrosine kinase inhibitors (TKI) belonging to two distinct groups (competitors at the ATP-binding site and the substrate-binding site, respectively) was performed, based on their effects on the contractility of rat mesenteric arteries. 2. Both the ATP-site competitors (genistein and its inactive analogue, daidzein) and the substrate-site competitors (tyrphostins A-23, A-47 and the inactive analogue, A-1) reversibly inhibited noradrenaline (NA, (10 microM)) and KCl (125 mM) induced contractions, concentration-dependently. Genistein was slightly but significantly more potent than daidzein; the tyrphostins were all less potent than genistein, and there were no significant differences between the individual potencies. The tyrosine kinase substrate-site inhibitor bis-tyrphostin had no inhibitory effect. 3. Genistein, daidzein, A-23 and A-47 each suppressed the contraction induced by Ca2+ (1 microM) in alpha-toxin permeabilized arteries. A-1 and bis-tyrphostin had little or no effect on contraction of the permeabilized arteries. 4. Genistein was significantly more potent than daidzein with respect to inhibition of the contraction induced by 200 nM Ca2+ in the presence of NA (100 microM) and GTP (3 microM). The effect of A-23, A-47, A-1 and bis-tyrphostin was similar in permeabilized arteries activated with Ca2+ (200 nM) + NA (100 microM) + GTP (3 microM) and permeabilized arteries activated with 1 microM Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7620718

  12. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

    PubMed Central

    Wilson, Timothy R.; Fridlyand, Jane; Yan, Yibing; Penuel, Elicia; Burton, Luciana; Chan, Emily; Peng, Jing; Lin, Eva; Wang, Yulei; Sosman, Jeff; Ribas, Antoni; Li, Jiang; Moffat, John; Sutherlin, Daniel P.; Koeppen, Hartmut; Merchant, Mark; Neve, Richard; Settleman, Jeff

    2013-01-01

    Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy1. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance2,3. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase4. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK)5. Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma6 or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-‘addicted’ human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases. PMID:22763448

  13. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors.

    PubMed

    Wilson, Timothy R; Fridlyand, Jane; Yan, Yibing; Penuel, Elicia; Burton, Luciana; Chan, Emily; Peng, Jing; Lin, Eva; Wang, Yulei; Sosman, Jeff; Ribas, Antoni; Li, Jiang; Moffat, John; Sutherlin, Daniel P; Koeppen, Hartmut; Merchant, Mark; Neve, Richard; Settleman, Jeff

    2012-07-26

    Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK). Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-'addicted' human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases. PMID:22763448

  14. A review of a novel, Bruton's tyrosine kinase inhibitor, ibrutinib.

    PubMed

    Lee, Chung-Shien; Rattu, Mohammad A; Kim, Sara S

    2016-02-01

    Ibrutinib, a Bruton's kinase inhibitor, was granted an accelerated approval by the US Food and Drug Administration in November, 2013, for the treatment of relapsed or refractory mantle cell lymphoma and subsequently for the treatment of relapsed refractory chronic lymphocytic leukemia in February, 2014. In the pivotal phase 2 study of 111 patients with relapsed or refractory mantle cell lymphoma, the overall response rate in patients who received ibrutinib 560 mg daily was 68%. The median progression-free survival was 13.9 months, and the overall survival was 58% at 18 months. In a recently published phase 3 trial (RESONATE) that compared ibrutinib and ofatumumab for the treatment of relapsed and refractory chronic lymphocytic leukemia or small lymphocytic lymphoma, ibrutinib at the daily dosage of 420 mg demonstrated a significantly higher overall response rate (43% in ibrutinib vs. 4% in ofatumumab) and a significantly improved overall survival at 12 months (90% ibrutinib vs. 81% ofatumumab). Similar clinical benefits were shown regardless of del (17 p). Ibrutinib was well tolerated, and dose-limiting toxicity was not observed. Ibrutinib has shown durable remission, improved progression-free survival and overall survival, and favorable safety profile in indolent B-cell lymphoid malignancies. Ibrutinib, as a monotherapy, is an effective treatment modality as a salvage therapy for treatment of mantle cell lymphoma and chronic lymphocytic leukemia / small lymphocytic lymphoma, particularly in older patients (age ≥70 years) who are not a candidate for intensive chemotherapy and/or those with del (17 p). In patients with chronic lymphocytic leukemia and del (17 p), the current practice guideline recommends ibrutinib as an upfront treatment option. Current on-going trials will further define its role as upfront therapy and/or as a combination therapy in indolent B-cell lymphoid malignancies. PMID:25425007

  15. Mice lacking the Raf-1 kinase inhibitor protein exhibit exaggerated hypoxia-induced pulmonary hypertension

    PubMed Central

    Morecroft, I; Doyle, B; Nilsen, M; Kolch, W; Mair, K; MacLean, MR

    2011-01-01

    BACKGROUND AND PURPOSE Increased pulmonary vascular remodelling, pulmonary arterial pressure and pulmonary vascular resistance characterize the development of pulmonary arterial hypertension (PAH). Activation of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)1/2 is thought to play an important role in PAH and Raf-1 kinase inhibitor protein (RKIP), negatively regulates this pathway. This study investigated whether genetic deletion of RKIP (and hence ERK1/2 up-regulation) resulted in a pulmonary hypertensive phenotype in mice and investigated a role for RKIP in mitogen-regulated proliferative responses in lung fibroblasts. EXPERIMENTAL APPROACH Pulmonary vascular haemodynamics and remodelling were assessed in mice genetically deficient in RKIP (RKIP−/−) after 2 weeks of either normoxia or hypoxia. Immunoblotting and immunohistochemistry were used to examine phosphorylation of Raf-1, RKIP and ERK1/2 in mouse pulmonary arteries. In vitro, RKIP inhibition of mitogen signalling was analysed in CCL39 hamster lung fibroblasts. KEY RESULTS RKIP−/− mice demonstrated elevated indices of PAH and ERK1/2 phosphorylation compared with wild-type (WT) mice. Hypoxic RKIP−/− mice exhibited exaggerated PAH indices. Hypoxia increased phosphorylation of Raf-1, RKIP and ERK1/2 in WT mouse pulmonary arteries and Raf-1 phosphorylation in RKIP−/− mouse pulmonary arteries. In CCL39 cells, inhibition of RKIP potentiated mitogen-induced proliferation and phosphorylation of RKIP, and Raf-1. CONCLUSIONS AND IMPLICATIONS The lack of RKIP protein resulted in a pulmonary hypertensive phenotype, exaggerated in hypoxia. Hypoxia induced phosphorylation of RKIP signalling elements in WT pulmonary arteries. RKIP inhibition potentiated mitogen-induced proliferation in lung fibroblasts. These results provide evidence for the involvement of RKIP in suppressing the development of hypoxia-induced PAH in mice. PMID:21385176

  16. Virtual screening of selective multitarget kinase inhibitors by combinatorial support vector machines.

    PubMed

    Ma, X H; Wang, R; Tan, C Y; Jiang, Y Y; Lu, T; Rao, H B; Li, X Y; Go, M L; Low, B C; Chen, Y Z

    2010-10-01

    Multitarget agents have been increasingly explored for enhancing efficacy and reducing countertarget activities and toxicities. Efficient virtual screening (VS) tools for searching selective multitarget agents are desired. Combinatorial support vector machines (C-SVM) were tested as VS tools for searching dual-inhibitors of 11 combinations of 9 anticancer kinase targets (EGFR, VEGFR, PDGFR, Src, FGFR, Lck, CDK1, CDK2, GSK3). C-SVM trained on 233-1,316 non-dual-inhibitors correctly identified 26.8%-57.3% (majority >36%) of the 56-230 intra-kinase-group dual-inhibitors (equivalent to the 50-70% yields of two independent individual target VS tools), and 12.2% of the 41 inter-kinase-group dual-inhibitors. C-SVM were fairly selective in misidentifying as dual-inhibitors 3.7%-48.1% (majority <20%) of the 233-1,316 non-dual-inhibitors of the same kinase pairs and 0.98%-4.77% of the 3,971-5,180 inhibitors of other kinases. C-SVM produced low false-hit rates in misidentifying as dual-inhibitors 1,746-4,817 (0.013%-0.036%) of the 13.56 M PubChem compounds, 12-175 (0.007%-0.104%) of the 168 K MDDR compounds, and 0-84 (0.0%-2.9%) of the 19,495-38,483 MDDR compounds similar to the known dual-inhibitors. C-SVM was compared to other VS methods Surflex-Dock, DOCK Blaster, kNN and PNN against the same sets of kinase inhibitors and the full set or subset of the 1.02 M Zinc clean-leads data set. C-SVM produced comparable dual-inhibitor yields, slightly better false-hit rates for kinase inhibitors, and significantly lower false-hit rates for the Zinc clean-leads data set. Combinatorial SVM showed promising potential for searching selective multitarget agents against intra-kinase-group kinases without explicit knowledge of multitarget agents. PMID:20712327

  17. Design, Synthesis, and Structure-Activity Relationships of Pyridine-Based Rho Kinase (ROCK) Inhibitors.

    PubMed

    Green, Jeremy; Cao, Jingrong; Bandarage, Upul K; Gao, Huai; Court, John; Marhefka, Craig; Jacobs, Marc; Taslimi, Paul; Newsome, David; Nakayama, Tomoko; Shah, Sundeep; Rodems, Steve

    2015-06-25

    The Rho kinases (ROCK1 and ROCK2) are highly homologous serine/threonine kinases that act on substrates associated with cellular motility, morphology, and contraction and are of therapeutic interest in diseases associated with cellular migration and contraction, such as hypertension, glaucoma, and erectile dysfunction. Beginning with compound 4, an inhibitor of ROCK1 identified through high-throughput screening, systematic exploration of SAR, and application of structure-based design, led to potent and selective ROCK inhibitors. Compound 37 represents significant improvements in inhibition potency, kinase selectivity, and CYP inhibition and possesses pharmacokinetics suitable for in vivo experimentation. PMID:26039570

  18. Bivalent Inhibitors of c-Src Tyrosine Kinase That Bind a Regulatory Domain.

    PubMed

    Johnson, Taylor K; Soellner, Matthew B

    2016-07-20

    We have developed a general methodology to produce bivalent kinase inhibitors for c-Src that interact with the SH2 and ATP binding pockets. Our approach led to a highly selective bivalent inhibitor of c-Src. We demonstrate impressive selectivity for c-Src over homologous kinases. Exploration of the unexpected high level of selectivity yielded insight into the inherent flexibility of homologous kinases. Finally, we demonstrate that our methodology is modular and both the ATP-competitive fragment and conjugation chemistry can be swapped. PMID:27266260

  19. Exploiting the repertoire of CK2 inhibitors to target DYRK and PIM kinases.

    PubMed

    Cozza, Giorgio; Sarno, Stefania; Ruzzene, Maria; Girardi, Cristina; Orzeszko, Andrzej; Kazimierczuk, Zygmunt; Zagotto, Giuseppe; Bonaiuto, Emanuela; Di Paolo, Maria Luisa; Pinna, Lorenzo A

    2013-07-01

    Advantage has been taken of the relative promiscuity of commonly used inhibitors of protein kinase CK2 to develop compounds that can be exploited for the selective inhibition of druggable kinases other than CK2 itself. Here we summarize data obtained by altering the scaffold of CK2 inhibitors to give rise to novel selective inhibitors of DYRK1A and to a powerful cell permeable dual inhibitor of PIM1 and CK2. In the former case one of the new compounds, C624 (naphto [1,2-b]benzofuran-5,9-diol) displays a potency comparable to that of the first-in-class DYRK1A inhibitor, harmine, lacking however the drawback of drastically inhibiting monoamine oxidase-A (MAO-A) as harmine does. On the other hand the promiscuous CK2 inhibitor 4,5,6,7-tetrabromo-1H-benzimidazole (TBI,TBBz) has been derivatized with a sugar moiety to generate a 1-(β-D-2'-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (TDB) compound which inhibits PIM1 and CK2 with comparably high efficacy (IC50 values<100nM) and remarkable selectivity. TDB, unlike other dual PIM1/CK2 inhibitors described in the literature is readily cell permeable and displays a cytotoxic effect on cancer cells consistent with concomitant inhibition of both its onco-kinase targets. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012). PMID:23360763

  20. Ruxolitinib: An Oral Janus Kinase 1 and Janus Kinase 2 Inhibitor in the Management of Myelofibrosis

    PubMed Central

    Verstovsek, Srdan

    2016-01-01

    Myelofibrosis (MF), polycythemia vera (PV), and essential thrombocythemia (ET) are referred to as the classic Philadelphia chromosome (BCR-ABL1)-negative myeloproliferative neoplasms. Although each has distinct pathologic features, all 3 display alterations in Janus kinase (JAK) signal transduction activator of transcription signaling. Myelofibrosis is the most serious of the 3, associated with shortened survival (median survival, 5–7 years); bone marrow failure with anemia; progressive splenomegaly; and chronic, burdensome symptoms, including fatigue, night sweats, itching, abdominal discomfort, loss of appetite/early satiety, unintentional weight loss, and bone, chest, and abdominal pain. Treatments for MF have been mainly palliative, with the exception of allogeneic stem cell transplantation, which, although potentially curative, is feasible only in a small subpopulation of patients. In November 2011, ruxolitinib, an inhibitor of JAK1 and JAK2, was approved by the US Food and Drug Administration for the treatment of intermediate- or high-risk MF, including primary MF, post-PV MF, and post-ET MF. In clinical trials, ruxolitinib was shown to reduce spleen volume and improve MF-related symptoms and quality-of-life measures. Evidence also suggests that ruxolitinib therapy has a survival advantage over placebo and best available therapy. Thrombocytopenia and anemia were the most common adverse events with treatment. Ongoing trials are assessing the efficacy and safety of ruxolitinib therapy in patients with PV and ET. PMID:23391678

  1. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site. PMID:17062013

  2. Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632.

    PubMed

    Yamaguchi, Hiroto; Miwa, Yukiko; Kasa, Miyuki; Kitano, Ken; Amano, Mutsuki; Kaibuchi, Kozo; Hakoshima, Toshio

    2006-09-01

    Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability. PMID:16891330

  3. The lack of target specificity of small molecule anticancer kinase inhibitors is correlated with their ability to damage myocytes in vitro

    SciTech Connect

    Hasinoff, Brian B. Patel, Daywin

    2010-12-01

    Many new targeted small molecule anticancer kinase inhibitors are actively being developed. However, the clinical use of some kinase inhibitors has been shown to result in cardiotoxicity. In most cases the mechanisms by which they exert their cardiotoxicity are not well understood. We have used large scale profiling data on 8 FDA-approved tyrosine kinase inhibitors and 10 other kinase inhibitors to a panel of 317 kinases in order to correlate binding constants and kinase inhibitor binding selectivity scores with kinase inhibitor-induced damage to neonatal rat cardiac myocytes. The 18 kinase inhibitors that were the subject of this study were: canertinib, dasatinib, dovitinib, erlotinib, flavopiridol, gefitinib, imatinib, lapatinib, midostaurin, motesanib, pazopanib, sorafenib, staurosporine, sunitinib, tandutinib, tozasertib, vandetanib and vatalanib. The combined tyrosine kinase and serine-threonine kinase selectivity scores were highly correlated with the myocyte-damaging effects of the kinase inhibitors. This result suggests that myocyte damage was due to a lack of target selectivity to binding of both tyrosine kinases and serine-threonine kinases, and was not due to binding to either group specifically. Finally, the strength of kinase inhibitor binding for 290 kinases was examined for correlations with myocyte damage. Kinase inhibitor binding was significantly correlated with myocyte damage for 12 kinases. Thus, myocyte damage may be multifactorial in nature with the inhibition of a number of kinases involved in producing kinase inhibitor-induced myocyte damage.

  4. The lack of target specificity of small molecule anticancer kinase inhibitors is correlated with their ability to damage myocytes in vitro.

    PubMed

    Hasinoff, Brian B; Patel, Daywin

    2010-12-01

    Many new targeted small molecule anticancer kinase inhibitors are actively being developed. However, the clinical use of some kinase inhibitors has been shown to result in cardiotoxicity. In most cases the mechanisms by which they exert their cardiotoxicity are not well understood. We have used large scale profiling data on 8 FDA-approved tyrosine kinase inhibitors and 10 other kinase inhibitors to a panel of 317 kinases in order to correlate binding constants and kinase inhibitor binding selectivity scores with kinase inhibitor-induced damage to neonatal rat cardiac myocytes. The 18 kinase inhibitors that were the subject of this study were: canertinib, dasatinib, dovitinib, erlotinib, flavopiridol, gefitinib, imatinib, lapatinib, midostaurin, motesanib, pazopanib, sorafenib, staurosporine, sunitinib, tandutinib, tozasertib, vandetanib and vatalanib. The combined tyrosine kinase and serine-threonine kinase selectivity scores were highly correlated with the myocyte-damaging effects of the kinase inhibitors. This result suggests that myocyte damage was due to a lack of target selectivity to binding of both tyrosine kinases and serine-threonine kinases, and was not due to binding to either group specifically. Finally, the strength of kinase inhibitor binding for 290 kinases was examined for correlations with myocyte damage. Kinase inhibitor binding was significantly correlated with myocyte damage for 12 kinases. Thus, myocyte damage may be multifactorial in nature with the inhibition of a number of kinases involved in producing kinase inhibitor-induced myocyte damage. PMID:20832415

  5. Targeting the RAS pathway by mitogen-activated protein kinase inhibitors.

    PubMed

    Kiessling, Michael K; Rogler, Gerhard

    2015-01-01

    Targeting of oncogenic driver mutations with small-molecule inhibitors resulted in powerful treatment options for cancer patients in recent years. The RAS (rat sarcoma) pathway is among the most frequently mutated pathways in human cancer. Whereas targeting mutant Kirsten RAS (KRAS) remains difficult, mutant B rapidly accelerated fibrosarcoma (BRAF) kinase is an established drug target in cancer. Now data show that neuroblastoma RAS (NRAS) and even Harvey RAS (HRAS) mutations could be predictive markers for treatment with mitogen-activated protein kinase (MEK) inhibitors. This review discusses recent preclinical and clinical studies of MEK inhibitors in BRAF and RAS mutant cancer. PMID:26691679

  6. Electrochemical screening of the indole/quinolone derivatives as potential protein kinase CK2 inhibitors.

    PubMed

    Martić, Sanela; Tackenburg, Stefanie; Bilokin, Yaroslav; Golub, Andriy; Bdzhola, Volodymyr; Yarmoluk, Sergiy; Kraatz, Heinz-Bernhard

    2012-02-15

    An electrochemical method based on the bioorganometallic Fc-ATP cosubstrate for kinase-catalyzed phosphorylation reactions was used for monitoring casein kinase 2 (CK2) phosphorylations in the absence and presence of five indole/quinolone-based potential inhibitors. Fc-phosphorylation of immobilized peptide RRRDDDSDDD on Au surfaces resulted in a current density at approximately 460 ± 10 mV. An electrochemical redox signal was significantly decreased in the presence of inhibitors. In addition, the electrochemical signal was concentration dependent with respect to the potential inhibitors 1 to 5, which proved to be viable CK2 drug targets with estimated IC₅₀ values in the nanomolar range. PMID:22178909

  7. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies.

    PubMed

    Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

    2015-01-01

    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated. PMID:25523586

  8. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  9. Discovery of mammalian target of rapamycin (mTOR) kinase inhibitor CC-223.

    PubMed

    Mortensen, Deborah S; Perrin-Ninkovic, Sophie M; Shevlin, Graziella; Zhao, Jingjing; Packard, Garrick; Bahmanyar, Sogole; Correa, Matthew; Elsner, Jan; Harris, Roy; Lee, Branden G S; Papa, Patrick; Parnes, Jason S; Riggs, Jennifer R; Sapienza, John; Tehrani, Lida; Whitefield, Brandon; Apuy, Julius; Bisonette, René R; Gamez, James C; Hickman, Matt; Khambatta, Godrej; Leisten, Jim; Peng, Sophie X; Richardson, Samantha J; Cathers, Brian E; Canan, Stacie S; Moghaddam, Mehran F; Raymon, Heather K; Worland, Peter; Narla, Rama Krishna; Fultz, Kimberly E; Sankar, Sabita

    2015-07-01

    We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in vivo efficacy. These efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of CC-223 for clinical development. PMID:26083478

  10. Targeting inhibition of extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) suppresses growth and angiogenesis of gastric cancer

    PubMed Central

    Gao, Jin-Hang; Wang, Chun-Hui; Tong, Huan; Wen, Shi-Lei; Huang, Zhi-Yin; Tang, Cheng-Wei

    2015-01-01

    AZD6244 (ARRY-142886), a highly selective MAPK-ERK kinase inhibitor, has shown excellent clinical efficacy in many tumors. However, the anti-tumor and anti-angiogenesis efficacy of AZD6244 on gastric cancer has not been well characterized. In this study, high p-ERK expression was associated with advanced TNM stage, increased lymphovascular invasion and poor survival. For absence of NRAS, KRAS and BRAF mutation, SGC7901 and BGC823 gastric cancer cells were relative resistance to AZD6244 in vitro. And such resistance was not attributed to the insufficient inhibition of ERK phosphorylation. However, tumor growth was significantly suppressed in SGC7901 xenografts by blockage of angiogenesis. This result was further supported by suppression of tube formation and migration in HUVEC cells after treatment with AZD6244. Moreover, the anti-angiogenesis effect of AZD6244 may predominantly attribute to its modulation on VEGF through p-ERK − c-Fos − HIF-1α integrated signal pathways. In conclusions, High p-ERK expression was associated with advanced TNM stage, increased lymphovascular invasion and poor survival. Targeting inhibition of p-ERK by AZD6244 suppress gastric cancer xenografts by blockage of angiogenesis without systemic toxicity. The anti-angiogenesis effect afford by AZD6244 may attribute to its modulation on p-ERK − c-Fos − HIF-1α − VEGF integrated signal pathways. PMID:26567773

  11. A derivative of chrysin suppresses two-stage skin carcinogenesis by inhibiting mitogen- and stress-activated kinase 1

    PubMed Central

    Liu, Haidan; Hwang, Joon-Sung; Li, Wei; Choi, Tae Woong; Liu, Kangdong; Huang, Zunnan; Jang, Jae-Hyuk; Thimmegowda, N. R.; Lee, Ki-Won; Ryoo, In-Ja; Ahn, Jong-Seog; Bode, Ann M.; Zhou, Xinmin; Yang, Yifeng; Erikson, Raymond L.; Kim, Bo-Yeon; Dong, Zigang

    2013-01-01

    Mitogen-activated and stress-activated kinase 1 (MSK1) is a nuclear serine/threonine protein kinase that acts downstream of both ERKs and p38 MAP kinases in response to stress or mitogenic extracellular stimuli. Increasing evidence has shown that MSK1 is closely associated with malignant transformation and cancer development. MSK1 should be an effective target for cancer chemoprevention and chemotherapy. However, very few MSK1 inhibitors, especially natural compounds, have been reported. We used virtual screening of a natural products database and the active conformation of the C-terminal kinase domain of MSK1 (PDB id 3KN) as the receptor structure to identify chrysin and its derivative, compound 69407, as inhibitors of MSK1. Compared with chrysin, compound 69407 more strongly inhibited proliferation and TPA-induced neoplastic transformation of JB6 P+ cells with lower cytotoxicity. Western blot data demonstrated that compound 69407 suppressed phosphorylation of the MSK1 downstream effector histone H3 in intact cells. Knocking down the expression of MSK1 effectively reduced the sensitivity of JB6 P+ cells to compound 69407. Moreover, topical treatment with compound 69407 prior to TPA application significantly reduced papilloma development in terms of number and size in a two-stage mouse skin carcinogenesis model. The reduction in papilloma development was accompanied by the inhibition of histone H3 phosphorylation at Ser10 in tumors extracted from mouse skin. The results indicated that compound 69407 exerts inhibitory effects on skin tumorigenesis by directly binding with MSK1 and attenuates the MSK1/histone H3 signaling pathway, which makes it an ideal chemopreventive agent against skin cancer. PMID:24169959

  12. Combined therapeutic potential of nuclear receptors with receptor tyrosine kinase inhibitors in lung cancer

    SciTech Connect

    Wairagu, Peninah M.; Park, Kwang Hwa; Kim, Jihye; Choi, Jong-Whan; Kim, Hyun-Won; Yeh, Byung-Il; Jung, Soon-Hee; Yong, Suk-Joong; Jeong, Yangsik

    2014-05-09

    Highlights: • The 48 NR genes and 48 biological anti-cancer targets are profiled in paired-cells. • Growth inhibition by NR ligands or TKIs is target receptor level-dependent. • T0901317 with gefitinib/PHA665752 shows additive growth inhibition in lung cells. - Abstract: Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where each pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs.

  13. Mangiferin induces apoptosis in multiple myeloma cell lines by suppressing the activation of nuclear factor kappa B-inducing kinase.

    PubMed

    Takeda, Tomoya; Tsubaki, Masanobu; Kino, Toshiki; Yamagishi, Misa; Iida, Megumi; Itoh, Tatsuki; Imano, Motohiro; Tanabe, Genzoh; Muraoka, Osamu; Satou, Takao; Nishida, Shozo

    2016-05-01

    Mangiferin is a naturally occurring glucosyl xanthone, which induces apoptosis in various cancer cells. However, the molecular mechanism underlying mangiferin-induced apoptosis has not been clarified thus far. Therefore, we examined the molecular mechanism underlying mangiferin-induced apoptosis in multiple myeloma (MM) cell lines. We found that mangiferin decreased the viability of MM cell lines in a concentration-dependent manner. We also observed an increased number of apoptotic cells, caspase-3 activation, and a decrease in the mitochondrial membrane potential. In addition, mangiferin inhibited the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated inhibitor kappa B (IκB) and increased the expression of IκB protein, whereas no changes were observed in the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase 1/2 (JNK1/2), and mammalian target of rapamycin (mTOR). The molecular mechanism responsible for mangiferin-induced inhibition of nuclear translocation of NF-κB was a decrease in the expression of phosphorylated NF-κB-inducing kinase (NIK). Moreover, mangiferin decreased the expression of X-linked inhibitor of apoptosis protein (XIAP), survivin, and Bcl-xL proteins. Knockdown of NIK expression showed results similar to those observed with mangiferin treatment. Our results suggest that mangiferin induces apoptosis through the inhibition of nuclear translocation of NF-κB by suppressing NIK activation in MM cell lines. Our results provide a new insight into the molecular mechanism of mangiferin-induced apoptosis. Importantly, since the number of reported NIK inhibitors is limited, mangiferin, which targets NIK, may be a potential anticancer agent for the treatment of MM. PMID:26996543

  14. Differential Sensitivity of Glioma- versus Lung Cancer-specific EGFR mutations to EGFR Kinase Inhibitors

    PubMed Central

    Vivanco, Igor; Robins, H. Ian; Rohle, Daniel; Campos, Carl; Grommes, Christian; Nghiemphu, Phioanh Leia; Kubek, Sara; Oldrini, Barbara; Chheda, Milan G.; Yannuzzi, Nicolas; Tao, Hui; Zhu, Shaojun; Iwanami, Akio; Kuga, Daisuke; Dang, Julie; Pedraza, Alicia; Brennan, Cameron W.; Heguy, Adriana; Liau, Linda M.; Lieberman, Frank; Yung, W.K. Alfred; Gilbert, Mark R.; Reardon, David A.; Drappatz, Jan; Wen, Patrick Y.; Lamborn, Kathleen R.; Chang, Susan M.; Prados, Michael D.; Fine, Howard A.; Horvath, Steve; Wu, Nian; Lassman, Andrew B.; DeAngelis, Lisa M.; Yong, William H.; Kuhn, John G.; Mischel, Paul S.; Mehta, Minesh P.; Cloughesy, Timothy F.; Mellinghoff, Ingo K.

    2012-01-01

    Activation of the epidermal growth factor receptor (EGFR) in glioblastoma (GBM) occurs through mutations or deletions in the extracellular (EC) domain. Unlike lung cancers with EGFR kinase domain (KD) mutations, GBMs respond poorly to the EGFR inhibitor erlotinib. Using RNAi, we show that GBM cells carrying EGFR EC mutations display EGFR addiction. In contrast to KD mutants found in lung cancer, glioma-specific EGFR EC mutants are poorly inhibited by EGFR inhibitors that target the active kinase conformation (e.g., erlotinib). Inhibitors which bind to the inactive EGFR conformation, on the other hand, potently inhibit EGFR EC mutants and induce cell death in EGFR mutant GBM cells. Our results provide first evidence for single kinase addiction in GBM, and suggest that the disappointing clinical activity of first-generation EGFR inhibitors in GBM versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these two cancer types. PMID:22588883

  15. Pharmacophore modeling studies of type I and type II kinase inhibitors of Tie2.

    PubMed

    Xie, Qing-Qing; Xie, Huan-Zhang; Ren, Ji-Xia; Li, Lin-Li; Yang, Sheng-Yong

    2009-02-01

    In this study, chemical feature based pharmacophore models of type I and type II kinase inhibitors of Tie2 have been developed with the aid of HipHop and HypoRefine modules within Catalyst program package. The best HipHop pharmacophore model Hypo1_I for type I kinase inhibitors contains one hydrogen-bond acceptor, one hydrogen-bond donor, one general hydrophobic, one hydrophobic aromatic, and one ring aromatic feature. And the best HypoRefine model Hypo1_II for type II kinase inhibitors, which was characterized by the best correlation coefficient (0.976032) and the lowest RMSD (0.74204), consists of two hydrogen-bond donors, one hydrophobic aromatic, and two general hydrophobic features, as well as two excluded volumes. These pharmacophore models have been validated by using either or both test set and cross validation methods, which shows that both the Hypo1_I and Hypo1_II have a good predictive ability. The space arrangements of the pharmacophore features in Hypo1_II are consistent with the locations of the three portions making up a typical type II kinase inhibitor, namely, the portion occupying the ATP binding region (ATP-binding-region portion, AP), that occupying the hydrophobic region (hydrophobic-region portion, HP), and that linking AP and HP (bridge portion, BP). Our study also reveals that the ATP-binding-region portion of the type II kinase inhibitors plays an important role to the bioactivity of the type II kinase inhibitors. Structural modifications on this portion should be helpful to further improve the inhibitory potency of type II kinase inhibitors. PMID:19138543

  16. Selective Serotonin Reuptake Inhibitor Suppression of HIV Infectivity and Replication

    PubMed Central

    Benton, Tami; Lynch, Kevin; Dubé, Benoit; Gettes, David R.; Tustin, Nancy B.; Lai, Jian Ping; Metzger, David S.; Blume, Joshua; Douglas, Steven D.; Evans, Dwight L.

    2010-01-01

    Objective To test the hypothesis that the selective serotonin reuptake inhibitor (SSRI) citalopram would down regulate HIV infectivity and that the greatest effects would be seen in people with depression. Depression is a risk factor for morbidity and mortality in HIV/AIDS. Serotonin (5-HT) neurotransmission has been implicated in the pathobiology of depression, and pharmacologic therapies for depression target this system. The 5-HT transporter and 5-HT receptors are widely distributed throughout the central nervous and immune systems. Depression has been associated with suppression of natural killer cells (NK) cells and CD8+ lymphocytes, key regulators of HIV infection. Methods Ex-vivo models for acute and chronic HIV infection were used to study the effects of citalopram on HIV viral infection and replication, in 48 depressed and non-depressed women. For both the acute and chronic infection models, HIV reverse transcriptase (RT) activity was measured in the citalopram treatment condition and the control condition. Results The SSRI significantly downregulated the RT response in both the acute and chronic infection models. Specifically, citalopram significantly decreased the acute HIV infectivity of macrophages. Citalopram also significantly decreased HIV viral replication in the latently infected T-cell line and in the latently infected macrophage cell line. There was no difference in down-regulation by depression status. Conclusions These studies suggest that an SSRI enhances NK/CD8 non-cytolytic HIV suppression in HIV/AIDS and decreases HIV viral infectivity of macrophages, ex vivo, suggesting the need for in vivo studies to determine a potential role for agents targeting serotonin in the host defense against HIV. PMID:20947783

  17. Global Effects of Kinase Inhibitors on Signaling Networks Revealed by Quantitative Phosphoproteomics*

    PubMed Central

    Pan, Cuiping; Olsen, Jesper V.; Daub, Henrik; Mann, Matthias

    2009-01-01

    Aberrant signaling causes many diseases, and manipulating signaling pathways with kinase inhibitors has emerged as a promising area of drug research. Most kinase inhibitors target the conserved ATP-binding pocket; therefore specificity is a major concern. Proteomics has previously been used to identify the direct targets of kinase inhibitors upon affinity purification from cellular extracts. Here we introduce a complementary approach to evaluate the effects of kinase inhibitors on the entire cell signaling network. We used triple labeling SILAC (stable isotope labeling by amino acids in cell culture) to compare cellular phosphorylation levels for control, epidermal growth factor stimulus, and growth factor combined with kinase inhibitors. Of thousands of phosphopeptides, less than 10% had a response pattern indicative of targets of U0126 and SB202190, two widely used MAPK inhibitors. Interestingly, 83% of the growth factor-induced phosphorylation events were affected by either or both inhibitors, showing quantitatively that early signaling processes are predominantly transmitted through the MAPK cascades. In contrast to MAPK inhibitors, dasatinib, a clinical drug directed against BCR-ABL, which is the cause of chronic myelogenous leukemia, affected nearly 1,000 phosphopeptides. In addition to the proximal effects on ABL and its immediate targets, dasatinib broadly affected the downstream MAPK pathways. Pathway mapping of regulated sites implicated a variety of cellular functions, such as chromosome remodeling, RNA splicing, and cytoskeletal organization, some of which have been described in the literature before. Our assay is streamlined and generic and could become a useful tool in kinase drug development. PMID:19651622

  18. Crystal structure of a human cyclin-dependent kinase 6 complexwith a flavonol inhibitor, Fisetin

    SciTech Connect

    Lu, Heshu; Chang, Debbie J.; Baratte, Blandine; Meijer, Laurent; Schulze-Gahmen, Ursula

    2005-01-10

    Cyclin-dependent kinases (CDKs) play a central role in cell cycle control, apoptosis, transcription and neuronal functions. They are important targets for the design of drugs with anti-mitotic and/or anti-neurodegenerative effects. CDK4 and CDK6 form a subfamily among the CDKs in mammalian cells, as defined by sequence similarities. Compared to CDK2 and CDK5, structural information on CDK4 and CDK6 is sparse. We describe here the crystal structure of human CDK6 in complex with a viral cyclin and a flavonol inhibitor, fisetin. Fisetin binds to the active form of CDK6, forming hydrogen bonds with the side chains of residues in the binding pocket that undergo large conformational changes during CDK activation by cyclin binding. The 4-keto group and the 3-hydroxyl group of fisetin are hydrogen bonded with the backbone in the hinge region between the N-terminal and C-terminal kinase domain, as has been observed for many CDK inhibitors. However, CDK2 and HCK kinase in complex with other flavone inhibitors such as quercetin and flavopiridol showed a different binding mode with the inhibitor rotated by about 180. The structural information of the CDK6-fisetin complex is correlated with the binding affinities of different flavone inhibitors for CDK6. This complex structure is the first description of an inhibitor complex with a kinase from the CDK4/6 subfamily and can provide a basis for selecting and designing inhibitor compounds with higher affinity and specificity.

  19. Ability of the Met Kinase Inhibitor Crizotinib and New Generation EGFR Inhibitors to Overcome Resistance to EGFR Inhibitors

    PubMed Central

    Nanjo, Shigeki; Yamada, Tadaaki; Nishihara, Hiroshi; Takeuchi, Shinji; Sano, Takako; Nakagawa, Takayuki; Ishikawa, Daisuke; Zhao, Lu; Ebi, Hiromichi; Yasumoto, Kazuo; Matsumoto, Kunio; Yano, Seiji

    2013-01-01

    Purpose Although EGF receptor tyrosine kinase inhibitors (EGFR-TKI) have shown dramatic effects against EGFR mutant lung cancer, patients ultimately develop resistance by multiple mechanisms. We therefore assessed the ability of combined treatment with the Met inhibitor crizotinib and new generation EGFR-TKIs to overcome resistance to first-generation EGFR-TKIs. Experimental Design Lung cancer cell lines made resistant to EGFR-TKIs by the gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression and mice with tumors induced by these cells were treated with crizotinib and a new generation EGFR-TKI. Results The new generation EGFR-TKI inhibited the growth of lung cancer cells containing the gatekeeper EGFR-T790M mutation, but did not inhibit the growth of cells with Met amplification or HGF overexpression. In contrast, combined therapy with crizotinib plus afatinib or WZ4002 was effective against all three types of cells, inhibiting EGFR and Met phosphorylation and their downstream molecules. Crizotinib combined with afatinib or WZ4002 potently inhibited the growth of mouse tumors induced by these lung cancer cell lines. However, the combination of high dose crizotinib and afatinib, but not WZ4002, triggered severe adverse events. Conclusions Our results suggest that the dual blockade of mutant EGFR and Met by crizotinib and a new generation EGFR-TKI may be promising for overcoming resistance to reversible EGFR-TKIs but careful assessment is warranted clinically. PMID:24386407

  20. A rapid assay for assessment of sphingosine kinase inhibitors and substrates

    PubMed Central

    Kharel, Yugesh; Mathews, Thomas P.; Kennedy, Andrew J.; Houck, Joseph D.; Macdonald, Timohy L.; Lynch, Kevin R.

    2011-01-01

    Sphingosine kinases catalyze the transfer of phosphate from ATP to sphingosine to generate sphingosine 1-phosphate, an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of sphingosine kinase enzymatic activity uses [γ-32P]ATP and sphingosine as substrates with the radiolabeled S1P product recovered by organic extraction, displayed by thin-layer chromatography and quantified by liquid scintillation counting. While this assay is sensitive and accurate, it is slow and labor intensive and thus precludes the simultaneous screening of more than a few inhibitor compounds. Herein we describe a 96 well assay for sphingosine kinases that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, post-reaction transfers and chromatography. Further, our assay enables assessment of both inhibitors and substrates, and can detect endogenous sphingosine kinase activity in cell and tissue extracts. The sphingosine kinase kinetic parameter, Km, and the Ki values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well suited for the screening of chemical libraries of sphingosine kinase inhibitors. PMID:21216217

  1. Eribulin synergizes with Polo-like kinase 1 inhibitors to induce apoptosis in rhabdomyosarcoma.

    PubMed

    Stehle, Angelika; Hugle, Manuela; Fulda, Simone

    2015-08-28

    Eribulin, a novel microtubule-interfering drug, was recently shown to exhibit high antitumor activity in vivo against various pediatric cancers. Here, we identify a novel synthetic lethal interaction of Eribulin together with Polo-like kinase 1 (PLK1) inhibitors against rhabdomyosarcoma (RMS) in vitro and in vivo. Eribulin and the PLK1 inhibitor BI 2536 at subtoxic concentrations synergize to induce apoptosis in RMS cells as confirmed by calculation of combination index (CI). Also, Eribulin/BI 2536 co-treatment is significantly more effective than monotherapy to reduce cell viability and inhibit colony formation of RMS cells. Similarly, Eribulin and BI 2536 act in concert to trigger apoptosis in a primary, patient-derived ARMS culture, underscoring the clinical relevance of this combination. Importantly, Eribulin and BI 2536 cooperate to suppress tumor growth in an in vivo model of RMS. On molecular grounds, Eribulin/BI 2536 co-treatment causes profound mitotic arrest, which is critically required for synergism, since inhibition of mitotic arrest by CDK1 inhibitor RO-3306 abolishes Eribulin/BI 2536-mediated apoptosis. Eribulin and BI 2536 cooperate to activate caspase-9, -3 and -8, which is necessary for apoptosis induction, since the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) reduces Eribulin/BI 2536-induced apoptosis significantly, yet partially. Intriguingly, knockdown of endonuclease G (ENDOG) also significantly inhibits Eribulin/BI 2536-triggered apoptosis, demonstrating the involvement of both caspase-dependent and -independent effector pathways. Synergistic induction of apoptosis is similarly found for Eribulin/BI 2536 co-treatment in neuroblastoma cells and for the combination of vincristine (another antimicrotubule chemotherapeutic) with Poloxin (another PLK1 inhibitor), thus pointing to a broader significance of this concomitant microtubule- and PLK1-targeting strategy for pediatric oncology. In

  2. Inhibition of sphingosine kinase prevents lipopolysaccharide-induced preterm birth and suppresses proinflammatory responses in a murine model.

    PubMed

    Vyas, Vibhuti; Ashby, Charles R; Olgun, Nicole S; Sundaram, Sruthi; Salami, Oluwabukola; Munnangi, Swapna; Pekson, Ryan; Mahajan, Prathamesh; Reznik, Sandra E

    2015-03-01

    Premature delivery occurs in 12% of all births, and accounts for nearly half of long-term neurological morbidity, and 60% to 80% of perinatal mortality. Despite advances in obstetrics and neonatology, the rate of premature delivery has increased approximately 12% since 1990. The single most common cause of spontaneous preterm birth is infection. Several lines of evidence have demonstrated the role of endothelin-1 as both a constrictor of uterine myometrial smooth muscle and a proinflammatory mediator. Endothelin-1 activates the phospholipase C pathway, leading to activation of protein kinase C and, in turn, sphingosine kinase (SphK). The inhibition of SphK has been recently shown to control the proinflammatory response associated with sepsis. We show herein, for the first time, that SphK inhibition prevents inflammation-associated preterm birth in a murine model. Rescue of pups from premature abortion with an SphK inhibitor occurs by suppression of the proinflammatory cytokines tumor necrosis factor α, Il-1β, and Il-6 and attenuation of polymorphonuclear inflammatory cells into the placental labyrinth. Moreover, we postulate that inhibition of SphK leads to suppression of endothelin-converting enzyme-1 expression, indicating the presence of an endothelin-converting enzyme 1/endothelin 1-SphK positive feedback loop. This work introduces a novel approach for the control of infection-triggered preterm labor, a condition for which there is no effective treatment. PMID:25579843

  3. Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

    SciTech Connect

    Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian; Huber, Jochen; Tesmer, John J.G.

    2012-07-11

    G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

  4. Repurposing Kinase Inhibitors as Antiviral Agents to Control Influenza A Virus Replication.

    PubMed

    Perwitasari, Olivia; Yan, Xiuzhen; O'Donnell, Jason; Johnson, Scott; Tripp, Ralph A

    2015-12-01

    Influenza A virus (IAV) infection causes seasonal epidemics of contagious respiratory illness that causes substantial morbidity and some mortality. Regular vaccination is the principal strategy for controlling influenza virus, although vaccine efficacy is variable. IAV antiviral drugs are available; however, substantial drug resistance has developed to two of the four currently FDA-approved antiviral drugs. Thus, new therapeutic approaches are being sought to reduce the burden of influenza-related disease. A high-throughput screen using a human kinase inhibitor library was performed targeting an emerging IAV strain (H7N9) in A549 cells. The inhibitor library contained 273 structurally diverse, active cell permeable kinase inhibitors with known bioactivity and safety profiles, many of which are at advanced stages of clinical development. The current study shows that treatment of human A549 cells with kinase inhibitors dinaciclib, flavopiridol, or PIK-75 exhibits potent antiviral activity against H7N9 IAV as well as other IAV strains. Thus, targeting host kinases can provide a broad-spectrum therapeutic approach against IAV. These findings provide a path forward for repurposing existing kinase inhibitors safely as potential antivirals, particularly those that can be tested in vivo and ultimately for clinical use. PMID:26192013

  5. Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold

    PubMed Central

    Huang, Yen-Hua; Henriques, Sónia T.; Wang, Conan K.; Thorstholm, Louise; Daly, Norelle L.; Kaas, Quentin; Craik, David J.

    2015-01-01

    The constitutively active tyrosine kinase BCR-ABL is the underlying cause of chronic myeloid leukemia (CML). Current CML treatments rely on the long-term use of tyrosine kinase inhibitors (TKIs), which target the ATP binding site of BCR-ABL. Over the course of treatment, 20–30% of CML patients develop TKI resistance, which is commonly attributed to point mutations in the drug-binding region. We design a new class of peptide inhibitors that target the substrate-binding site of BCR-ABL by grafting sequences derived from abltide, the optimal substrate of Abl kinase, onto a cell-penetrating cyclotide MCoTI-II. Three grafted cyclotides show significant Abl kinase inhibition in vitro in the low micromolar range using a novel kinase inhibition assay. Our work also demonstrates that a reengineered MCoTI-II with abltide sequences grafted in both loop 1 and 6 inhibits the activity of [T315I]Abl in vitro, a mutant Abl kinase harboring the “gatekeeper” mutation which is notorious for being multidrug resistant. Results from serum stability and cell internalization studies confirm that the MCoTI-II scaffold provides enzymatic stability and cell-penetrating properties to the lead molecules. Taken together, our study highlights that reengineered cyclotides incorporating abltide-derived sequences are promising substrate-competitive inhibitors for Abl kinase and the T315I mutant. PMID:26264857

  6. Targeting Cyclin-Dependent Kinases in Human Cancers: From Small Molecules to Peptide Inhibitors

    PubMed Central

    Peyressatre, Marion; Prével, Camille; Pellerano, Morgan; Morris, May C.

    2015-01-01

    Cyclin-dependent kinases (CDK/Cyclins) form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported. PMID:25625291

  7. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2004-10-12

    The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  8. BRAF kinase inhibitor exerts anti-tumor activity against breast cancer cells via inhibition of FGFR2

    PubMed Central

    Zhang, Zong Xin; Jin, Wen Jun; Yang, Sheng; Ji, Cun Li

    2016-01-01

    Most anti-angiogenic therapies currently being evaluated in clinical trials targetvascular endothelial growth factor (VEGF) pathway; however, the tumor vasculature can acquire resistance to VEGF-targeted therapy by shifting to other angiogenesis mechanisms. Therefore, other potential therapeutic agents that block non-VEGF angiogenic pathways need to be evaluated. Here we identified BRAF kinase inhibitor, vemurafenibas an agent with potential anti-angiogenic and anti-breast cancer activities. Vemurafenib demonstrated inhibition of endothelial cell proliferation, migration, and tube formation in response to basic fibroblast growth factor (bFGF). In ex vivo and in vivo angiogenesis assays, vemurafenib suppressed bFGF-induced microvessel sprouting of rat aortic rings and angiogenesis in vivo. To understand the underlying molecular basis, we examined the effects of vemurafenib on different molecular components in treated endothelial cell, and found that vemurafenib suppressed bFGF-triggered activation of FGFR2 and protein kinase B (AKT). Moreover, vemurafenib directly inhibited proliferation and blocked the oncogenic signaling pathways in breast cancer cell. In vivo, using xenograft models of breast cancer cells MDA-MB-231, vemurafenib showed growth-inhibitory activity associated with inhibition of tumor angiogenesis. Taken together, our results indicate that vemurafenib targets the FGFR2-mediated AKT signaling pathway in endothelial cells, leading to the suppression of tumor growth and angiogenesis. PMID:27293997

  9. BIM expression in treatment naïve cancers predicts responsiveness to kinase inhibitors

    PubMed Central

    Faber, Anthony; Corcoran, Ryan B.; Ebi, Hiromichi; Sequist, Lecia V.; Waltman, Belinda A.; Chung, Euiheon; Incio, Joao; Digumarthy, Subba R.; Pollack, Sarah F.; Song, Youngchul; Muzikansky, Alona; Lifshits, Eugene; Roberge, Sylvie; Coffman, Erik J.; Benes, Cyril; Gómez, Henry; Baselga, Jose; Arteaga, Carlos L.; Rivera, Miguel N.; Dias-Santagata, Dora; Jain, Rakesh K.; Engelman, Jeffrey A.

    2011-01-01

    Cancers with specific genetic mutations are susceptible to selective kinase inhibitors. However, there is wide spectrum of benefit among cancers harboring the same sensitizing genetic mutations. Herein, we measured apoptotic rates among cell lines sharing the same driver oncogene following treatment with the corresponding kinase inhibitor. There was a wide range of kinase inhibitor-induced apoptosis despite comparable inhibition of the target and associated downstream signaling pathways. Surprisingly, pre-treatment RNA levels of the BH3-only pro-apoptotic BIM strongly predicted the capacity of EGFR, HER2, and PI3K inhibitors to induce apoptosis in EGFR mutant, HER2 amplified, and PIK3CA mutant cancers, respectively, but BIM levels did not predict responsiveness to standard chemotherapies. Furthermore, BIM RNA levels in EGFR mutant lung cancer specimens predicted response and duration of clinical benefit from EGFR inhibitors. These findings suggest assessment of BIM levels in treatment naïve tumor biopsies may indicate the degree of benefit from single-agent kinase inhibitors in multiple oncogene-addiction paradigms. PMID:22145099

  10. Antitumor activity of a small-molecule inhibitor of the histone kinase Haspin

    PubMed Central

    Huertas, D; Soler, M; Moreto, J; Villanueva, A; Martinez, A; Vidal, A; Charlton, M; Moffat, D; Patel, S; McDermott, J; Owen, J; Brotherton, D; Krige, D; Cuthill, S; Esteller, M

    2012-01-01

    The approval of histone deacetylase inhibitors for treatment of lymphoma subtypes has positioned histone modifications as potential targets for the development of new classes of anticancer drugs. Histones also undergo phosphorylation events, and Haspin is a protein kinase the only known target of which is phosphorylation of histone H3 at Thr3 residue (H3T3ph), which is necessary for mitosis progression. Mitotic kinases can be blocked by small drugs and several clinical trials are underway with these agents. As occurs with Aurora kinase inhibitors, Haspin might be an optimal candidate for the pharmacological development of these compounds. A high-throughput screening for Haspin inhibitors identified the CHR-6494 compound as being one promising such agent. We demonstrate that CHR-6494 reduces H3T3ph levels in a dose-dependent manner and causes a mitotic catastrophe characterized by metaphase misalignment, spindle abnormalities and centrosome amplification. From the cellular standpoint, the identified small-molecule Haspin inhibitor causes arrest in G2/M and subsequently apoptosis. Importantly, ex vivo assays also demonstrate its anti-angiogenetic features; in vivo, it shows antitumor potential in xenografted nude mice without any observed toxicity. Thus, CHR-6494 is a first-in-class Haspin inhibitor with a wide spectrum of anticancer effects that merits further preclinical research as a new member of the family of mitotic kinase inhibitors. PMID:21804608

  11. Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form*

    PubMed Central

    Gould, Christine M.; Antal, Corina E.; Reyes, Gloria; Kunkel, Maya T.; Adams, Ryan A.; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C.

    2011-01-01

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C. PMID:21715334

  12. Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors

    PubMed Central

    Davare, Monika A.; Vellore, Nadeem A.; Wagner, Jacob P.; Eide, Christopher A.; Goodman, James R.; Drilon, Alexander; Deininger, Michael W.; O’Hare, Thomas; Druker, Brian J.

    2015-01-01

    Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib’s dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1G2032R mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure–function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies. PMID:26372962

  13. Prediction of kinase inhibitor response using activity profiling, in vitro screening, and elastic net regression

    PubMed Central

    2014-01-01

    Background Many kinase inhibitors have been approved as cancer therapies. Recently, libraries of kinase inhibitors have been extensively profiled, thus providing a map of the strength of action of each compound on a large number of its targets. These profiled libraries define drug-kinase networks that can predict the effectiveness of untested drugs and elucidate the roles of specific kinases in different cellular systems. Predictions of drug effectiveness based on a comprehensive network model of cellular signalling are difficult, due to our partial knowledge of the complex biological processes downstream of the targeted kinases. Results We have developed the Kinase Inhibitors Elastic Net (KIEN) method, which integrates information contained in drug-kinase networks with in vitro screening. The method uses the in vitro cell response of single drugs and drug pair combinations as a training set to build linear and nonlinear regression models. Besides predicting the effectiveness of untested drugs, the KIEN method identifies sets of kinases that are statistically associated to drug sensitivity in a given cell line. We compared different versions of the method, which is based on a regression technique known as elastic net. Data from two-drug combinations led to predictive models, and we found that predictivity can be improved by applying logarithmic transformation to the data. The method was applied to the A549 lung cancer cell line, and we identified specific kinases known to have an important role in this type of cancer (TGFBR2, EGFR, PHKG1 and CDK4). A pathway enrichment analysis of the set of kinases identified by the method showed that axon guidance, activation of Rac, and semaphorin interactions pathways are associated to a selective response to therapeutic intervention in this cell line. Conclusions We have proposed an integrated experimental and computational methodology, called KIEN, that identifies the role of specific kinases in the drug response of a given

  14. Recent advances in the development of Aurora kinases inhibitors in hematological malignancies

    PubMed Central

    Choudary, Iqra; Barr, Paul M.; Friedberg, Jonathan

    2015-01-01

    Over the last two decades, since the discovery of Drosophila mutants in 1995, much effort has been made to understand Aurora kinase biology. Three mammalian subtypes have been identified thus far which include the Aurora A, B and C kinases. These regulatory proteins specifically work at the cytoskeleton and chromosomal structures between the kinetochores and have vital functions in the early phases of the mitotic cell cycle. Today, there are multiple phase I and phase II clinical trials as well as numerous preclinical studies taking place looking at Aurora kinase inhibitors in both hematologic and solid malignancies. This review focuses on the preclinical and clinical development of Aurora kinase inhibitors in hematological malignancy and discusses their therapeutic potential. PMID:26622997

  15. AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides*

    PubMed Central

    Ishibashi, Yohei; Hirabayashi, Yoshio

    2015-01-01

    The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer. PMID:26048992

  16. Method for distinguishing normal and transformed cells using G1 kinase inhibitors

    DOEpatents

    Crissman, Harry A.; Gadbois, Donna M.; Tobey, Robert A.; Bradbury, E. Morton

    1993-01-01

    A G.sub.1 phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G.sub.1 phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G.sub.1 cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G.sub.1 phase, suggesting that such G.sub.1 phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

  17. Method for distinguishing normal and transformed cells using G1 kinase inhibitors

    SciTech Connect

    Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Bradbury, E.M.

    1991-12-31

    A G{sub 1} phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G{sub 1} phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G{sub 1} cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G{sub 1} phase, suggesting that such G{sub 1} phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

  18. Benzofuran Small Molecules as Potential Inhibitors of Human Protein Kinases. A Review.

    PubMed

    Kwiecień, Halina; Goszczyńska, Agata; Rokosz, Paulina

    2016-01-01

    Kinases are known to regulate the majority of human cellular processes such as communication, division, metabolism, survival and apoptosis therefore they can be promising targets in cancer diseases, viral infection and in other disorders. Small molecules acting as selective human protein kinase inhibitors are very attractive pharmacological targets. This review presents a number of examples of biologically active natural and synthetic benzo[b]furans and their derivatives, such as benzo[b]furan-2- and 3-ones, benzo[b]furan-2- and 3-carboxylic acids, as well as benzo[c]furans as potential inhibitors of various human protein kinases. The pathways of function and implication of the inhibitors in cancer and other diseases are discussed. PMID:26648467

  19. Method for distinguishing normal and transformed cells using G1 kinase inhibitors

    DOEpatents

    Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Bradbury, E.M.

    1993-02-09

    A G[sub 1] phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G[sub 1] phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G[sub 1] cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G[sub 1] phase, suggesting that such G[sub 1] phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

  20. Cyclin-dependent kinases inhibitors as potential anticancer, antineurodegenerative, antiviral and antiparasitic agents.

    PubMed

    Meijer, Laurent

    2000-04-01

    Cyclin-dependent kinases (CDKs) play a key role in the cell division cycle, in neuronal functions, in transcription and in apoptosis. Intensive screening with these kinases as targets has lead to the identification of highly selective and potent small - molecule inhibitors. Co-crystallization with CDK2 shows that these flat heterocyclic hydrophobic compounds bind through two or three hydrogen bonds with the side chains of two amino acids located in the ATP-binding pocket of the kinase. These inhibitors are anti-proliferative; they arrest cells in G1 and in G2/M phase. Furthermore they facilitate or even trigger apoptosis in proliferating cells while they protect neuronal cells and thymocytes from apoptosis. The potential use of these inhibitors is being extensively evaluated for cancer chemotherapy and also in other therapeutic areas: neurology (Alzheimer's disease), cardiovascular (restenosis, angiogenesis), nephrology (glomerulonephritis), parasitology (Plasmodium, Trypanosoma, Toxoplasma, etc.) and virology (cytomegalovirus, HIV, herpes virus). Copyright 2000 Harcourt Publishers Ltd. PMID:11498372

  1. A Covalent Cysteine-Targeting Kinase Inhibitor of Ire1 Permits Allosteric Control of Endoribonuclease Activity.

    PubMed

    Waller, Daniel D; Jansen, Gregor; Golizeh, Makan; Martel-Lorion, Chloe; Dejgaard, Kurt; Shiao, Tze Chieh; Mancuso, John; Tsantrizos, Youla S; Roy, René; Sebag, Michael; Sleno, Lekha; Thomas, David Y

    2016-05-01

    The unfolded protein response (UPR) initiated by the transmembrane kinase/ribonuclease Ire1 has been implicated in a variety of diseases. Ire1, with its unique position in the UPR, is an ideal target for the development of therapies; however, the identification of specific kinase inhibitors is challenging. Recently, the development of covalent inhibitors has gained great momentum because of the irreversible deactivation of the target. We identified and determined the mechanism of action of the Ire1-inhibitory compound UPRM8. MS analysis revealed that UPRM8 inhibition occurs by covalent adduct formation at a conserved cysteine at the regulatory DFG+2 position in the Ire1 kinase activation loop. Mutational analysis of the target cysteine residue identified both UPRM8-resistant and catalytically inactive Ire1 mutants. We describe a novel covalent inhibition mechanism of UPRM8, which can serve as a lead for the rational design and optimization of inhibitors of human Ire1. PMID:26792008

  2. Raf-kinase inhibitor protein attenuates microglia inflammation in an in vitro model of intracerebral hemorrhage.

    PubMed

    Wang, J; Du, J; Miao, C; Lian, H

    2016-01-01

    Microglia mediated neuroinflammation plays a crucial role in intracerebral hemorrhage (ICH). Raf kinase inhibitor protein (RKIP), a member of the phosphatidylethanolamine-binding protein (PEBP) family, is a negative regulator of inflammatory responses. However, the expression and anti-inflammatory effects of RKIP in microglia after ICH have not been reported. Therefore, in the current study, we investigated the effects of RKIP on inflammatory responses in erythrocyte lysate-treated BV2 microglia. Furthermore, we analyzed the detailed molecular mechanisms underlying the anti-inflammatory effects of RKIP in microglia. Our results showed that the expression level of RKIP was significantly decreased by erythrocyte lysate treatment in BV2 microglia. Overexpression of RKIP inhibited the production of pro-inflammatory molecules. In addition, overexpression of RKIP attenuated neuronal cell death induced by activated microglia. Moreover, RKIP suppressed the activation of NF-κB signaling pathway in erythrocyte lysis-treated BV2 cells. In conclusion, these data suggest that overexpression of RKIP attenuated microglia inflammation through inhibiting the NF-κB signaling pathway in erythrocyte lysis-treated BV2 cells. The present study provides evidence that RKIP may be used as an effective molecular target for the treatment of ICH. PMID:27262809

  3. Suppression of BRCA1 sensitizes cells to proteasome inhibitors

    PubMed Central

    Gu, Y; Bouwman, P; Greco, D; Saarela, J; Yadav, B; Jonkers, J; Kuznetsov, S G

    2014-01-01

    BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity. PMID:25522274

  4. Suppression of BRCA1 sensitizes cells to proteasome inhibitors.

    PubMed

    Gu, Y; Bouwman, P; Greco, D; Saarela, J; Yadav, B; Jonkers, J; Kuznetsov, S G

    2014-01-01

    BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity. PMID:25522274

  5. Synthesis and biological evaluation of 4-quinazolinones as Rho kinase inhibitors.

    PubMed

    Fang, Xingang; Chen, Yen Ting; Sessions, E Hampton; Chowdhury, Sarwat; Vojkovsky, Tomas; Yin, Yan; Pocas, Jennifer R; Grant, Wayne; Schröter, Thomas; Lin, Li; Ruiz, Claudia; Cameron, Michael D; LoGrasso, Philip; Bannister, Thomas D; Feng, Yangbo

    2011-03-15

    Rho kinase (ROCK) is an attractive therapeutic target for various diseases including glaucoma, hypertension, and spinal cord injury. Herein, we report the development of a series of ROCK-II inhibitors based on 4-quinazolinone and quinazoline scaffolds. SAR studies at three positions of the quinazoline core led to the identification of analogs with high potency against ROCK-II and good selectivity over protein kinase A (PKA). PMID:21349713

  6. Slow Inhibition and Conformation Selective Properties of Extracellular Signal-Regulated Kinase 1 and 2 Inhibitors

    PubMed Central

    Rudolph, Johannes; Xiao, Yao; Pardi, Arthur; Ahn, Natalie G.

    2016-01-01

    The mitogen-activated protein (MAP) kinase pathway is a target for anticancer therapy, validated using inhibitors of B-Raf and MAP kinase kinase (MKK) 1 and 2. Clinical outcomes show a high frequency of acquired resistance in patient tumors, involving upregulation of activity of the MAP kinase, extracellular signal-regulated kinase (ERK) 1 and 2. Thus, inhibitors for ERK1/2 are potentially important for targeted therapeutics against cancer. The structures and potencies of different ERK inhibitors have been published, but their kinetic mechanisms have not been characterized. Here we perform enzyme kinetic studies on six representative ERK inhibitors, with potencies varying from 100 pM to 20 μM. Compounds with significant biological activity (IC50 < 100 nM) that inhibit in the subnanomolar range (Vertex-11e and SCH772984) display slow-onset inhibition and represent the first inhibitors of ERK2 known to demonstrate slow dissociation rate constants (values of 0.2 and 1.1 h−1, respectively). Furthermore, we demonstrate using kinetic competition assays that Vertex-11e binds with differing affinities to ERK2 in its inactive, unphosphorylated and active, phosphorylated forms. Finally, two-dimensional heteronuclear multiple-quantum correlation nuclear magnetic resonance experiments reveal that distinct conformational states are formed in complexes of Vertex-11e with inactive and active ERK2. Importantly, two conformers interconvert in equilibrium in the active ERK2 apoenzyme, but Vertex-11e strongly shifts the equilibrium completely to one conformer. Thus, a high-affinity, slow dissociation inhibitor stabilizes different enzyme conformations depending on the activity state of ERK2 and reveals properties of conformational selection toward the active kinase. PMID:25350931

  7. Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors.

    PubMed

    Klaeger, Susan; Gohlke, Bjoern; Perrin, Jessica; Gupta, Vipul; Heinzlmeir, Stephanie; Helm, Dominic; Qiao, Huichao; Bergamini, Giovanna; Handa, Hiroshi; Savitski, Mikhail M; Bantscheff, Marcus; Médard, Guillaume; Preissner, Robert; Kuster, Bernhard

    2016-05-20

    Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise, but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compound's target space is desirable for a better understanding of its biological effects. The enzyme ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question of whether FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib, and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to a loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since the genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggest that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the preclinical molecular toxicology package for the development of kinase inhibitors. PMID:26863403

  8. Targeting kinases with anilinopyrimidines: discovery of N-phenyl-N’-[4-(pyrimidin-4-ylamino)phenyl]urea derivatives as selective inhibitors of class III receptor tyrosine kinase subfamily

    PubMed Central

    Gandin, Valentina; Ferrarese, Alessandro; Dalla Via, Martina; Marzano, Cristina; Chilin, Adriana; Marzaro, Giovanni

    2015-01-01

    Kinase inhibitors are attractive drugs/drug candidates for the treatment of cancer. The most recent literature has highlighted the importance of multi target kinase inhibitors, although a correct balance between specificity and non-specificity is required. In this view, the discovery of multi-tyrosine kinase inhibitors with subfamily selectivity is a challenging goal. Herein we present the synthesis and the preliminary kinase profiling of a set of novel 4-anilinopyrimidines. Among the synthesized compounds, the N-phenyl-N’-[4-(pyrimidin-4-ylamino)phenyl]urea derivatives selectively targeted some members of class III receptor tyrosine kinase family. Starting from the structure of hit compound 19 we synthesized a further compound with an improved affinity toward the class III receptor tyrosine kinase members and endowed with a promising antitumor activity both in vitro and in vivo in a murine solid tumor model. Molecular modeling simulations were used in order to rationalize the behavior of the title compounds. PMID:26568452

  9. Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-κB–regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-κBα kinase, leading to potentiation of apoptosis

    PubMed Central

    Sung, Bokyung; Pandey, Manoj K.; Ahn, Kwang Seok; Yi, Tingfang; Chaturvedi, Madan M.; Liu, Mingyao

    2008-01-01

    Anacardic acid (6-pentadecylsalicylic acid) is derived from traditional medicinal plants, such as cashew nuts, and has been linked to anticancer, anti-inflammatory, and radiosensitization activities through a mechanism that is not yet fully understood. Because of the role of nuclear factor-κB (NF-κB) activation in these cellular responses, we postulated that anacardic acid might interfere with this pathway. We found that this salicylic acid potentiated the apoptosis induced by cytokine and chemotherapeutic agents, which correlated with the down-regulation of various gene products that mediate proliferation (cyclin D1 and cyclooxygenase-2), survival (Bcl-2, Bcl-xL, cFLIP, cIAP-1, and survivin), invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1), and angiogenesis (vascular endothelial growth factor), all known to be regulated by the NF-κB. We found that anacardic acid inhibited both inducible and constitutive NF-κB activation; suppressed the activation of IκBα kinase that led to abrogation of phosphorylation and degradation of IκBα; inhibited acetylation and nuclear translocation of p65; and suppressed NF-κB–dependent reporter gene expression. Down-regulation of the p300 histone acetyltransferase gene by RNA interference abrogated the effect of anacardic acid on NF-κB suppression, suggesting the critical role of this enzyme. Overall, our results demonstrate a novel role for anacardic acid in potentially preventing or treating cancer through modulation of NF-κB signaling pathway. PMID:18349320

  10. Selective Phosphorylation Inhibitor of Delta Protein Kinase C-Pyruvate Dehydrogenase Kinase Protein-Protein Interactions: Application for Myocardial Injury in Vivo.

    PubMed

    Qvit, Nir; Disatnik, Marie-Hélène; Sho, Eiketsu; Mochly-Rosen, Daria

    2016-06-22

    Protein kinases regulate numerous cellular processes, including cell growth, metabolism, and cell death. Because the primary sequence and the three-dimensional structure of many kinases are highly similar, the development of selective inhibitors for only one kinase is challenging. Furthermore, many protein kinases are pleiotropic, mediating diverse and sometimes even opposing functions by phosphorylating multiple protein substrates. Here, we set out to develop an inhibitor of a selective protein kinase phosphorylation of only one of its substrates. Focusing on the pleiotropic delta protein kinase C (δPKC), we used a rational approach to identify a distal docking site on δPKC for its substrate, pyruvate dehydrogenase kinase (PDK). We reasoned that an inhibitor of PDK's docking should selectively inhibit the phosphorylation of only PDK without affecting phosphorylation of the other δPKC substrates. Our approach identified a selective inhibitor of PDK docking to δPKC with an in vitro Kd of ∼50 nM and reducing cardiac injury IC50 of ∼5 nM. This inhibitor, which did not affect the phosphorylation of other δPKC substrates even at 1 μM, demonstrated that PDK phosphorylation alone is critical for δPKC-mediated injury by heart attack. The approach we describe is likely applicable for the identification of other substrate-specific kinase inhibitors. PMID:27218445

  11. Oncogenic roles of TOPK and MELK, and effective growth suppression by small molecular inhibitors in kidney cancer cells

    PubMed Central

    Kato, Taigo; Inoue, Hiroyuki; Imoto, Seiya; Tamada, Yoshinori; Miyamoto, Takashi; Matsuo, Yo; Nakamura, Yusuke; Park, Jae-Hyun

    2016-01-01

    T–lymphokine-activated killer cell–originated protein kinase (TOPK) and maternal embryonic leucine zipper kinase (MELK) have been reported to play critical roles in cancer cell proliferation and maintenance of stemness. In this study, we investigated possible roles of TOPK and MELK in kidney cancer cells and found their growth promotive effect as well as some feedback mechanism between these two molecules. Interestingly, the blockade of either of these two kinases effectively caused downregulation of forkhead box protein M1 (FOXM1) activity which is known as an oncogenic transcriptional factor in various types of cancer cells. Small molecular compound inhibitors against TOPK (OTS514) and MELK (OTS167) effectively suppressed the kidney cancer cell growth, and the combination of these two compounds additively worked and showed the very strong growth suppressive effect on kidney cancer cells. Collectively, our results suggest that both TOPK and MELK are promising molecular targets for kidney cancer treatment and that dual blockade of OTS514 and OTS167 may bring additive anti-tumor effects with low risk of side effects. PMID:26933922

  12. A computational workflow for the design of irreversible inhibitors of protein kinases.

    PubMed

    Del Rio, Alberto; Sgobba, Miriam; Parenti, Marco Daniele; Degliesposti, Gianluca; Forestiero, Rosetta; Percivalle, Claudia; Conte, Pier Franco; Freccero, Mauro; Rastelli, Giulio

    2010-03-01

    Design of irreversible inhibitors is an emerging and relatively less explored strategy for the design of protein kinase inhibitors. In this paper, we present a computational workflow that was specifically conceived to assist such design. The workflow takes the form of a multi-step procedure that includes: the creation of a database of already known reversible inhibitors of protein kinases, the selection of the most promising scaffolds that bind one or more desired kinase templates, the modification of the scaffolds by introduction of chemically reactive groups (suitable cysteine traps) and the final evaluation of the reversible and irreversible protein-ligand complexes with molecular dynamics simulations and binding free energy predictions. Most of these steps were automated. In order to prove that this is viable, the workflow was tested on a database of known inhibitors of ERK2, a protein kinase possessing a cysteine in the ATP site. The modeled ERK2-ligand complexes and the values of the estimated binding free energies of the putative ligands provide useful indicators of their aptitude to bind reversibly and irreversibly to the protein kinase. Moreover, the computational data are used to rank the ligands according to their computed binding free energies and their ability to bind specific protein residues in the reversible and irreversible complexes, thereby providing a useful decision-making tool for each step of the design. In this work we present the overall procedure and the first proof of concept results. PMID:20306284

  13. Suppression of protein kinase C and nuclear oncogene expression as possible action mechanisms of cancer chemoprevention by Curcumin.

    PubMed

    Lin, Jen-Kun

    2004-07-01

    Curcumin (diferuloylmethane) is a major naturally-occurring polyphenol of Curcuma species, which is commonly used as a yellow coloring and flavoring agent in foods. Curcumin has shown anti-carcinogenic activity in animal models. Curcumin possesses anti-inflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes such as lipoxygenase/cyclooxygenase, xanthine dehydrogenase/oxidase and inducible nitric oxide synthase; and an effective inducer of heme oxygenase-1. Curcumin is also a potent inhibitor of protein kinase C (PKC), EGF(Epidermal growth factor)-receptor tyrosine kinase and IkappaB kinase. Subsequently, curcumin inhibits the activation of NF(nucleor factor)kappaB and the expressions of oncogenes including c-jun, c-fos, c-myc, NIK, MAPKs, ERK, ELK, PI3K, Akt, CDKs and iNOS. It is proposed that curcumin may suppress tumor promotion through blocking signal transduction pathways in the target cells. The oxidant tumor promoter TPA activates PKC by reacting with zinc thiolates present within the regulatory domain, while the oxidized form of cancer chemopreventive agent such as curcumin can inactivate PKC by oxidizing the vicinal thiols present within the catalytic domain. Recent studies indicated that proteasome-mediated degradation of cell proteins play a pivotal role in the regulation of several basic cellular processes including differentiation, proliferation, cell cycling, and apoptosis. It has been demonstrated that curcumin-induced apoptosis is mediated through the impairment of ubiquitin-proteasome pathway. Curcumin was first biotransformed to dihydrocurcumin and tetrahydrocurcumin and that these compounds subsequently were converted to monoglucuronide conjugates. These results suggest that curcumin-glucuronide, dihydrocurcumin-glucuronide, tetrahydrocurcumin-glucuronide and tetrahydrocurcumin are the major metabolites of curcumin in mice, rats and humans. PMID:15356994

  14. Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

    SciTech Connect

    Nifuji, Akira; Ideno, Hisashi; Ohyama, Yoshio; Takanabe, Rieko; Araki, Ryoko; Abe, Masumi; Noda, Masaki; Shibuya, Hiroshi

    2010-04-15

    Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.

  15. Glycogen synthase kinasesuppresses polyglutamine aggregation by inhibiting Vaccinia-related kinase 2 activity

    PubMed Central

    Lee, Eunju; Ryu, Hye Guk; Kim, Sangjune; Lee, Dohyun; Jeong, Young-Hun; Kim, Kyong-Tai

    2016-01-01

    Huntington’s disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the N-terminal of huntingtin. The amount of aggregate-prone protein is controlled by various mechanisms, including molecular chaperones. Vaccinia-related kinase 2 (VRK2) is known to negatively regulate chaperonin TRiC, and VRK2-facilitated degradation of TRiC increases polyQ protein aggregation, which is involved in HD. We found that VRK2 activity was negatively controlled by glycogen synthase kinase 3β (GSK3β). GSK3β directly bound to VRK2 and inhibited the catalytic activity of VRK2 in a kinase activity-independent manner. Furthermore, GSK3β increased the stability of TRiC and decreased the formation of HttQ103-GFP aggregates by inhibiting VRK2. These results indicate that GSK3β signaling may be a regulatory mechanism of HD progression and suggest targets for further therapeutic trials for HD. PMID:27377031

  16. Glycogen synthase kinasesuppresses polyglutamine aggregation by inhibiting Vaccinia-related kinase 2 activity.

    PubMed

    Lee, Eunju; Ryu, Hye Guk; Kim, Sangjune; Lee, Dohyun; Jeong, Young-Hun; Kim, Kyong-Tai

    2016-01-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the N-terminal of huntingtin. The amount of aggregate-prone protein is controlled by various mechanisms, including molecular chaperones. Vaccinia-related kinase 2 (VRK2) is known to negatively regulate chaperonin TRiC, and VRK2-facilitated degradation of TRiC increases polyQ protein aggregation, which is involved in HD. We found that VRK2 activity was negatively controlled by glycogen synthase kinase 3β (GSK3β). GSK3β directly bound to VRK2 and inhibited the catalytic activity of VRK2 in a kinase activity-independent manner. Furthermore, GSK3β increased the stability of TRiC and decreased the formation of HttQ103-GFP aggregates by inhibiting VRK2. These results indicate that GSK3β signaling may be a regulatory mechanism of HD progression and suggest targets for further therapeutic trials for HD. PMID:27377031

  17. De novo design of VEGFR-2 tyrosine kinase inhibitors based on a linked-fragment approach.

    PubMed

    Liu, Yi-Zhou; Wang, Xiao-Li; Wang, Xin-Ying; Yu, Ri-Lei; Liu, Dong-Qing; Kang, Cong-Min

    2016-09-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors have been demonstrated to possess substantial antitumor activity. VEGFR-2 tyrosine kinase inhibitors are crucial for development of antitumor drugs. Based on the crystal structure of VEGFR-2 tyrosine kinase, a linked-fragment strategy was employed to design novel VEGFR-2 tyrosine kinase inhibitors, and 1000 compounds were generated in this process. Absorption, distribution, metabolism, excretion and toxicity (ADMET) were used to screen the 1000 compounds, and 59 compounds were acceptable. Scaffold hopping was then used for further screening, and only four compounds were obtained in this way. Then, the binding energy of the four molecules to VEGFR-2 tyrosine kinase was calculated using molecular docking, and their values were found to be lower than that of Sorafenib. Finally, molecular dynamics simulations were performed on the complex of the compound with the lowest binding energy with VEGFR-2 tyrosine kinase, and the binding model was analyzed. At the end, four chemical entities with novel structures were obtained, and were suggested for experimental testing in future studies. PMID:27558799

  18. Structural Bioinformatics-Based Prediction of Exceptional Selectivity of p38 MAP Kinase Inhibitor PH-797804

    SciTech Connect

    Xing, Li; Shieh, Huey S.; Selness, Shaun R.; Devraj, Rajesh V.; Walker, John K.; Devadas, Balekudru; Hope, Heidi R.; Compton, Robert P.; Schindler, John F.; Hirsch, Jeffrey L.; Benson, Alan G.; Kurumbail, Ravi G.; Stegeman, Roderick A.; Williams, Jennifer M.; Broadus, Richard M.; Walden, Zara; Monahan, Joseph B.; Pfizer

    2009-07-24

    PH-797804 is a diarylpyridinone inhibitor of p38{alpha} mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38{alpha} inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38{alpha} kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180{sup o} rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38{alpha} kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38{alpha} kinase inhibitors.

  19. The effects of ripasudil (K-115), a Rho kinase inhibitor, on activation of human conjunctival fibroblasts.

    PubMed

    Futakuchi, Akiko; Inoue, Toshihiro; Fujimoto, Tomokazu; Inoue-Mochita, Miyuki; Kawai, Motofumi; Tanihara, Hidenobu

    2016-08-01

    The most common cause of glaucoma surgery failure is scar formation induced by activation of wound-healing responses and resultant fibrosis at the surgical site. We investigated the effects of ripasudil, a Rho kinase inhibitor, on activation of human conjunctival fibroblasts (HConF). HConF were pretreated with different concentrations of ripasudil for 1 h before addition of transforming growth factor (TGF)-β2, followed by incubation for 48 h. TGF-β2-treated fibroblasts exhibited a significant increase in expression of α-smooth muscle actin (α-SMA), a marker of fibroblast-to-myofibroblast differentiation, and this increase was significantly suppressed, in a dose-dependent manner, by pretreatment with ripasudil. Ripasudil pretreatment also significantly attenuated TGF-β2-induced fibronectin production and collagen gel contraction. TGF-β2 increased both the number of viable cells and the number of cells in the G2/M phase of the cell cycle; these effects were attenuated by pretreatment with ripasudil. In addition, we explored the effects of ripasudil on stimulation of HConF by activated macrophages. Human monocytic cell line THP-1 cells were differentiated into M1 or M2 macrophage-like cells, and HConF were treated with conditioned media derived from these macrophages in the presence or absence of ripasudil. Conditioned medium from M2 macrophage-like cells induced a significant increase in α-SMA expression, viable cell numbers, and gel contraction, all of which were significantly suppressed by ripasudil. Thus, overall, ripasudil attenuated activation of human conjunctival fibroblasts. Ripasudil may be of therapeutic utility, preventing excessive scarring after glaucoma filtration surgery. PMID:27394186

  20. Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase.

    PubMed

    Johnson, Christopher N; Adelinet, Christophe; Berdini, Valerio; Beke, Lijs; Bonnet, Pascal; Brehmer, Dirk; Calo, Frederick; Coyle, Joseph E; Day, Phillip J; Frederickson, Martyn; Freyne, Eddy J E; Gilissen, Ron A H J; Hamlett, Christopher C F; Howard, Steven; Meerpoel, Lieven; Mevellec, Laurence; McMenamin, Rachel; Pasquier, Elisabeth; Patel, Sahil; Rees, David C; Linders, Joannes T M

    2015-01-01

    A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein-ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK. PMID:25589926

  1. Structure-Based Design of Type II Inhibitors Applied to Maternal Embryonic Leucine Zipper Kinase

    PubMed Central

    2014-01-01

    A novel Type II kinase inhibitor chemotype has been identified for maternal embryonic leucine zipper kinase (MELK) using structure-based ligand design. The strategy involved structural characterization of an induced DFG-out pocket by protein–ligand X-ray crystallography and incorporation of a slender linkage capable of bypassing a large gate-keeper residue, thus enabling design of molecules accessing both hinge and induced pocket regions. Optimization of an initial hit led to the identification of a low-nanomolar, cell-penetrant Type II inhibitor suitable for use as a chemical probe for MELK. PMID:25589926

  2. Preclinical testing of selective Aurora kinase inhibitors on a medullary thyroid carcinoma-derived cell line.

    PubMed

    Tuccilli, Chiara; Baldini, Enke; Prinzi, Natalie; Morrone, Stefania; Sorrenti, Salvatore; Filippini, Angelo; Catania, Antonio; Alessandrini, Stefania; Rendina, Roberta; Coccaro, Carmela; D'Armiento, Massimino; Ulisse, Salvatore

    2016-05-01

    Deregulated expression of the Aurora kinases (Aurora-A, B, and C) is thought to be involved in cell malignant transformation and genomic instability in several cancer types. Over the last decade, a number of small-molecule inhibitors of Aurora kinases have been developed, which have proved to efficiently restrain malignant cell growth and tumorigenicity. Regarding medullary thyroid carcinoma (MTC), we previously showed the efficacy of a pan-Aurora kinase inhibitor (MK-0457) in impairing growth and survival of the MTC-derived cell line TT. In the present study, we sought to establish if one of the Aurora kinases might represent a preferential target for MTC therapy. The effects of selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) were analyzed on TT cell proliferation, apoptosis, cell cycle, and ploidy. The two inhibitors reduced TT cell proliferation in a time- and dose-dependent manner, with IC50 of 19.0 ± 2.4 nM for MLN8237 and 401.6 ± 44.1 nM for AZD1152. Immunofluorescence experiments confirmed that AZD1152 inhibited phosphorylation of histone H3 (Ser10) by Aurora-B, while it did not affect Aurora-A autophosphorylation. MLN8237 inhibited Aurora-A autophosphorylation as expected, but at concentrations required to achieve the maximum antiproliferative effects it also abolished H3 (Ser10) phosphorylation. Cytofluorimetry experiments showed that both inhibitors induced accumulation of cells in G2/M phase and increased the subG0/G1 fraction and polyploidy. Finally, both inhibitors triggered apoptosis. We demonstrated that inhibition of either Aurora-A or Aurora-B has antiproliferative effects on TT cells, and thus it would be worthwhile to further investigate the therapeutical potential of Aurora kinase inhibitors in MTC treatment. PMID:26215279

  3. Are Accurins the cure for Aurora kinase inhibitors?

    PubMed

    Bearss, David J

    2016-02-10

    A nanoparticle formulation of an Aurora B inhibitor increases antitumor efficacy and reduces toxicity, which may be a precedent for the use of this technology with other small molecules (Ashton et al., this issue). PMID:26865564

  4. Inhibitors of cellular kinases with broad-spectrum antiviral activity for hemorrhagic fever viruses.

    PubMed

    Mohr, Emma L; McMullan, Laura K; Lo, Michael K; Spengler, Jessica R; Bergeron, Éric; Albariño, César G; Shrivastava-Ranjan, Punya; Chiang, Cheng-Feng; Nichol, Stuart T; Spiropoulou, Christina F; Flint, Mike

    2015-08-01

    Host cell kinases are important for the replication of a number of hemorrhagic fever viruses. We tested a panel of kinase inhibitors for their ability to block the replication of multiple hemorrhagic fever viruses. OSU-03012 inhibited the replication of Lassa, Ebola, Marburg and Nipah viruses, whereas BIBX 1382 dihydrochloride inhibited Lassa, Ebola and Marburg viruses. BIBX 1382 blocked both Lassa and Ebola virus glycoprotein-dependent cell entry. These compounds may be used as tools to understand conserved virus-host interactions, and implicate host cell kinases that may be targets for broad spectrum therapeutic intervention. PMID:25986249

  5. Selective inhibitors of Cyclin-G associated kinase (GAK) as anti-HCV agents

    PubMed Central

    Kovackova, Sona; Chang, Lei; Bekerman, Elena; Neveu, Gregory; Barouch-Bentov, Rina; Chaikuad, Apirat; Heroven, Christina; Šála, Michal; De Jonghe, Steven; Knapp, Stefan; Einav, Shirit; Herdewijn, Piet

    2015-01-01

    Cyclin-G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Co-crystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV lifecycle (i.e. viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer and Parkinson's disease). PMID:25822739

  6. Tyrosine Kinase Inhibitors and Vascular Toxicity: Impetus for a Classification System?

    PubMed

    Herrmann, Joerg

    2016-06-01

    The introduction of molecularly targeted therapies with tyrosine kinase inhibitors has revolutionized cancer therapy and has contributed to a steady decline in cancer-related mortality since the late 1990s. However, not only cardiac but also vascular toxicity has been reported for these agents, some as expected on-target effects (e.g., VEGF receptor inhibitors) and others as unanticipated events (e.g., BCR-Abl inhibitors). A sound understanding of these cardiovascular toxic effects is critical to advance mechanistic insight into vascular disease and clinical care. From a conceptual standpoint, there might be value in defining type I (permanent) and type II (transient) vascular toxicity. This review will focus on the tyrosine kinase inhibitors in current clinical use and their associated vascular side effects. PMID:27099141

  7. Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

    DOEpatents

    Gray, Nathanael S. , Schultz, Peter , Wodicka, Lisa , Meijer, Laurent , Lockhart, David J.

    2003-06-03

    The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

  8. mTOR kinase inhibitors synergize with histone deacetylase inhibitors to kill B-cell acute lymphoblastic leukemia cells.

    PubMed

    Beagle, Brandon R; Nguyen, Duc M; Mallya, Sharmila; Tang, Sarah S; Lu, Mengrou; Zeng, Zhihong; Konopleva, Marina; Vo, Thanh-Trang; Fruman, David A

    2015-02-10

    High activity of the mechanistic target of rapamycin (mTOR) is associated with poor prognosis in pre-B-cell acute lymphoblastic leukemia (B-ALL), suggesting that inhibiting mTOR might be clinically useful. However, emerging data indicate that mTOR inhibitors are most effective when combined with other target agents. One strategy is to combine with histone deacetylase (HDAC) inhibitors, since B-ALL is often characterized by epigenetic changes that silence the expression of pro-apoptotic factors. Here we tested combinations of mTOR and pan-HDAC inhibitors on B-ALL cells, including both Philadelphia chromosome-positive (Ph+) and non-Ph cell lines. We found that mTOR kinase inhibitors (TOR-KIs) synergize with HDAC inhibitors to cause apoptosis in B-ALL cells and the effect is greater when compared to rapamycin plus HDAC inhibitors. The combination of TOR-KIs with the clinically approved HDAC inhibitor vorinostat increased apoptosis in primary pediatric B-ALL cells in vitro. Mechanistically, TOR-KI and HDAC inhibitor combinations increased expression of pro-death genes, including targets of the Forkhead Box O (FOXO) transcription factors, and increased sensitivity to apoptotic triggers at the mitochondria. These findings suggest that targeting epigenetic factors can unmask the cytotoxic potential of TOR-KIs towards B-ALL cells. PMID:25576920

  9. The Src-family tyrosine kinase inhibitor PP1 interferes with the activation of ribosomal protein S6 kinases.

    PubMed Central

    Shah, O Jameel; Kimball, Scot R; Jefferson, Leonard S

    2002-01-01

    Considerable biochemical and pharmacological evidence suggests that the activation of ribosomal protein S6 kinases (S6Ks) by activated receptor tyrosine kinases involves multiple co-ordinated input signals. However, the identities of many of these inputs remain poorly described, and their precise involvement in S6K activation has been the subject of great investigative effort. In the present study, we have shown that 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), a selective inhibitor of the Src family of non-receptor tyrosine kinases, interferes with the activation of 70 and 85 kDa S6K gene products (p70S6K1 and p85S6K1) by insulin, insulin-like growth factor 1, sodium orthovanadate and activated alleles of phosphoinositide 3-kinase and H-Ras. PP1 also impedes the activation of AKT/protein kinase B and the extracellular signal-regulated protein kinases 1 and 2 by these various stimuli. Insulin-like growth factor 1 was observed to induce a sustained increase in c-Src autophosphorylation as revealed using anti-phospho-Y416 antisera, but this effect was absent from the cells treated with PP1. To conclude, an activated allele of p70S6K1 is compared with the wild-type allele, resistant to inhibition by PP1 when co-expressed with phosphoinositide-dependent kinase 1 (PDK1), suggesting that PP1 affects p70S6K1 via a PDK1-independent pathway. Thus activation of Src may supply a necessary signal for the activation of p70S6K1 and possibly other S6Ks. PMID:12014987

  10. Structural differences between wild type and double mutant EGFR modulated by third-generation kinase inhibitors

    PubMed Central

    Lowder, Melissa A.; Doerner, Amy E.; Schepartz, Alanna

    2015-01-01

    Mutations in the EGFR kinase domain are implicated in non-small cell lung cancer. Of particular interest is the drug-resistant double mutant (L858R/T790M, DM EGFR), which is not inhibited selectively by any approved kinase inhibitor. Here we apply bipartite tetracysteine display to demonstrate that DM and WT EGFR differ in structure outside the kinase domain. The structural difference is located within the cytoplasmic juxtamembrane segment (JM) that links the kinase domain with the extracellular and transmembrane regions and is essential for EGFR activation. We show further that third-generation DM EGFR-selective TKIs alter JM structure via allostery to restore the conformation found when WT EGFR is activated by the growth factors EGF and HB-EGF. This work suggests that the oncogenic activity of DM EGFR may extend beyond kinase activity per se to include kinase-independent activities. As JM structure may provide a biomarker for these kinase-independent functions, these insights could guide the development of allosteric, DM-selective inhibitors. PMID:25973741

  11. Development of Specific, Irreversible Inhibitors for a Receptor Tyrosine Kinase EphB3.

    PubMed

    Kung, Alvin; Chen, Ying-Chu; Schimpl, Marianne; Ni, Feng; Zhu, Jianfa; Turner, Maurice; Molina, Henrik; Overman, Ross; Zhang, Chao

    2016-08-24

    Erythropoietin-producing human hepatocellular carcinoma (Eph) receptor tyrosine kinases (RTKs) regulate a variety of dynamic cellular events, including cell protrusion, migration, proliferation, and cell-fate determination. Small-molecule inhibitors of Eph kinases are valuable tools for dissecting the physiological and pathological roles of Eph. However, there is a lack of small-molecule inhibitors that are selective for individual Eph isoforms due to the high homology within the family. Herein, we report the development of the first potent and specific inhibitors of a single Eph isoform, EphB3. Through structural bioinformatic analysis, we identified a cysteine in the hinge region of the EphB3 kinase domain, a feature that is not shared with any other human kinases. We synthesized and characterized a series of electrophilic quinazolines to target this unique, reactive feature in EphB3. Some of the electrophilic quinazolines selectively and potently inhibited EphB3 both in vitro and in cells. Cocrystal structures of EphB3 in complex with two quinazolines confirmed the covalent linkage between the protein and the inhibitors. A "clickable" version of an optimized inhibitor was created and employed to verify specific target engagement in the whole proteome and to probe the extent and kinetics of target engagement of existing EphB3 inhibitors. Furthermore, we demonstrate that the autophosphorylation of EphB3 within the juxtamembrane region occurs in trans using a specific inhibitor. These exquisitely specific inhibitors will facilitate the dissection of EphB3's role in various biological processes and disease contribution. PMID:27478969

  12. Extracellular Signal-Regulated Kinases Modulate DNA Damage Response - A Contributing Factor to Using MEK Inhibitors in Cancer Therapy

    PubMed Central

    Wei, F; Yan, J; Tang, D

    2011-01-01

    The Raf-MEK-ERK pathway is commonly activated in human cancers, largely attributable to the extracellular signal-regulated kinases (ERKs) being a common downstream target of growth factor receptors, Ras, and Raf. Elevation of these up-stream signals occurs frequently in a variety of malignancies and ERK kinases play critical roles in promoting cell proliferation. Therefore, inhibition of MEK-mediated ERK activation is very appealing in cancer therapy. Consequently, numerous MEK inhibitors have been developed over the years. However, clinical trials have yet to produce overwhelming support for using MEK inhibitors in cancer therapy. Although complex reasons may have contributed to this outcome, an alternative possibility is that the MEK-ERK pathway may not solely provide proliferation signals to malignancies, the central scientific rationale in developing MEK inhibitors for cancer therapy. Recent developments may support this alternative possibility. Accumulating evidence now demonstrated that the MEK-ERK pathway contributes to the proper execution of cellular DNA damage response (DDR), a major pathway of tumor suppression. During DDR, the MEK-ERK pathway is commonly activated, which facilitates the proper activation of DDR checkpoints to prevent cell division. Inhibition of MEK-mediated ERK activation, therefore, compromises checkpoint activation. As a result, cells may continue to proliferate in the presence of DNA lesions, leading to the accumulation of mutations and thereby promoting tumorigenesis. Alternatively, reduction in checkpoint activation may prevent efficient repair of DNA damages, which may cause apoptosis or cell catastrophe, thereby enhancing chemotherapy’s efficacy. This review summarizes our current understanding of the participation of the ERK kinases in DDR. PMID:22087839

  13. An integrin-targeted, pan-isoform, phosphoinositide-3 kinase inhibitor, SF1126, has activity against multiple myeloma in vivo

    PubMed Central

    De, Pradip; Dey, Nandini; Terakedis, Breanne; Bersagel, Leif; Li, Zhi Hua; Mahadevan, Daruka; Garlich, Joseph R.; Trudel, Suzanne; Makale, Milan T.; Durden, Donald L.

    2013-01-01

    Purpose Multiple reports point to an important role for the phosphoinositide-3 kinase (PI3K) and AKT signaling pathways in tumor survival and chemoresistance in multiple myeloma (MM). The goals of our study were: (1) to generate the preclinical results necessary to justify a Phase I clinical trial of SF1126 in hematopoietic malignancies including multiple myeloma, and (2) to begin combining pan PI-3 kinase inhibitors with other agents to augment antitumor activity of this class of agent in preparation for combination therapy in Phase I/II trials. Methods We determined the in vitro activity of SF1126 with16 human MM cell lines. In vivo tumor growth suppression was determined with human myeloma (MM.1R) xenografts in athymic mice. In addition, we provide evidence that SF1126 has pharmacodynamic activity in the treatment of patients with MM. Results SF1126 was cytotoxic to all tested MM lines and potency was augmented by the addition of bortezomib. SF1126 affected MM.1R cell line signaling in vitro, inhibiting phospho-AKT, phospho-ERK, and the hypoxic stabilization of HIF1α. Tumor growth was 94% inhibited, with a marked decrease in both cellular proliferation (PCNA immunostaining) and angiogenesis (tumor microvessel density via CD31 immunostaining). Our clinical results demonstrate pharmacodynamic knockdown of p-AKT in primary patient derived MM tumor cells in vivo. Conclusions Our results establish three important points: (1) SF1126, a pan PI-3 kinase inhibitor has potent antitumor activity against multiple myeloma in vitro and in vivo, (2) SF1126 displays augmented antimyeloma activity when combined with proteasome inhibitor, bortezomib/Velcade®, and (3) SF1126 blocks the IGF-1 induced activation of AKT in primary MM tumor cells isolated from SF1126 treated patients The results support the ongoing early Phase I clinical trial in MM and suggest a future Phase I trial in combination with bortezomib in hematopoietic malignancies. PMID:23355037

  14. Hepatocyte growth factor reduces sensitivity to the epidermal growth factor receptor-tyrosine kinase inhibitor, gefitinib, in lung adenocarcinoma cells harboring wild-type EGFR

    PubMed Central

    Yang, Hua; Wang, Rong; Peng, Shunli; Chen, Longhua; Li, Qi; Wang, Wei

    2016-01-01

    Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy is an option for lung cancers harboring wild-type EGFR when chemotherapeutic reagents have failed. In this study, we found that the EGFR-TKI, gefitinib, modestly suppressed proliferation of the lung cancer cell lines, A549 and H358, which both harbor wild-type EGFR. Treatment with hepatocyte growth factor (HGF) reduced the sensitivity to gefitinib, whereas sensitivity was restored by treatment with an HGF antibody, a MET inhibitor, or depletion of MET but not ErbB3 gene. Moreover, both PI3K/mTOR inhibitors and MEK inhibitors suppressed proliferation of A549 cells, whereas only PI3K/mTOR inhibitors effectively suppressed cell viability of EGFR mutant PC-9 cells. Our findings suggest that HGF reduced the gefitinib sensitivity through MET and downstream PI3K and MAPK pathways. Combined use of EGFR-TKI and MET inhibitors or inhibition of downstream signaling molecules might be a better second or third line choice for a group of patients with advanced lung cancer harboring wild-type EGFR. PMID:26919104

  15. A Pentacyclic Aurora Kinase Inhibitor (AKI-001) With High in Vivo Potency And Oral Bioavailability

    SciTech Connect

    Rawson, T.E.; Ruth, M.; Blackwood, E.; Burdick, D.; Corson, L.; Dotson, J.; Drummond, J.; Fields, C.; Georges, G.J.; Goller, B.; Halladay, J.; Hunsaker, T.; Kleinheinz, T.; Krell, H.-W.; Li, J.; Liang, J.; Limberg, A.; McNutt, A.; Moffat, J.; Phillips, G.; Ran, Y.

    2009-05-21

    Aurora kinase inhibitors have attracted a great deal of interest as a new class of antimitotic agents. We report a novel class of Aurora inhibitors based on a pentacyclic scaffold. A prototype pentacyclic inhibitor 32 (AKI-001) derived from two early lead structures improves upon the best properties of each parent and compares favorably to a previously reported Aurora inhibitor, 39 (VX-680). The inhibitor exhibits low nanomolar potency against both Aurora A and Aurora B enzymes, excellent cellular potency (IC{sub 50} < 100 nM), and good oral bioavailability. Phenotypic cellular assays show that both Aurora A and Aurora B are inhibited at inhibitor concentrations sufficient to block proliferation. Importantly, the cellular activity translates to potent inhibition of tumor growth in vivo. An oral dose of 5 mg/kg QD is well tolerated and results in near stasis (92% TGI) in an HCT116 mouse xenograft model.

  16. Structural Mechanism of the Pan-BCR-ABL Inhibitor Ponatinib (AP24534): Lessons for Overcoming Kinase Inhibitor Resistance

    SciTech Connect

    Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng; Wang, Yihan; Thomas, Mathew; Keats, Jeff; Xu, Qihong; Rivera, Victor M.; Shakespeare, William C.; Clackson, Tim; Dalgarno, David C.; Zhu, Xiaotian

    2012-01-20

    The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

  17. Discovery and Characterization of a Biologically Active Non-ATP-Competitive p38 MAP Kinase Inhibitor.

    PubMed

    Wilson, Brice A P; Alam, Muhammad S; Guszczynski, Tad; Jakob, Michal; Shenoy, Shilpa R; Mitchell, Carter A; Goncharova, Ekaterina I; Evans, Jason R; Wipf, Peter; Liu, Gang; Ashwell, Jonathan D; O'Keefe, Barry R

    2016-03-01

    Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer. A number of p38 inhibitors have been tested in clinical trials, with none receiving regulatory approval. One characteristic shared by all of the compounds that failed clinical trials is that they are all adenosine triphosphate (ATP)-competitive p38 inhibitors. Seeing this lack of mechanistic diversity as an opportunity, we screened ~32,000 substances in search of novel p38 inhibitors. Among the inhibitors discovered is a compound that is both non-ATP competitive and biologically active in cell-based models for p38 activity. This is the first reported discovery of a non-ATP-competitive p38 inhibitor that is active in cells and, as such, may enable new pharmacophore designs for both therapeutic and basic research to better understand and exploit non-ATP-competitive inhibitors of p38 activity. PMID:26538432

  18. LIM kinase inhibitors disrupt mitotic microtubule organization and impair tumor cell proliferation

    PubMed Central

    Mardilovich, Katerina; Baugh, Mark; Crighton, Diane; Kowalczyk, Dominika; Gabrielsen, Mads; Munro, June; Croft, Daniel R.; Lourenco, Filipe; James, Daniel; Kalna, Gabriella; McGarry, Lynn; Rath, Oliver; Shanks, Emma; Garnett, Mathew J.; McDermott, Ultan; Brookfield, Joanna; Charles, Mark; Hammonds, Tim; Olson, Michael F.

    2015-01-01

    The actin and microtubule cytoskeletons are critically important for cancer cell proliferation, and drugs that target microtubules are widely-used cancer therapies. However, their utility is compromised by toxicities due to dose and exposure. To overcome these issues, we characterized how inhibition of the actin and microtubule cytoskeleton regulatory LIM kinases could be used in drug combinations to increase efficacy. A previously-described LIMK inhibitor (LIMKi) induced dose-dependent microtubule alterations that resulted in significant mitotic defects, and increased the cytotoxic potency of microtubule polymerization inhibitors. By combining LIMKi with 366 compounds from the GSK Published Kinase Inhibitor Set, effective combinations were identified with kinase inhibitors including EGFR, p38 and Raf. These findings encouraged a drug discovery effort that led to development of CRT0105446 and CRT0105950, which potently block LIMK1 and LIMK2 activity in vitro, and inhibit cofilin phosphorylation and increase αTubulin acetylation in cells. CRT0105446 and CRT0105950 were screened against 656 cancer cell lines, and rhabdomyosarcoma, neuroblastoma and kidney cancer cells were identified as significantly sensitive to both LIMK inhibitors. These large-scale screens have identified effective LIMK inhibitor drug combinations and sensitive cancer types. In addition, the LIMK inhibitory compounds CRT0105446 and CRT0105950 will enable further development of LIMK-targeted cancer therapy. PMID:26540348

  19. Protein-Protein Interaction for the De Novo Design of Cyclin-Dependent Kinase Peptide Inhibitors.

    PubMed

    Arumugasamy, Karthiga; Tripathi, Sunil Kumar; Singh, Poonam; Singh, Sanjeev Kumar

    2016-01-01

    The homology of the inhibitor binding site regions on the surface of cyclin-dependent kinases (CDKs) makes actual CDK inhibitors unable to bind specifically to their molecular targets. Most of them are ATP competitive inhibitors with low specificity that also affect the phosphorylation mechanisms of other nontarget kinases giving rise to harmful side effects. So, the search of specific and potent inhibitors able to bind to the desired CDK target is still a pending issue. Structure based drug design minimized the erroneous binding and increased the affinity of the inhibitor interaction. In the case of CDKs their activation and regulation mechanisms mainly depend on protein-protein interactions (PPIs). The design of drugs targeting these PPIs makes feasible and promising towards the discovery of new and specific CDK inhibitors. Development of peptide inhibitors for a target protein is an emerging approach in computer aided drug designing. This chapter describes in detail methodology for use of the VitAL-Viterbi algorithm for de novo peptide design of CDK2 inhibitors. PMID:26231708

  20. Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNF{alpha} in human epidermal keratinocytes (HaCaT)

    SciTech Connect

    Onda, Kenji . E-mail: knjond@ps.toyaku.ac.jp; Nagashima, Masahiro; Kawakubo, Yo; Inoue, Shota; Hirano, Toshihiko; Oka, Kitaro

    2006-12-08

    Glucocorticoids (GCs) are essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) {alpha} reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNF{alpha} was not accompanied by changes in mRNA expressions of GR isoforms ({alpha} or {beta}). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNF{alpha}. Additionally, we observed that TNF{alpha} reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNF{alpha}-mediated GC insensitivity. Our data suggest that overexpression of TNF{alpha} leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.

  1. Identification of ponatinib and other known kinase inhibitors with potent MEKK2 inhibitory activity.

    PubMed

    Ahmad, Syed; Johnson, Gary L; Scott, John E

    2015-08-01

    The kinase MEKK2 (MAP3K2) may play an important role in tumor growth and metastasis for several cancer types. Thus, targeting MEKK2 may represent a novel strategy for developing more effective therapies for cancer. In order to identify small molecules with MEKK2 inhibitory activity, we screened a collection of known kinase inhibitors using a high throughput MEKK2 intrinsic ATPase enzyme assay and confirmed activity of the most potent hits with this primary assay. We also confirmed activities of these known kinase inhibitors with an MEKK2 transphosphorylation slot blot assay using MKK6 as a substrate. We observed a good correlation in potencies between the two orthogonal MEKK2 kinase activity assay formats for this set of inhibitors. We report that ponatinib, AT9283, AZD7762, JNJ-7706621, PP121 and hesperadin had potent MEKK2 enzyme inhibitory activities ranging from 4.7 to 60 nM IC50. Ponatinib is an FDA-approved drug that potently inhibited MEKK2 enzyme activity with IC50 values of 10-16 nM. AT9283 is currently in clinical trials and produced MEKK2 IC50 values of 4.7-18 nM. This set of known kinase inhibitors represents some of the most potent in vitro MEKK2 inhibitors reported to date and may be useful as research tools. Although these compounds are not selective for MEKK2, the structures of these compounds give insight into pharmacophores that potently inhibit MEKK2 and could be used as initial leads to design highly selective inhibitors of MEKK2. PMID:26056008

  2. Pharmacophore modeling study based on known spleen tyrosine kinase inhibitors together with virtual screening for identifying novel inhibitors.

    PubMed

    Xie, Huan-Zhang; Li, Lin-Li; Ren, Ji-Xia; Zou, Jun; Yang, Li; Wei, Yu-Quan; Yang, Sheng-Yong

    2009-04-01

    In this investigation, chemical features based 3D pharmacophore models were developed based on the known inhibitors of Spleen tyrosine kinase (Syk) with the aid of hiphop and hyporefine modules within catalyst. The best quantitative pharmacophore model, Hypo1, was used as a 3D structural query for retrieving potential inhibitors from chemical databases including Specs, NCI, MayBridge, and Chinese Nature Product Database (CNPD). The hit compounds were subsequently subjected to filtering by Lipinski's rule of five and docking studies to refine the retrieved hits. Finally 30 compounds were selected from the top ranked hit compounds and conducted an in vitro kinase inhibitory assay. Six compounds showed a good inhibitory potency against Syk, which have been selected for further investigation. PMID:19254842

  3. Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

    SciTech Connect

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu . E-mail: dxliu001@yahoo.com

    2007-07-13

    Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.

  4. The cardiotoxicity and myocyte damage caused by small molecule anticancer tyrosine kinase inhibitors is correlated with lack of target specificity

    SciTech Connect

    Hasinoff, Brian B.

    2010-04-15

    The use of the new anticancer tyrosine kinase inhibitors (TKI) has revolutionized the treatment of certain cancers. However, the use of some of these results in cardiotoxicity. Large-scale profiling data recently made available for the binding of 7 of the 9 FDA-approved tyrosine kinase inhibitors to a panel of 317 kinases has allowed us to correlate kinase inhibitor binding selectivity scores with TKI-induced damage to neonatal rat cardiac myocytes. The tyrosine kinase selectivity scores, but not the serine-threonine kinase scores, were highly correlated with the myocyte damaging effects of the TKIs. Additionally, we showed that damage to myocytes gave a good rank order correlation with clinical cardiotoxicity. Finally, strength of TKI binding to colony-stimulating factor 1 receptor (CSF1R) was highly correlated with myocyte damage, thus possibly implicating this kinase in contributing to TKI-induced cardiotoxicity.

  5. Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer.

    PubMed

    Chung, Suyoun; Suzuki, Hanae; Miyamoto, Takashi; Takamatsu, Naofumi; Tatsuguchi, Ayako; Ueda, Koji; Kijima, Kyoko; Nakamura, Yusuke; Matsuo, Yo

    2012-12-01

    We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also reported to be highly upregulated in multiple types of human cancer. It was implied to play indispensable roles in cancer cell survival and indicated its involvement in the maintenance of tumor-initiating cells. We conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC₅₀ of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin-like), which we identified as novel MELK substrates and are important for stem-cell characteristics and invasiveness. The compound suppressed mammosphere formation of breast cancer cells and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. This MELK inhibitor should be a promising compound possibly to suppress the growth of tumor-initiating cells and be applied for treatment of a wide range of human cancer. PMID:23283305

  6. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  7. Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.

    PubMed

    Hegemann, L; Wevers, A; Bonnekoh, B; Mahrle, G

    1992-03-01

    Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes. PMID:1376142

  8. FMS-like tyrosine kinase 3 (FLT3) inhibitors: Molecular docking and experimental studies.

    PubMed

    Mashkani, Baratali; Tanipour, Mohammad Hossein; Saadatmandzadeh, Mohammad; Ashman, Leonie K; Griffith, Renate

    2016-04-01

    Activating mutations in FMS-like tyrosine kinase 3 (FLT3) occur in 25% of acute lymphoid and 30% of acute myeloid leukaemia cases. Therefore, FLT3 is a potential therapeutic target for small molecule kinase inhibitors. In this study, protein-ligand interactions between FLT3 and kinase inhibitors (CEP701, PKC412, sunitinib, imatinib and dasatinib) were obtained through homology modelling and molecular docking. A cellular system for experimental testing of the inhibitors was also established by expressing wildtype and internal tandem duplication mutant FLT3 (FLT3-WT and FLT3-ITD) in FDC-P1 cells. Imatinib and dasatinib could not be docked into any of the FLT3 models, consistent with their lack of activity in the experimental assays. CEP701, PKC412 and sunitinib interacted with the ATP-binding pocket of FLT3, forming H-bonds with Cys694 and Glu692. Based on the EC50 values in the cell proliferation assay, CEP701 was the most potent inhibitor; sunitinib and PKC412 were ranked second and third, respectively. Sunitinib was the most selective inhibitor, followed by PKC421 and CEP701. The potency of sunitinib and to a lesser extent CEP701 in inhibition of FLT3 autophosphorylation was lower than the cell proliferation inhibition, indicating that inhibition of FLT3 downstream proteins may contribute to the cellular effects. It was shown in this study that the docking procedure was able to differentiate FLT3 inhibitors from ineffective compounds. Additionally, interaction with the phosphate binding region in the ATP-binding pocket increased potency at the cost of selectivity. These findings can be applied in designing highly effective and selective inhibitors for FLT3 and other related kinases. PMID:26896780

  9. Molecular Pathways: Molecular Basis for Sensitivity and Resistance to JAK Kinase Inhibitors

    PubMed Central

    Meyer, Sara C.; Levine, Ross L.

    2014-01-01

    Janus kinases (JAK) are the mediators of a variety of cytokine signals via their cognate receptors that result in activation of intracellular signaling pathways. Alterations in JAK1, JAK2, JAK3 and TYK2 signaling contribute to different disease states, and dysregulated JAK-STAT signaling is associated with hematological malignancies, autoimmune disorders and immune-deficient conditions. Genetic alterations of JAK2 occur in the majority of patients with myeloproliferative neoplasms (MPN) and occur in a subset of patients with acute leukemias. JAK-mediated signaling critically relies on STAT transcription factors, and on activation of the MAPK and PI3K/Akt signaling axes. Hyperactive JAK at the apex of these potent oncogenic signaling pathways therefore represents an important target for small molecule kinase inhibitors in different disease states. The JAK1/2 inhibitor ruxolitinib and the JAK3 inhibitor tofacitinib were recently approved for the treatment of myelofibrosis and rheumatoid arthritis, respectively and additional ATP-competitive JAK inhibitors are in clinical development. Although these agents show clinical activity, the ability of these JAK inhibitors to induce clinical/molecular remissions in hematological malignancies appears limited and resistance upon chronic drug exposure is seen. Alternative modes of targeting JAK2 such as allosteric kinase inhibition or HSP-90 inhibition are under evaluation as is the use of histone deacetylase inhibitors. Combination therapy approaches integrating inhibition of STAT, PI3K/Akt and MAPK pathways with JAK kinase inhibitors might be critical to overcome malignancies characterized by dysregulated JAK signaling. PMID:24583800

  10. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    SciTech Connect

    Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung; Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon; Cho, Dong-Hyung

    2011-05-13

    Highlights: {yields} We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. {yields} Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. {yields} Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. {yields} Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  11. Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway

    SciTech Connect

    Yu, Zhiwei; Cui, Binbin; Jin, Yinghu; Chen, Haipeng; Wang, Xishan

    2011-08-12

    Highlights: {yields} This article described the effects of the EGFR tyrosine kinase inhibitor on the cell proliferation and the apoptosis induction of the colon carcinoma cell lines. {yields} Demonstrated that 326474 is a more potent EGFR inhibitor on colon cancer cells than other three TKIs. {yields} It can be important when considering chemotherapy for colonic cancer patients. -- Abstract: Background: Epidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. Methods: In this study, using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. Results: Among the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. Conclusion: Our studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.

  12. Rapid evolution of 6-phenylpurine inhibitors of protein kinase B through structure-based design.

    PubMed

    Donald, Alastair; McHardy, Tatiana; Rowlands, Martin G; Hunter, Lisa-Jane K; Davies, Thomas G; Berdini, Valerio; Boyle, Robert G; Aherne, G Wynne; Garrett, Michelle D; Collins, Ian

    2007-05-17

    6-phenylpurines were identified as novel, ATP-competitive inhibitors of protein kinase B (PKB/Akt) from a fragment-based screen and were rapidly progressed to potent compounds using iterative protein-ligand crystallography with a PKA-PKB chimeric protein. An elaborated lead compound showed cell growth inhibition and effects on cellular signaling pathways characteristic of PKB inhibition. PMID:17451235

  13. Design of Targeted Inhibitors of Polo-like Kinase 1 (Plk1)

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    2011-03-01

    Computational design of small molecule inhibitors of Polo-like Kinase 1 (Plk1) is presented. Plk1, which regulates cell cycle, is often overexpressed in cancers. Its downregulation was shown to inhibit cancer progression. Most inhibitors of kinases' interact with the highly conserved ATP binding site. This makes the development of Plk1-specific inhibitors challenging, since different kinases have similar ATP sites. However, Plk1 also contains the polo-box domain (PBD), which is absent from other kinases. In this study, the PBD site was used as a target for designed Plk1 inhibitors. Common structural features of experimentally known Plk1 ligands were first identified. The information was used to design putative small molecules that specifically bonded Plk1. Druglikeness and possible toxicities of the designed molecules were determined. Molecules with no implied toxicities and optimal druglikeness were used for docking studies. The docking studies identified several molecules that made stable complexes with the Plk1 PBD site. Possible utilization of the designed molecules in drugs against cancers with overexpressed Plk1 is discussed.

  14. Inhibitors of Glycogen Synthase Kinase 3 with Exquisite Kinome-Wide Selectivity and Their Functional Effects.

    PubMed

    Wagner, Florence F; Bishop, Joshua A; Gale, Jennifer P; Shi, Xi; Walk, Michelle; Ketterman, Joshua; Patnaik, Debasis; Barker, Doug; Walpita, Deepika; Campbell, Arthur J; Nguyen, Shannon; Lewis, Michael; Ross, Linda; Weïwer, Michel; An, W Frank; Germain, Andrew R; Nag, Partha P; Metkar, Shailesh; Kaya, Taner; Dandapani, Sivaraman; Olson, David E; Barbe, Anne-Laure; Lazzaro, Fanny; Sacher, Joshua R; Cheah, Jaime H; Fei, David; Perez, Jose; Munoz, Benito; Palmer, Michelle; Stegmaier, Kimberly; Schreiber, Stuart L; Scolnick, Edward; Zhang, Yan-Ling; Haggarty, Stephen J; Holson, Edward B; Pan, Jen Q

    2016-07-15

    The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3's inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3β, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitro systems or in vivo models. A nonconventional analysis of data from a GSK3β inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652 and BRD0209, which demonstrated in vivo efficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders. PMID:27128528

  15. HALOACETIC ACIDS AND KINASE INHIBITORS PERTURB MOUSE NEURAL CREST CELLS IN VITRO

    EPA Science Inventory

    HUNTER, E.S.1, J. SMITH2, J. ANDREWS1. 1 Reproductive Toxicology Division, NHEERL, US EPA, Research Triangle Park and 2 Department of Cell and Developmental Biology, UNC-CH, Chapel Hill, North Carolina. Haloacetic acids and kinase inhibitors perturb mouse neural crest cells in vi...

  16. COMPARATIVE PATHOGENESIS OF HALOACETIC ACID AND PROTEIN KINASE INHIBITOR EMBRYOTOXICITY IN MOUSE WHOLE EMBRYO CULTURE

    EPA Science Inventory

    Comparative pathogenesis of haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.

    Ward KW, Rogers EH, Hunter ES 3rd.

    Curriculum in Toxicology, University of North Carolina at Chapel Hill, 27599-7270, USA.

    Haloacetic acids ...

  17. Structure-based design of isoquinoline-5-sulfonamide inhibitors of protein kinase B.

    PubMed

    Collins, Ian; Caldwell, John; Fonseca, Tatiana; Donald, Alastair; Bavetsias, Vassilios; Hunter, Lisa-Jane K; Garrett, Michelle D; Rowlands, Martin G; Aherne, G Wynne; Davies, Thomas G; Berdini, Valerio; Woodhead, Steven J; Davis, Deborah; Seavers, Lisa C A; Wyatt, Paul G; Workman, Paul; McDonald, Edward

    2006-02-15

    Structure-based drug design of novel isoquinoline-5-sulfonamide inhibitors of PKB as potential antitumour agents was investigated. Constrained pyrrolidine analogues that mimicked the bound conformation of linear prototypes were identified and investigated by co-crystal structure determinations with the related protein PKA. Detailed variation in the binding modes between inhibitors with similar overall conformations was observed. Potent PKB inhibitors from this series inhibited GSK3beta phosphorylation in cellular assays, consistent with inhibition of PKB kinase activity in cells. PMID:16249095

  18. A High-Throughput Screen Reveals New Small-Molecule Activators and Inhibitors of Pantothenate Kinases

    PubMed Central

    2016-01-01

    Pantothenate kinase (PanK) is a regulatory enzyme that controls coenzyme A (CoA) biosynthesis. The association of PanK with neurodegeneration and diabetes suggests that chemical modifiers of PanK activity may be useful therapeutics. We performed a high throughput screen of >520000 compounds from the St. Jude compound library and identified new potent PanK inhibitors and activators with chemically tractable scaffolds. The HTS identified PanK inhibitors exemplified by the detailed characterization of a tricyclic compound (7) and a preliminary SAR. Biophysical studies reveal that the PanK inhibitor acts by binding to the ATP–enzyme complex. PMID:25569308

  19. Novel morpholin-3-one fused quinazoline derivatives as EGFR tyrosine kinase inhibitors.

    PubMed

    Qin, Xuemei; Lv, Yongjuan; Liu, Peng; Li, Zhipeng; Hu, Liming; Zeng, Chengchu; Yang, Leifu

    2016-03-15

    A series of novel morpholin-3-one-fused quinazoline derivatives were designed, synthesized and evaluated as EGFR tyrosine kinase inhibitors. Nineteen compounds showed significant inhibitory activities against EGFR(wt) kinase (IC50<1 μM). Compound a8 demonstrated the most potent inhibitory activity toward EGFR(wt) (IC50=53.1 nM). Compound a7 and a8 showed excellent inhibitory activities against mutant EGFR(T790M/L858R) and strong antiproliferative activity against H358 and A549 cell lines. Finally, molecular docking studies were performed to predict the possible binding mode of the target compounds. It is believed that this work would be very useful for designing a new series of tyrosine kinase inhibitors targeting EGFR. PMID:26879314

  20. Risk of Infectious Complications in Hemato-Oncological Patients Treated with Kinase Inhibitors

    PubMed Central

    Reinwald, Mark; Boch, Tobias; Hofmann, Wolf-Karsten; Buchheidt, Dieter

    2015-01-01

    Infectious complications are a major cause of morbidity and mortality in patients with hemato-oncological diseases. Although disease-related immunosuppression represents one factor, aggressive treatment regimens, such as chemotherapy, stem cell transplantation, or antibody treatment, account for a large proportion of infectious side effects. With the advent of targeted therapies affecting specific kinases in malignant diseases, the outcome of patients has further improved. Nonetheless, dependent on the specific pathway targeted or off-target activity of the kinase inhibitor, therapy-associated infectious complications may occur. We review the most common and approved kinase inhibitors targeting a variety of hemato-oncological malignancies for their immunosuppressive potential and evaluate their risk of infectious side effects based on preclinical evidence and clinical data in order to raise awareness of the potential risks involved. PMID:27127405

  1. Phosphoproteomics data classify hematological cancer cell lines according to tumor type and sensitivity to kinase inhibitors

    PubMed Central

    2013-01-01

    Background Tumor classification based on their predicted responses to kinase inhibitors is a major goal for advancing targeted personalized therapies. Here, we used a phosphoproteomic approach to investigate biological heterogeneity across hematological cancer cell lines including acute myeloid leukemia, lymphoma, and multiple myeloma. Results Mass spectrometry was used to quantify 2,000 phosphorylation sites across three acute myeloid leukemia, three lymphoma, and three multiple myeloma cell lines in six biological replicates. The intensities of the phosphorylation sites grouped these cancer cell lines according to their tumor type. In addition, a phosphoproteomic analysis of seven acute myeloid leukemia cell lines revealed a battery of phosphorylation sites whose combined intensities correlated with the growth-inhibitory responses to three kinase inhibitors with remarkable correlation coefficients and fold changes (> 100 between the most resistant and sensitive cells). Modeling based on regression analysis indicated that a subset of phosphorylation sites could be used to predict response to the tested drugs. Quantitative analysis of phosphorylation motifs indicated that resistant and sensitive cells differed in their patterns of kinase activities, but, interestingly, phosphorylations correlating with responses were not on members of the pathway being targeted; instead, these mainly were on parallel kinase pathways. Conclusion This study reveals that the information on kinase activation encoded in phosphoproteomics data correlates remarkably well with the phenotypic responses of cancer cells to compounds that target kinase signaling and could be useful for the identification of novel markers of resistance or sensitivity to drugs that target the signaling network. PMID:23628362

  2. A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance.

    PubMed

    Ouellette, Steven B; Noel, Brett M; Parker, Laurie L

    2016-01-01

    Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments. PMID:27598410

  3. Anti-proliferative effects of protein kinase C inhibitors in human keratinocytes.

    PubMed

    Hegemann, L; Bonnekoh, B; van Rooijen, L A; Mahrle, G

    1992-07-01

    Various lines of evidence indicate that protein kinase C, a key enzyme in transmembraneous signal transduction, is involved in the regulation of keratinocyte proliferation. In the present study we have investigated the effects of various structurally unrelated protein kinase C inhibitors on the proliferation of HaCa T cells, a non-tumorigenic human keratinocyte cell line. All protein kinase C inhibitors dose-dependently inhibited cell proliferation as assessed by the incorporation of radioactively labelled thymidine and amino acids as well as the increase in total protein content in keratinocytes. The potencies of the drugs to inhibit cell proliferation were strongly correlated to their inhibitory potency on purified protein kinase C, displaying a correlation coefficient of 0.97. Methotrexate, an anti-proliferative drug, was found not to inhibit protein kinase C. Therefore, our data provide evidence that protein kinase C is crucially involved in the regulation of keratinocyte proliferation but is not the only target of anti-proliferative drug action. PMID:1390454

  4. AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies.

    PubMed

    Zabludoff, Sonya D; Deng, Chun; Grondine, Michael R; Sheehy, Adam M; Ashwell, Susan; Caleb, Benjamin L; Green, Stephen; Haye, Heather R; Horn, Candice L; Janetka, James W; Liu, Dongfang; Mouchet, Elizabeth; Ready, Shannon; Rosenthal, Judith L; Queva, Christophe; Schwartz, Gary K; Taylor, Karen J; Tse, Archie N; Walker, Graeme E; White, Anne M

    2008-09-01

    Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings. PMID:18790776

  5. Protein kinase C-δ inhibitor, Rottlerin inhibits growth and survival of mycobacteria exclusively through Shikimate kinase.

    PubMed

    Pandey, Sapna; Chatterjee, Aditi; Jaiswal, Swati; Kumar, Sanjay; Ramachandran, Ravishankar; Srivastava, Kishore K

    2016-09-16

    The molecular bases of disease provide exceptional prospect to translate research findings into new drugs. Nevertheless, to develop new and novel chemical entities takes huge amount of time and efforts, mainly due to the stringent processes. Therefore, drug repurposing is one of such strategies which is being used in recent times to identify new pharmacophores. The essential first step in discovery of the specific inhibitor with low toxicity is the identification and elucidation of pathways exclusive to target pathogen. One such target is the shikimate pathway, which is essential for algae, higher plants, bacteria and fungi. Since, this enzyme system is absent in higher eukaryotes and in mammals, the enzymes involved in the pathway provide an attractive target for the development of potentially selective and non toxic antimicrobial agents. Since, so far there is no specific inhibitor which is able to restrain mycobacterial shikimate pathway; we expanded the use of a known kinase inhibitor; Rottlerin, in order to predict the prototype in discovering the specific molecules against this enzyme. For the first time we have shown that Rottlerin inhibits extracellular mycobacteria by affecting Shikimate Kinase (SK) and this effect is further enhanced during the intracellular infection due to the added effect of PKC- δ down-regulation. The molecular docking of Rottlerin with both the mycobacterial SKs, corroborated the inhibition data, and revealed that the effects of SK, in slow and in fast grower mycobacteria are due to the changes in affinity of binding with the drug. PMID:27498028

  6. Protein kinase C betaII peptide inhibitor exerts cardioprotective effects in rat cardiac ischemia/reperfusion injury.

    PubMed

    Omiyi, Didi; Brue, Richard J; Taormina, Philip; Harvey, Margaret; Atkinson, Norrell; Young, Lindon H

    2005-08-01

    Ischemia followed by reperfusion (I/R) in the presence of polymorphonuclear leukocytes (PMNs) results in a marked cardiac contractile dysfunction. A cell-permeable protein kinase C (PKC) betaII peptide inhibitor was used to test the hypothesis that PKC betaII inhibition could attenuate PMN-induced cardiac dysfunction by suppression of superoxide production from PMNs and increase NO release from vascular endothelium. The effects of the PKC betaII peptide inhibitor were examined in isolated ischemic (20 min) and reperfused (45 min) rat hearts with PMNs. The PKC betaII inhibitor (10 microM; n = 7) significantly attenuated PMN-induced cardiac dysfunction compared with I/R hearts (n = 9) receiving PMNs alone in left ventricular developed pressure (LVDP) and the maximal rate of LVDP (+dP/dt(max)) cardiac function indices (p < 0.01). The PKC betaII inhibitor at 10 microM significantly increased endothelial NO release from a basal value of 1.85 +/- 0.18 pmol NO/mg tissue to 3.49 +/- 0.62 pmol NO/mg tissue from rat aorta. It also significantly inhibited superoxide release (i.e., absorbance) from N-formyl-L-methionyl-L-leucyl-L-phenylalanine-stimulated rat PMNs from 0.13 +/- 0.01 to 0.02 +/- 0.004 (p < 0.01) at 10 microM. Histological analysis of the left ventricle of representative rat hearts from each group showed that the PKC betaII peptide inhibitor-treated hearts experienced a marked reduction in PMN vascular adherence and infiltration into the postreperfused cardiac tissue compared with I/R + PMN hearts (p < 0.01). These results suggest that the PKC betaII peptide inhibitor attenuates PMN-induced post-I/R cardiac contractile dysfunction by increasing endothelial NO release and by inhibiting superoxide release from PMNs. PMID:15878997

  7. Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity

    SciTech Connect

    Lin, Yen -Lin; Meng, Yilin; Huang, Lei; Roux, Benoît

    2014-10-22

    Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a “conformational selection” mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to the DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.

  8. Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity

    DOE PAGESBeta

    Lin, Yen -Lin; Meng, Yilin; Huang, Lei; Roux, Benoît

    2014-10-22

    Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a “conformational selection” mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to themore » DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.« less

  9. Discovery of a Selective Inhibitor of Oncogenic B-Raf Kinase With Potent Antimelanoma Activity

    SciTech Connect

    Tsai, J.; Lee, J.T.; Wang, W.; Zhang, J.; Cho, H.; Mamo, S.; Bremer, R.; Gillette, S.; Kong, J.; Haass, N.K.; Sproesser, K.; Li, L.; Smalley, K.S.M.; Fong, D.; Zhu, Y.-L.; Marimuthu, A.; Nguyen, H.; Lam, B.; Liu, J.; Cheung, I.; Rice, J.

    2009-05-26

    BRAF{sup V600E} is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting 'active' protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf{sup V600E} with an IC{sub 50} of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf{sup V600E} kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf{sup V600E}-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf{sup V600E}-positive cells. In B-Raf{sup V600E}-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf{sup V600E}-driven tumors.

  10. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity

    PubMed Central

    Tsai, James; Lee, John T.; Wang, Weiru; Zhang, Jiazhong; Cho, Hanna; Mamo, Shumeye; Bremer, Ryan; Gillette, Sam; Kong, Jun; Haass, Nikolas K.; Sproesser, Katrin; Li, Ling; Smalley, Keiran S. M.; Fong, Daniel; Zhu, Yong-Liang; Marimuthu, Adhirai; Nguyen, Hoa; Lam, Billy; Liu, Jennifer; Cheung, Ivana; Rice, Julie; Suzuki, Yoshihisa; Luu, Catherine; Settachatgul, Calvin; Shellooe, Rafe; Cantwell, John; Kim, Sung-Hou; Schlessinger, Joseph; Zhang, Kam Y. J.; West, Brian L.; Powell, Ben; Habets, Gaston; Zhang, Chao; Ibrahim, Prabha N.; Hirth, Peter; Artis, Dean R.; Herlyn, Meenhard; Bollag, Gideon

    2008-01-01

    BRAFV600E is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting “active” protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-RafV600E with an IC50 of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-RafV600E kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-RafV600E-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-RafV600E-positive cells. In B-RafV600E-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-RafV600E-driven tumors. PMID:18287029

  11. A Novel Triazolopyridine-Based Spleen Tyrosine Kinase Inhibitor That Arrests Joint Inflammation

    PubMed Central

    Ferguson, Gregory D.; Delgado, Mercedes; Plantevin-Krenitsky, Veronique; Jensen-Pergakes, Kristen; Bates, R. J.; Torres, Sanaa; Celeridad, Maria; Brown, Heather; Burnett, Kelven; Nadolny, Lisa; Tehrani, Lida; Packard, Garrick; Pagarigan, Barbra; Haelewyn, Jason; Nguyen, Trish; Xu, Li; Tang, Yang; Hickman, Matthew; Baculi, Frans; Pierce, Steven; Miyazawa, Keiji; Jackson, Pilgrim; Chamberlain, Philip; LeBrun, Laurie; Xie, Weilin; Bennett, Brydon; Blease, Kate

    2016-01-01

    Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1α in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA. PMID:26756335

  12. Effects of BP-14, a novel cyclin-dependent kinase inhibitor, on anaplastic thyroid cancer cells.

    PubMed

    Allegri, Lorenzo; Baldan, Federica; Mio, Catia; Puppin, Cinzia; Russo, Diego; Kryštof, Vladimir; Damante, Giuseppe

    2016-04-01

    Anaplastic thyroid carcinoma (ATC) is an extremely aggressive human malignancy characterized by a marked degree of invasiveness, absense of features of thyroid differentiation and resistance to current medical treatment. It is well known that ATCs are characterized by deregulation of genes related to cell cycle regulation, i.e., cyclin-dependent kinases (CDKs) and endogenous cyclin-dependent kinase inhibitors (CDKIs). Therefore, in the present study, the effect of a novel exogenous cyclin-dependent kinase inhibitor, BP-14, was investigated in three human ATC cell lines. The ATC-derived cell lines FRO, SW1736 and 8505C were treated with BP-14 alone or in combination with the mTOR inhibitor everolimus. In all ATC cell lines, treatment with BP-14 decreased cell viability and, in two of them, BP-14 modified expression of genes involved in epithelial-mesenchymal transition. Thus, our data indicate that BP-14 is a potential new compound effective against ATC. Combined treatment with BP-14 and the mTOR inhibitor everolimus had a strong synergistic effect on cell viability in all three cell lines, suggesting that the combined used of CDK and mTOR inhibitors may be a useful strategy for ATC treatment. PMID:26884249

  13. An Aminopyridazine Inhibitor of Death Associated Protein Kinase Attenuates Hypoxia-Ischemia Induced Brain Damage

    SciTech Connect

    Velentza, A.V.; Wainwright, M.S.; Zasadzki, M.; Mirzoeva, S.; Haiech, J.; Focia, P.J.; Egli, M.; Watterson, D.M.

    2010-03-08

    Death associated protein kinase (DAPK) is a calcium and calmodulin regulated enzyme that functions early in eukaryotic programmed cell death, or apoptosis. To validate DAPK as a potential drug discovery target for acute brain injury, the first small molecule DAPK inhibitor was synthesized and tested in vivo. A single injection of the aminopyridazine-based inhibitor administered 6 h after injury attenuated brain tissue or neuronal biomarker loss measured, respectively, 1 week and 3 days later. Because aminopyridazine is a privileged structure in neuropharmacology, we determined the high-resolution crystal structure of a binary complex between the kinase domain and a molecular fragment of the DAPK inhibitor. The co-crystal structure describes a structural basis for interaction and provides a firm foundation for structure-assisted design of lead compounds with appropriate molecular properties for future drug development.

  14. 3-Phosphoinositide-Dependent protein Kinase-1 (PDK1) inhibitors: A review from patent literature

    PubMed Central

    Barile, Elisa; De, Surya K.; Pellecchia, Maurizio

    2016-01-01

    PDK1 (3-Phosphoinositide-dependent kinase 1) is a key member of the AGC protein kinase family. It plays an important role in a variety of cellular functions, leading to the activation of the PI3K signaling pathway, an event often associated with the onset and progression of several human cancers. Numerous recent observations suggest that PDK1 inhibitors may provide novel opportunities for the development of effective classes of therapeutics. On these premises, recent years have witnessed an increased effort by medicinal chemists to develop novel scaffolds to derive potent and selective PDK1 inhibitors. The intent of this review is to update the reader on the recent patent literature covering applications published between June 2008 and September 2011 that report on PDK1 inhibitors. PMID:24236780

  15. Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes

    PubMed Central

    Yoon, Jeong-Hwan; Jung, Su Myung; Park, Seok Hee; Kato, Mitsuyasu; Yamashita, Tadashi; Lee, In-Kyu; Sudo, Katsuko; Nakae, Susumu; Han, Jin Soo; Kim, Ok-Hee; Oh, Byung-Chul; Sumida, Takayuki; Kuroda, Masahiko; Ju, Ji-Hyeon; Jung, Kyeong Cheon; Park, Seong Hoe; Kim, Dae-Kee; Mamura, Mizuko

    2013-01-01

    Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8+ T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8+ T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation. PMID:24127404

  16. Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes.

    PubMed

    Yoon, Jeong-Hwan; Jung, Su Myung; Park, Seok Hee; Kato, Mitsuyasu; Yamashita, Tadashi; Lee, In-Kyu; Sudo, Katsuko; Nakae, Susumu; Han, Jin Soo; Kim, Ok-Hee; Oh, Byung-Chul; Sumida, Takayuki; Kuroda, Masahiko; Ju, Ji-Hyeon; Jung, Kyeong Cheon; Park, Seong Hoe; Kim, Dae-Kee; Mamura, Mizuko

    2013-11-01

    Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8(+) T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8(+) T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation. PMID:24127404

  17. Identification of a Dual Inhibitor of Janus Kinase 2 (JAK2) and p70 Ribosomal S6 Kinase1 (S6K1) Pathways.

    PubMed

    Byun, Sanguine; Lim, Semi; Mun, Ji Young; Kim, Ki Hyun; Ramadhar, Timothy R; Farrand, Lee; Shin, Seung Ho; Thimmegowda, N R; Lee, Hyong Joo; Frank, David A; Clardy, Jon; Lee, Sam W; Lee, Ki Won

    2015-09-25

    Bioactive phytochemicals can suppress the growth of malignant cells, and investigation of the mechanisms responsible can assist in the identification of novel therapeutic strategies for cancer therapy. Ginger has been reported to exhibit potent anti-cancer effects, although previous reports have often focused on a narrow range of specific compounds. Through a direct comparison of various ginger compounds, we determined that gingerenone A selectively kills cancer cells while exhibiting minimal toxicity toward normal cells. Kinase array screening revealed JAK2 and S6K1 as the molecular targets primarily responsible for gingerenone A-induced cancer cell death. The effect of gingerenone A was strongly associated with relative phosphorylation levels of JAK2 and S6K1, and administration of gingerenone A significantly suppressed tumor growth in vivo. More importantly, the combined inhibition of JAK2 and S6K1 by commercial inhibitors selectively induced apoptosis in cancer cells, whereas treatment with either agent alone did not. These findings provide rationale for dual targeting of JAK2 and S6K1 in cancer for a combinatorial therapeutic approach. PMID:26242912

  18. Transcription and translation are primary targets of Pim kinase inhibitor SGI-1776 in mantle cell lymphoma

    PubMed Central

    Yang, Qingshan; Chen, Lisa S.; Neelapu, Sattva S.; Miranda, Roberto N.; Medeiros, L. Jeffrey

    2012-01-01

    Proviral integration site for Moloney murine leukemia virus (Pim) kinases are serine/threonine/tyrosine kinases and oncoproteins that promote tumor progression. Three isoforms of Pim kinases have been identified and are known to phosphorylate numerous substrates, with regulatory functions in transcription, translation, cell cycle, and survival pathways. These kinases are involved in production, proliferation, and survival of normal B cells and are overexpressed in B-cell malignancies such as mantle cell lymphoma (MCL). SGI-1776 is a small mol-ecule and Pim kinase inhibitor with selectivity for Pim-1. We hypothesize that Pim kinase function can be inhibited by SGI-1776 in MCL and that inhibition of phosphorylation of downstream substrates will disrupt transcriptional, translational, and cell cycle processes and promote cell death. SGI-1776 treatment in 4 MCL cell lines resulted in apoptosis induction. Phosphorylation of transcription (c-Myc) and translation targets (4E-BP1), tested in Jeko-1 and Mino, was declined. Consistent with these data, Mcl-1 and cyclin D1 protein levels were decreased. Importantly, similar to cell line data, MCL primary cells but not normal cells showed similar inhibition of substrate phosphorylation and cytotoxicity from SGI-1776 treatment. Genetic knockdown of Pim-1/Pim-2 affected similar proteins in MCL cell lines. Collectively these data demonstrate Pim kinases as therapeutic targets in MCL. PMID:22955922

  19. TARGETING SPHINGOSINE KINASE 1 INHIBITS AKT SIGNALING, INDUCES APOPTOSIS, AND SUPPRESSES GROWTH OF HUMAN GLIOBLASTOMA CELLS AND XENOGRAFTS

    PubMed Central

    Kapitonov, Dmitri; Allegood, Jeremy C.; Mitchell, Clint; Hait, Nitai C.; Almenara, Jorge A.; Adams, Jeffrey K.; Zipkin, Robert E.; Dent, Paul; Kordula, Tomasz; Milstien, Sheldon; Spiegel, Sarah

    2009-01-01

    Sphingosine-1-phosphate (S1P) is a potent sphingolipid mediator of diverse processes important for brain tumors, including cell growth, survival, migration, invasion, and angiogenesis. Sphingosine kinase 1 (SphK1), one of the two isoenzymes that produce S1P, is upregulated in glioblastoma and has been linked to poor prognosis in patients with glioblastoma multiforme (GBM). In the present study, we found that a potent isotype-specific SphK1 inhibitor, SK1-I, suppressed growth of LN229 and U373 glioblastoma cell lines and non-established human GBM6 cells. SK1-I also enhanced GBM cell death and inhibited their migration and invasion. SK1-I rapidly reduced phosphorylation of Akt but had no significant effect on activation of ERK1/2, another important survival pathway for GBM. Inhibition of the concomitant activation of the JNK pathway induced by SK1-I attenuated death of GBM cells. Importantly, SK1-I markedly reduced tumor growth rate of glioblastoma xenografts, inducing apoptosis and reducing tumor vascularization and enhanced the survival of mice harboring LN229 intracranial tumors. Our results support the notion that SphK1 may be an important factor in GBM and suggest that an isozyme-specific inhibitor of SphK1 deserves consideration as a new therapeutic agent for this disease. PMID:19723667

  20. Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

    PubMed Central

    Ito, Genta; Katsemonova, Kristina; Tonelli, Francesca; Lis, Pawel; Baptista, Marco A.S.; Shpiro, Natalia; Duddy, Graham; Wilson, Steve; Ho, Philip Wing-Lok; Ho, Shu-Leong; Reith, Alastair D.; Alessi, Dario R.

    2016-01-01

    Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway. PMID:27474410

  1. Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.

    PubMed

    Ito, Genta; Katsemonova, Kristina; Tonelli, Francesca; Lis, Pawel; Baptista, Marco A S; Shpiro, Natalia; Duddy, Graham; Wilson, Steve; Ho, Philip Wing-Lok; Ho, Shu-Leong; Reith, Alastair D; Alessi, Dario R

    2016-09-01

    Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1-2 min, markedly more rapidly than the Ser(935) and Ser(1292) biomarker sites that require 40-80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser(910) and Ser(935) and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway. PMID:27474410

  2. Kinase Inhibitors that Increase the Sensitivity of Methicillin Resistant Staphylococcus aureus to β-Lactam Antibiotics

    PubMed Central

    Vornhagen, Jay; Burnside, Kellie; Whidbey, Christopher; Berry, Jessica; Qin, Xuan; Rajagopal, Lakshmi

    2015-01-01

    Staphylococcus aureus are Gram-positive bacteria that are the leading cause of recurrent infections in humans that include pneumonia, bacteremia, osteomyelitis, arthritis, endocarditis, and toxic shock syndrome. The emergence of methicillin resistant S. aureus strains (MRSA) has imposed a significant concern in sustained measures of treatment against these infections. Recently, MRSA strains deficient in expression of a serine/threonine kinase (Stk1 or PknB) were described to exhibit increased sensitivity to β-lactam antibiotics. In this study, we screened a library consisting of 280 drug-like, low-molecular-weight compounds with the ability to inhibit protein kinases for those that increased the sensitivity of wild-type MRSA to β-lactams and then evaluated their toxicity in mice. We report the identification of four kinase inhibitors, the sulfonamides ST085384, ST085404, ST085405, and ST085399 that increased sensitivity of WT MRSA to sub-lethal concentrations of β-lactams. Furthermore, these inhibitors lacked alerting structures commonly associated with toxic effects, and toxicity was not observed with ST085384 or ST085405 in vivo in a murine model. These results suggest that kinase inhibitors may be useful in therapeutic strategies against MRSA infections. PMID:26506394

  3. ACTIVATION OF PERK KINASE IN NEURAL CELLS BY PROTEASOME INHIBITOR TREATMENT

    PubMed Central

    Zhang, Le; Ebenezer, Philip J; Dasuri, Kalavathi; Bruce-Keller, Annadora J.; Fernandez-Kim, Sun Ok; Liu, Ying; Keller, Jeffrey N.

    2010-01-01

    Inhibition of the proteasome proteolytic pathway occurs as the result of normal aging, as well as in a variety of neurodegenerative conditions, and is believed to promote cellular toxicity in each of these conditions through diverse mechanisms. In the present study we examined whether proteasome inhibition alters the protein kinase (PKR)-like ER kinase (PERK). Our studies demonstrate that proteasome inhibitors induce the transient activation of PERK in both primary rat neurons as well as the N2a neural cell line. Experiments with siRNA to PERK demonstrated that the modulation of PERK was not significant involved in regulating toxicity, ubiquitinated protein levels, or ribosome perturbations in response to proteasome inhibitor treatment. Surprisingly, PERK was observed to be involved in the upregulation of p38 kinase following proteasome inhibitor treatment. Taken together, these data demonstrate the ability of proteasome inhibition to activate PERK and demonstrate evidence for novel cross talk between PERK and the activation of p38 kinase in neural cells following proteasome inhibition. Taken together, these data have implications for understanding the basis by which proteasome inhibition alters neural homeostasis, and the basis by which cell signaling cascades are regulated by proteasome inhibition. PMID:19860852

  4. Kinase Inhibitors that Increase the Sensitivity of Methicillin Resistant Staphylococcus aureus to β-Lactam Antibiotics.

    PubMed

    Vornhagen, Jay; Burnside, Kellie; Whidbey, Christopher; Berry, Jessica; Qin, Xuan; Rajagopal, Lakshmi

    2015-01-01

    Staphylococcus aureus are Gram-positive bacteria that are the leading cause of recurrent infections in humans that include pneumonia, bacteremia, osteomyelitis, arthritis, endocarditis, and toxic shock syndrome. The emergence of methicillin resistant S. aureus strains (MRSA) has imposed a significant concern in sustained measures of treatment against these infections. Recently, MRSA strains deficient in expression of a serine/threonine kinase (Stk1 or PknB) were described to exhibit increased sensitivity to β-lactam antibiotics. In this study, we screened a library consisting of 280 drug-like, low-molecular-weight compounds with the ability to inhibit protein kinases for those that increased the sensitivity of wild-type MRSA to β-lactams and then evaluated their toxicity in mice. We report the identification of four kinase inhibitors, the sulfonamides ST085384, ST085404, ST085405, and ST085399 that increased sensitivity of WT MRSA to sub-lethal concentrations of β-lactams. Furthermore, these inhibitors lacked alerting structures commonly associated with toxic effects, and toxicity was not observed with ST085384 or ST085405 in vivo in a murine model. These results suggest that kinase inhibitors may be useful in therapeutic strategies against MRSA infections. PMID:26506394

  5. Characterization of interactions and pharmacophore development for DFG-out inhibitors to RET tyrosine kinase.

    PubMed

    Gao, Chunxia; Grøtli, Morten; Eriksson, Leif A

    2015-07-01

    RET (rearranged during transfection) tyrosine kinase is a promising target for several human cancers. Abt-348, Birb-796, Motesanib and Sorafenib are DFG-out multi-kinase inhibitors that have been reported to inhibit RET activity with good IC50 values. Although the DFG-out conformation has attracted great interest in the design of type II inhibitors, the structural requirements for binding to the RET DFG-out conformation remains unclear. Herein, the DFG-out conformation of RET was determined by homology modelling, the four inhibitors were docked, and the binding modes investigated by molecular dynamics simulation. Binding free energies were calculated using the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method. The trends in predicted binding free affinities correlated well with experimental data and were used to explain the activity difference of the studied inhibitors. Per-residue energy decomposition analyses provided further information on specific interaction properties. Finally, we also conducted a detailed e-pharmacophore modelling of the different RET-inhibitor complexes, explaining the common and specific pharmacophore features of the different complexes. The results reported herein will be useful in future rational design of novel DFG-out RET inhibitors. PMID:26044359

  6. Naturally Occurring Mutations in the MPS1 Gene Predispose Cells to Kinase Inhibitor Drug Resistance.

    PubMed

    Gurden, Mark D; Westwood, Isaac M; Faisal, Amir; Naud, Sébastien; Cheung, Kwai-Ming J; McAndrew, Craig; Wood, Amy; Schmitt, Jessica; Boxall, Kathy; Mak, Grace; Workman, Paul; Burke, Rosemary; Hoelder, Swen; Blagg, Julian; Van Montfort, Rob L M; Linardopoulos, Spiros

    2015-08-15

    Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells. PMID:26202014

  7. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    SciTech Connect

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G.

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  8. Identification and structure-function analysis of subfamily selective G protein-coupled receptor kinase inhibitors.

    PubMed

    Homan, Kristoff T; Larimore, Kelly M; Elkins, Jonathan M; Szklarz, Marta; Knapp, Stefan; Tesmer, John J G

    2015-01-16

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson's disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors. PMID:25238254

  9. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    SciTech Connect

    Okabe, Seiichi Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  10. Discovery of Pyrrolopyridine−Pyridone Based Inhibitors of Met Kinase: Synthesis, X-ray Crystallographic Analysis, and Biological Activities

    SciTech Connect

    Kim, Kyoung Soon; Zhang, Liping; Schmidt, Robert; Cai, Zhen-Wei; Wei, Donna; Williams, David K.; Lombardo, Louis J.; Trainor, George L.; Xie, Dianlin; Zhang, Yaquan; An, Yongmi; Sack, John S.; Tokarski, John S.; Darienzo, Celia; Kamath, Amrita; Marathe, Punit; Zhang, Yueping; Lippy, Jonathan; Jeyaseelan, Sr., Robert; Wautlet, Barri; Henley, Benjamin; Gullo-Brown, Johnni; Manne, Veeraswamy; Hunt, John T.; Fargnoli, Joseph; Borzilleri, Robert M.

    2008-10-02

    Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2 kinases with IC{sub 50} values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft model.

  11. The discovery of 2-substituted phenol quinazolines as potent RET kinase inhibitors with improved KDR selectivity.

    PubMed

    Newton, Rebecca; Bowler, Katherine A; Burns, Emily M; Chapman, Philip J; Fairweather, Emma E; Fritzl, Samantha J R; Goldberg, Kristin M; Hamilton, Niall M; Holt, Sarah V; Hopkins, Gemma V; Jones, Stuart D; Jordan, Allan M; Lyons, Amanda J; Nikki March, H; McDonald, Neil Q; Maguire, Laura A; Mould, Daniel P; Purkiss, Andrew G; Small, Helen F; Stowell, Alexandra I J; Thomson, Graeme J; Waddell, Ian D; Waszkowycz, Bohdan; Watson, Amanda J; Ogilvie, Donald J

    2016-04-13

    Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor using vandetanib 1 as the starting point for structure-based drug design. Phenolic anilinoquinazolines exemplified by 6 showed improved affinities towards RET but, unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte stability, but could also impart a significant gain in selectivity. This culminated in the identification of 36; a potent RET inhibitor with much improved selectivity against KDR. PMID:26874741

  12. QSAR and molecular docking studies on oxindole derivatives as VEGFR-2 tyrosine kinase inhibitors.

    PubMed

    Kang, Cong-Min; Liu, Dong-Qing; Zhao, Xu-Hao; Dai, Ying-Jie; Cheng, Jia-Gao; Lv, Ying-Tao

    2016-01-01

    The three-dimensional quantitative structure-activity relationships (3D-QSAR) were established for 30 oxindole derivatives as vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors by using comparative molecular field analysis (CoMFA) and comparative similarity indices analysis comparative molecular similarity indices analysis (CoMSIA) techniques. With the CoMFA model, the cross-validated value (q(2)) was 0.777, the non-cross-validated value (R(2)) was 0.987, and the external cross-validated value ([Formula: see text]) was 0.72. And with the CoMSIA model, the corresponding q(2), R(2) and [Formula: see text] values were 0.710, 0.988 and 0.78, respectively. Docking studies were employed to bind the inhibitors into the active site to determine the probable binding conformation. The binding mode obtained by molecular docking was in good agreement with the 3D-QSAR results. Based on the QSAR models and the docking binding mode, a set of new VEGFR-2 tyrosine kinase inhibitors were designed, which showed excellent predicting inhibiting potencies. The result revealed that both QSAR models have good predictive capability to guide the design and structural modification of homologic compounds. It is also helpful for further research and development of new VEGFR-2 tyrosine kinase inhibitors. PMID:26416217

  13. Treatment of cultured human astrocytes and vascular endothelial cells with protein kinase CK2 inhibitors induces early changes in cell shape and cytoskeleton.

    PubMed

    Kramerov, A A; Golub, A G; Bdzhola, V G; Yarmoluk, S M; Ahmed, K; Bretner, M; Ljubimov, A V

    2011-03-01

    Ubiquitous protein kinase CK2 is a key regulator of cell migration, proliferation and tumor growth. CK2 is abundant in retinal astrocytes, and its inhibition suppresses retinal neovascularization in a mouse retinopathy model. In human astrocytes, CK2 co-distributes with GFAP-containing intermediate filaments, which implies its association with cytoskeleton. Contrary to astrocytes, CK2 is co-localized in microvascular endothelial cells (HBMVEC) with microtubules and actin stress fibers, but not with vimentin-containing intermediate filaments. Specific CK2 inhibitors (TBB, TBI, TBCA and DMAT) and nine novel CK2 inhibiting compounds (TID43, TID46, Quinolone-7, Quinolone-39, FNH28, FNH62, FNH64, FNH68 and FNH74) were tested at 10-200 μM for their ability to induce morphological alterations in cultured human astrocytes (HAST-40), and HBMVEC (For explanation of the inhibitor names, see "Methods" section). CK2 inhibitors caused dramatic changes in shape of cultured cells with effective inhibitor concentrations between 50 and 100 μM. Attached cells retracted, acquired shortened processes, and eventually rounded up and detached. CK2 inhibitor-induced morphological alterations were completely reversible and were not blocked by caspase inhibition. However, longer treatment or higher inhibitor concentration did cause apoptosis. The speed and potency of the CK2 inhibitors effects on cell shape and adhesion were inversely correlated with serum concentration. Western analyses showed that TBB and TBCA elicited a significant (about twofold) increase in the activation of p38 and ERK1/2 MAP kinases that may be involved in cytoskeleton regulation. This novel early biological cell response to CK2 inhibition may underlie the anti-angiogenic effect of CK2 suppression in the retina. PMID:21125314

  14. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

    PubMed Central

    Miller, J. Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H. James; Huband, Michael D.; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y.; Mehrens, Shawn; Mueller, W. Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J. V. N. Vara; Shelly, John A.; Skerlos, Laura; Sulavik, Mark; Thomas, V. Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C. Kendall

    2009-01-01

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity. PMID:19164768

  15. Design, validation and efficacy of bisubstrate inhibitors specifically affecting ecto-CK2 kinase activity.

    PubMed

    Cozza, Giorgio; Zanin, Sofia; Sarno, Stefania; Costa, Elena; Girardi, Cristina; Ribaudo, Giovanni; Salvi, Mauro; Zagotto, Giuseppe; Ruzzene, Maria; Pinna, Lorenzo A

    2015-11-01

    By derivatizing the purely competitive CK2 inhibitor N1-(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)-propane-1,3-diamine (K137) at its 3-amino position with a peptidic fragment composed of three or four glutamic or aspartic acid residues, a new family of bisubstrate inhibitors has been generated whose ability to simultaneously interact with both the ATP and the phosphoacceptor substrate-binding sites has been probed by running mixed competition kinetics and by mutational mapping of the kinase residues implicated in substrate recognition. The most effective bisubstrate inhibitor, K137-E4, interacts with three functional regions of the kinase: the hydrophobic pocket close to the ATP-binding site, the basic residues of the p+1 loop that recognizes the acidic determinant at position n+1 and the basic residues of α-helixC that recognize the acidic determinant at position n+3. Compared with the parent inhibitor (K137), K137-E4 is severalfold more potent (IC50 25 compared with 130 nM) and more selective, failing to inhibit any other kinase as drastically as CK2 out of 140 enzymes, whereas 35 kinases are inhibited more potently than CK2 by K137. K137-E4 is unable to penetrate the cell and to inhibit endogenous CK2, its pro-apoptotic efficacy being negligible compared with cell-permeant inhibitors; however, it readily inhibits ecto-CK2 on the outer cell surface, reducing the phosphorylation of several external phosphoproteins. Inhibition of ecto-CK2 by K137-E4 is accompanied by a slower migration of cancer cells as judged by wound healing assays. On the basis of the cellular responses to K137-E4, we conclude that ecto-CK2 is implicated in cell motility, whereas its contribution to the pro-survival role of CK2 is negligible. PMID:26349539

  16. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

    SciTech Connect

    Miller, J. Richard; Dunham, Steve; Mochalkin, Igor; Banotai, Craig; Bowman, Matthew; Buist, Susan; Dunkle, Bill; Hanna, Debra; Harwood, H. James; Huband, Michael D.; Karnovsky, Alla; Kuhn, Michael; Limberakis, Chris; Liu, Jia Y.; Mehrens, Shawn; Mueller, W. Thomas; Narasimhan, Lakshmi; Ogden, Adam; Ohren, Jeff; Prasad, J.V.N. Vara; Shelly, John A.; Skerlos, Laura; Sulavik, Mark; Thomas, V. Hayden; VanderRoest, Steve; Wang, LiAnn; Wang, Zhigang; Whitton, Amy; Zhu, Tong; Stover, C. Kendall

    2009-06-25

    As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidines target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.

  17. Using ovality to predict nonmutagenic, orally efficacious pyridazine amides as cell specific spleen tyrosine kinase inhibitors.

    PubMed

    Lucas, Matthew C; Bhagirath, Niala; Chiao, Eric; Goldstein, David M; Hermann, Johannes C; Hsu, Pei-Yuan; Kirchner, Stephan; Kennedy-Smith, Joshua J; Kuglstatter, Andreas; Lukacs, Christine; Menke, John; Niu, Linghao; Padilla, Fernando; Peng, Ying; Polonchuk, Liudmila; Railkar, Aruna; Slade, Michelle; Soth, Michael; Xu, Daigen; Yadava, Preeti; Yee, Calvin; Zhou, Mingyan; Liao, Cheng

    2014-03-27

    Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented. PMID:24520947

  18. Araguspongine C induces autophagic death in breast cancer cells through suppression of c-Met and HER2 receptor tyrosine kinase signaling.

    PubMed

    Akl, Mohamed R; Ayoub, Nehad M; Ebrahim, Hassan Y; Mohyeldin, Mohamed M; Orabi, Khaled Y; Foudah, Ahmed I; El Sayed, Khalid A

    2015-01-01

    Receptor tyrosine kinases are key regulators of cellular growth and proliferation. Dysregulations of receptor tyrosine kinases in cancer cells may promote tumorigenesis by multiple mechanisms including enhanced cell survival and inhibition of cell death. Araguspongines represent a group of macrocyclic oxaquinolizidine alkaloids isolated from the marine sponge Xestospongia species. This study evaluated the anticancer activity of the known oxaquinolizidine alkaloids araguspongines A, C, K and L, and xestospongin B against breast cancer cells. Araguspongine C inhibited the proliferation of multiple breast cancer cell lines in vitro in a dose-dependent manner. Interestingly, araguspongine C-induced autophagic cell death in HER2-overexpressing BT-474 breast cancer cells was characterized by vacuole formation and upregulation of autophagy markers including LC3A/B, Atg3, Atg7, and Atg16L. Araguspongine C-induced autophagy was associated with suppression of c-Met and HER2 receptor tyrosine kinase activation. Further in-silico docking studies and cell-free Z-LYTE assays indicated the potential of direct interaction between araguspongine C and the receptor tyrosine kinases c-Met and HER2 at their kinase domains. Remarkably, araguspongine C treatment resulted in the suppression of PI3K/Akt/mTOR signaling cascade in breast cancer cells undergoing autophagy. Induction of autophagic death in BT-474 cells was also associated with decreased levels of inositol 1,4,5-trisphosphate receptor upon treatment with effective concentration of araguspongine C. In conclusion, results of this study are the first to reveal the potential of araguspongine C as an inhibitor to receptor tyrosine kinases resulting in the induction of autophagic cell death in breast cancer cells. PMID:25580621

  19. Identification and Validation of Inhibitor-Responsive Kinase Substrates using a New Paradigm to Measure Kinase-Specific Protein Phosphorylation Index

    PubMed Central

    Li, Xiang; Rao, Varsha; Jin, Jin; Guan, Bin; Anderes, Kenna L.; Bieberich, Charles J.

    2012-01-01

    Regulation of all cellular processes requires dynamic regulation of protein phosphorylation. We have developed an unbiased system to globally quantify the phosphorylation index for substrates of a specific kinase by independently quantifying phosphorylated and total substrate molecules in a reverse in-gel kinase assay. Non-phosphorylated substrate molecules are first quantified in the presence and absence of a specific stimulus. Total substrate molecules are then measured after complete chemical de-phosphorylation, and a ratio of phosphorylated to total substrate is derived. To demonstrate the utility of this approach, we profiled and quantified changes in phosphorylation index for Protein Kinase CK2 substrates that respond to a small-molecule inhibitor. A broad range of inhibitor-induced changes in phosphorylation was observed in cultured cells. Differences among substrates in the kinetics of phosphorylation change were also revealed. Comparison of CK2 inhibitor-induced changes in phosphorylation in cultured cells and in mouse peripheral blood lymphocytes in vivo revealed distinct kinetic and depth-of-response profiles. This technology provides a new approach to facilitate functional analyses of kinase-specific phosphorylation events. This strategy can be used to dissect the role of phosphorylation in cellular events, to facilitate kinase inhibitor target validation studies, and to inform in vivo analyses of kinase inhibitor drug efficacy. PMID:22663298

  20. Metabolism and bioactivation of famitinib, a novel inhibitor of receptor tyrosine kinase, in cancer patients

    PubMed Central

    Xie, Cen; Zhou, Jialan; Guo, Zitao; Diao, Xingxing; Gao, Zhiwei; Zhong, Dafang; Jiang, Haoyuan; Zhang, Lijia; Chen, Xiaoyan

    2013-01-01

    Background and Purpose Famitinib is a novel multi-targeted receptor tyrosine kinase inhibitor under development for cancer treatment. This study aims to characterize the metabolic and bioactivation pathways of famitinib. Experimental Approach The metabolites in human plasma, urine and feces were identified via ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry and confirmed using synthetic standards. Biotransformation and bioactivation mechanisms were investigated using microsomes, recombinant metabolic enzymes and hepatocytes. Key Results Famitinib was extensively metabolized after repeated oral administrations. Unchanged famitinib was the major circulating material, followed by N-desethylfaminitib (M3), whose steady-state exposure represented 7.2 to 7.5% that of the parent drug. Metabolites in the excreta were mainly from oxidative deamination (M1), N-desethylation (M3), oxidative defluorination (M7), indolylidene hydroxylation (M9-1 and M9-5) and secondary phase-II conjugations. CYP3A4/5 was the major contributor to M3 formation, CYP3A4/5 and aldehyde dehydrogenase to M1 formation and CYP1A1/2 to M7, M9-1 and M9-5 formations. Minor cysteine conjugates were observed in the plasma, urine and feces, implying the formation of reactive intermediate(s). In vitro microsomal studies proved that famitinib was bioactivated through epoxidation at indolylidene by CYP1A1/2 and spontaneously defluorinated rearrangement to afford a quinone-imine species. A correlation between famitinib hepatotoxicity and its bioactivation was observed in the primary human hepatocytes. Conclusion and Implications Famitinib is well absorbed and extensively metabolized in cancer patients. Multiple enzymes, mainly CYP3A4/5 and CYP1A1/2, are involved in famitinib metabolic clearance. The quinone-imine intermediate formed through bioactivation may be associated with famitinib hepatotoxicity. Co-administered CYP1A1/2 inducers or inhibitors may potentiate or

  1. Bruton's tyrosine kinase inhibitors for the treatment of rheumatoid arthritis.

    PubMed

    Whang, Jennifer A; Chang, Betty Y

    2014-08-01

    The function and role of Bruton's tyrosine kinase (BTK) in human B cell development was demonstrated by its association with X-linked agammaglobulinemia (XLA) manifested by a substantial reduction in immunoglobulins and B cells. BTK has a crucial role in pre-B cell receptor (BCR) and BCR signaling during normal B cell development and activation. Aberrant BCR signaling is associated with autoimmune diseases, such as rheumatoid arthritis (RA). In addition, BTK is also expressed in myeloid cell populations, including monocytes, macrophages, neutrophils and mast cells. These innate cells infiltrate the synovial cavity and produce inflammatory cytokines, aggravating arthritic symptoms. In myeloid cell populations, BTK functions downstream of the Fcγ receptors (FcγR) and Fcɛ receptors (FcɛR). In the absence of BTK, FcR-mediated functions, such as cytokine production, are impaired. In addition, Xid mice, which have a mutation in BTK, have decreased susceptibility to developing collagen-induced arthritis (CIA). Given that BTK is involved in multiple signaling pathways downstream of the BCR and FcR, it is an attractive therapeutic target for RA. PMID:24721226

  2. Role of tyrosine-kinase inhibitors in myeloproliferative neoplasms: comparative lessons learned.

    PubMed

    Pinilla-Ibarz, Javier; Sweet, Kendra L; Corrales-Yepez, Gabriela M; Komrokji, Rami S

    2016-01-01

    An important pathogenetic distinction in the classification of myeloproliferative neoplasms (MPNs) is the presence or absence of the BCR-ABL fusion gene, which encodes a unique oncogenic tyrosine kinase. The BCR-ABL fusion, caused by the formation of the Philadelphia chromosome (Ph) through translocation, constitutes the disease-initiating event in chronic myeloid leukemia. The development of successive BCR-ABL-targeted tyrosine-kinase inhibitors has led to greatly improved outcomes in patients with chronic myeloid leukemia, including high rates of complete hematologic, cytogenetic, and molecular responses. Such levels of treatment success have long been elusive for patients with Ph-negative MPNs, because of the difficulties in identifying specific driver proteins suitable as drug targets. However, in recent years an improved understanding of the complex pathobiology of classic Ph-negative MPNs, characterized by variable, overlapping multimutation profiles, has prompted the development of better and more broadly targeted (to pathway rather than protein) treatment options, particularly JAK inhibitors. In classic Ph-negative MPNs, overactivation of JAK-dependent signaling pathways is a central pathogenic mechanism, and mutually exclusive mutations in JAK2, MPL, and CALR linked to aberrant JAK activation are now recognized as key drivers of disease progression in myelofibrosis (MF). In clinical trials, the JAK1/JAK2 inhibitor ruxolitinib - the first therapy approved for MF worldwide - improved disease-related splenomegaly and symptoms independent of JAK2 (V617F) mutational status, and prolonged survival compared with placebo or standard therapy in patients with advanced MF. In separate trials, ruxolitinib also provided comprehensive hematologic control in patients with another Ph-negative MPN - polycythemia vera. However, complete cytogenetic or molecular responses with JAK inhibitors alone are normally not observed, underscoring the need for novel combination

  3. A Coiled-Coil Enabled Split-Luciferase Three-Hybrid System: Applied Toward Profiling Inhibitors of Protein Kinases

    PubMed Central

    Jester, Benjamin W.; Cox, Kurt J.; Gaj, Alicia; Shomin, Carolyn D.; Porter, Jason R.; Ghosh, Indraneel

    2010-01-01

    The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors. PMID:20669947

  4. Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors

    PubMed Central

    Canning, Peter; Ruan, Qui; Schwerd, Tobias; Hrdinka, Matous; Maki, Jenny L.; Saleh, Danish; Suebsuwong, Chalada; Ray, Soumya; Brennan, Paul E.; Cuny, Gregory D.; Uhlig, Holm H.; Gyrd-Hansen, Mads; Degterev, Alexei; Bullock, Alex N.

    2015-01-01

    Summary RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers. PMID:26320862

  5. Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors

    PubMed Central

    Marusiak, Anna A.; Edwards, Zoe C.; Hugo, Willy; Trotter, Eleanor W.; Girotti, Maria R.; Stephenson, Natalie L.; Kong, Xiangju; Gartside, Michael G.; Fawdar, Shameem; Hudson, Andrew; Breitwieser, Wolfgang; Hayward, Nicholas K.; Marais, Richard; Lo, Roger S.; Brognard, John

    2014-01-01

    RAF inhibitor therapy yields significant reductions in tumour burden in the majority of V600E-positive melanoma patients; however, resistance occurs within 2–18 months. Here we demonstrate that the mixed lineage kinases (MLK1–4) are MEK kinases that reactivate the MEK/ERK pathway in the presence of RAF inhibitors. Expression of MLK1–4 mediates resistance to RAF inhibitors and promotes survival in V600E-positive melanoma cell lines. Furthermore, we observe upregulation of the MLKs in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In summary, our data demonstrate a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors. PMID:24849047

  6. Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors.

    PubMed

    Canning, Peter; Ruan, Qui; Schwerd, Tobias; Hrdinka, Matous; Maki, Jenny L; Saleh, Danish; Suebsuwong, Chalada; Ray, Soumya; Brennan, Paul E; Cuny, Gregory D; Uhlig, Holm H; Gyrd-Hansen, Mads; Degterev, Alexei; Bullock, Alex N

    2015-09-17

    RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers. PMID:26320862

  7. Crystal Structure of Checkpoint Kinase 2 in Complex with Nsc 109555, a Potent and Selective Inhibitor

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Zhang, Di; Jobson, Andrew G.; Pommier, Yves; Shoemaker, Robert H.; Waugh, David S.

    2009-03-05

    Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA-damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC{sub 50} = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATP-binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.

  8. Potent, selective and orally bioavailable leucine-rich repeat kinase 2 (LRRK2) inhibitors.

    PubMed

    Greshock, Thomas J; Sanders, John M; Drolet, Robert E; Rajapakse, Hemaka A; Chang, Ronald K; Kim, Boyoung; Rada, Vanessa L; Tiscia, Heather E; Su, Hua; Lai, Ming-Tain; Sur, Sylvie M; Sanchez, Rosa I; Bilodeau, Mark T; Renger, John J; Kern, Jonathan T; McCauley, John A

    2016-06-01

    Familial Parkinson's disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinson's disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules. PMID:27106707

  9. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.

    PubMed Central

    Brunn, G J; Williams, J; Sabers, C; Wiederrecht, G; Lawrence, J C; Abraham, R T

    1996-01-01

    The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002. Images PMID:8895571

  10. Epidermal growth factor receptor tyrosine kinase inhibitors for non-small cell lung cancer

    PubMed Central

    Asami, Kazuhiro; Atagi, Shinji

    2014-01-01

    First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, have proven to be highly effective agents for advanced non-small cell lung cancer (NSCLC) in patients harboring an activating EGFR mutation such as the exon 19 deletion mutation and L858R. Although those reversible small molecular targeted agents provide a significant response and survival benefit, all responders eventually acquire resistance. Second-generation EGFR-targeting agents, such as irreversible EGFR/HER2 tyrosine kinase inhibitors and pan-HER TKIs, may improve survival further and be useful for patients who acquired resistance to first-generation EGFR-TKIs. This review discusses novel therapeutic strategies for EGFR-mutated advanced NSCLC using first- and second-generation EGFR-TKIs. PMID:25302168

  11. A Caged Ret Kinase Inhibitor and its Effect on Motoneuron Development in Zebrafish Embryos

    PubMed Central

    Bliman, David; Nilsson, Jesper R.; Kettunen, Petronella; Andréasson, Joakim; Grøtli, Morten

    2015-01-01

    Proto-oncogene tyrosine-protein kinase receptor RET is implicated in the development and maintenance of neurons of the central and peripheral nervous systems. Attaching activity-compromising photocleavable groups (caging) to inhibitors could allow for external spatiotemporally controlled inhibition using light, potentially providing novel information on how these kinase receptors are involved in cellular processes. Here, caged RET inhibitors were obtained from 3-substituted pyrazolopyrimidine-based compounds by attaching photolabile groups to the exocyclic amino function. The most promising compound displayed excellent inhibitory effect in cell-free, as well as live-cell assays upon decaging. Furthermore, inhibition could be efficiently activated with light in vivo in zebrafish embryos and was shown to effect motoneuron development. PMID:26300345

  12. Treating inflammation with the Janus kinase inhibitor CP-690,550.

    PubMed

    Vijayakrishnan, Lalitha; Venkataramanan, R; Gulati, Palak

    2011-01-01

    Commonly used immunosuppressants possess several significant dose-limiting toxicities, prompting the search for agents whose mechanisms of action are limited to immune cells. Inhibition of Janus Kinase 3 (JAK3), a hematopoetic cell-restricted tyrosine kinase, represents an attractive target for immunosuppression owing to its limited distribution in tissue and specific role in lymphoid homeostasis. CP-690,550, a JAK3 inhibitor undergoing clinical trials for the treatment of transplant rejection and autoimmune disorders, has shown efficacy similar to comparator immunosuppressants. However, its inhibition of the more ubiquitous JAK family members, JAK1 and JAK2, is a probable cause of drug-related adverse events (e.g. overt immunosuppression, anemia). Here, we argue that CP-690,550 represents only a starting point in the search for a safer small molecule immunosuppressant, and that an isozyme-selective JAK3 inhibitor identified by rational drug design might be substantially safer. PMID:21144599

  13. N,N-Dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphingosine 1-phosphate and ceramide.

    PubMed

    Edsall, L C; Van Brocklyn, J R; Cuvillier, O; Kleuser, B; Spiegel, S

    1998-09-15

    Sphingosine 1-phosphate (SPP), a lipid second messenger formed by the action of sphingosine kinase, has been implicated in regulating diverse biological processes, including growth, survival, and differentiation. N,N-Dimethylsphingosine (DMS) inhibits sphingosine kinase and has been used to investigate the biological roles of SPP; however, little is known of the mechanism of inhibition of sphingosine kinase by DMS. In addition, DMS has been shown to inhibit protein kinase C in vitro. Here we report that DMS is a competitive inhibitor of sphingosine kinase from U937 monoblastic leukemia cells, Swiss 3T3 fibroblasts, and PC12 pheochromocytoma cells. DMS decreases basal levels of SPP and prevents increases in SPP in response to physiological stimuli known to activate sphingosine kinase. DMS also effectively increases cellular levels of ceramide in a variety of cell types, and resetting of the ceramide/SPP rheostat may account for the pro-apoptotic effects of DMS. Moreover, DMS, at concentrations which effectively inhibit sphingosine kinase, has no effect on protein kinase C activity or its membrane translocation. Thus, DMS acts as a specific competitive inhibitor of sphingosine kinase in diverse cell types and is a useful tool to elucidate the role of SPP as an intracellular second messenger. PMID:9737868

  14. Effect of various protein kinase inhibitors on the induction of milk protein gene expression by prolactin.

    PubMed

    Bayat-Sarmadi, M; Houdebine, L M

    1993-03-01

    Prolactin has many known functions and one of them is to induce the expression of milk protein gene expression in the mammary gland. Specific membrane receptors have been recently characterized but the transduction mechanism involved in the transfer of the prolactin signal to milk protein genes remains unknown. In the present work, it is shown that several protein kinase inhibitors block prolactin action on milk protein genes. Primary rabbit mammary cells were cultured for several days on floating collagen gel in a serum-free medium. Prolactin and the inhibitors of protein kinase were then added to the culture medium. After 1 day, the concentration of alpha s1-casein in the culture medium was measured using a specific radioimmunoassay. The concentration of several mRNAs in cell extracts was also evaluated using Northern blot analysis. alpha s1-Casein secretion and alpha s1-casein mRNA accumulation were induced by prolactin. This induction was blocked by staurosporine, sphingosine, quercetin, genistein and to some extent by o-hydroxyphenyl acetate, but not by H7, polymyxin B, benzylsuccinate and lavendustin A. The concentration of the mRNA coding for transferrin, which is abundantly secreted in rabbit milk independently of prolactin action, was only moderately altered by the inhibitors. The concentration of two house-keeping mRNAs, beta-actin and glyceraldehyde 3-phosphate dehydrogenase, was lowered only by genistein after 1 day but not after 4 h of culture. These data show for the first time that a Ser/Thre kinase, which is not kinase C, and possibly a tyrosine kinase is involved in the transduction of the prolactin message from the receptor to the milk protein genes. PMID:8472863

  15. Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors.

    PubMed

    Luo, Guanglin; Chen, Ling; Burton, Catherine R; Xiao, Hong; Sivaprakasam, Prasanna; Krause, Carol M; Cao, Yang; Liu, Nengyin; Lippy, Jonathan; Clarke, Wendy J; Snow, Kimberly; Raybon, Joseph; Arora, Vinod; Pokross, Matt; Kish, Kevin; Lewis, Hal A; Langley, David R; Macor, John E; Dubowchik, Gene M

    2016-02-11

    GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics. PMID:26751161

  16. Anilinoquinazoline inhibitors of the RET kinase domain-Elaboration of the 7-position.

    PubMed

    Jordan, Allan M; Begum, Habiba; Fairweather, Emma; Fritzl, Samantha; Goldberg, Kristin; Hopkins, Gemma V; Hamilton, Niall M; Lyons, Amanda J; March, H Nikki; Newton, Rebecca; Small, Helen F; Vishwanath, Swamy; Waddell, Ian D; Waszkowycz, Bohdan; Watson, Amanda J; Ogilvie, Donald J

    2016-06-01

    We have previously reported a series of anilinoquinazoline derivatives as potent and selective biochemical inhibitors of the RET kinase domain. However, these derivatives displayed diminished cellular potency. Herein we describe further optimisation of the series through modification of their physicochemical properties, delivering improvements in cell potency. However, whilst cellular selectivity against key targets could be maintained, combining cell potency and acceptable pharmacokinetics proved challenging. PMID:27086121

  17. Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties.

    PubMed

    Sloman, David L; Noucti, Njamkou; Altman, Michael D; Chen, Dapeng; Mislak, Andrea C; Szewczak, Alexander; Hayashi, Mansuo; Warren, Lee; Dellovade, Tammy; Wu, Zhenhua; Marcus, Jacob; Walker, Deborah; Su, Hua-Poo; Edavettal, Suzanne C; Munshi, Sanjeev; Hutton, Michael; Nuthall, Hugh; Stanton, Matthew G

    2016-09-01

    Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer's disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility. PMID:27491711

  18. Tyrosine kinase inhibitor-associated syndrome of inappropriate secretion of anti-diuretic hormone.

    PubMed

    Hill, Jordan; Shields, Jenna; Passero, Vida

    2016-10-01

    Hyponatremia is a common complication among cancer patients. Certain antineoplastic agents have been associated with syndrome of inappropriate secretion of anti-diuretic hormone-induced hyponatremia. The most common agents associated with secretion of anti-diuretic hormone are vinca alkaloids, platinum compounds, and alkylating agents. We report a case of secretion of anti-diuretic hormone associated with tyrosine kinase inhibitors. PMID:26089312

  19. In Vitro High Throughput Screening, What Next? Lessons from the Screening for Aurora Kinase Inhibitors

    PubMed Central

    Hoang, Thi-My-Nhung; Vu, Hong-Lien; Le, Ly-Thuy-Tram; Nguyen, Chi-Hung; Molla, Annie

    2014-01-01

    Based on in vitro assays, we performed a High Throughput Screening (HTS) to identify kinase inhibitors among 10,000 small chemical compounds. In this didactic paper, we describe step-by-step the approach to validate the hits as well as the major pitfalls encountered in the development of active molecules. We propose a decision tree that could be adapted to most in vitro HTS. PMID:24833340

  20. In Vitro Interactions between Target of Rapamycin Kinase Inhibitor and Antifungal Agents against Aspergillus Species.

    PubMed

    Gao, Lujuan; Ding, Xiaozhen; Liu, Zhun; Wu, Qingzhi; Zeng, Tongxiang; Sun, Yi

    2016-06-01

    In vitro interactions of INK128, a target of rapamycin (TOR) kinase inhibitor, and antifungals, including itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin, against Aspergillus spp. were assessed with the broth microdilution checkerboard technique. Our results suggested synergistic effects between INK128 and all azoles tested, against multiple Aspergillus fumigatus and Aspergillus flavus isolates. However, no synergistic effects were observed when INK128 was combined with amphotericin B or caspofungin. No antagonism was observed for any combination. PMID:26976874

  1. Fragment-based discovery of type I inhibitors of maternal embryonic leucine zipper kinase.

    PubMed

    Johnson, Christopher N; Berdini, Valerio; Beke, Lijs; Bonnet, Pascal; Brehmer, Dirk; Coyle, Joseph E; Day, Phillip J; Frederickson, Martyn; Freyne, Eddy J E; Gilissen, Ron A H J; Hamlett, Christopher C F; Howard, Steven; Meerpoel, Lieven; McMenamin, Rachel; Patel, Sahil; Rees, David C; Sharff, Andrew; Sommen, François; Wu, Tongfei; Linders, Joannes T M

    2015-01-01

    Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 μM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology. PMID:25589925

  2. Fragment-Based Discovery of Type I Inhibitors of Maternal Embryonic Leucine Zipper Kinase

    PubMed Central

    2014-01-01

    Fragment-based drug design was successfully applied to maternal embryonic leucine zipper kinase (MELK). A low affinity (160 μM) fragment hit was identified, which bound to the hinge region with an atypical binding mode, and this was optimized using structure-based design into a low-nanomolar and cell-penetrant inhibitor, with a good selectivity profile, suitable for use as a chemical probe for elucidation of MELK biology. PMID:25589925

  3. The discovery of novel vascular endothelial growth factor receptor tyrosine kinases inhibitors: pharmacophore modeling, virtual screening and docking studies.

    PubMed

    Yu, Hui; Wang, Zhanli; Zhang, Liangren; Zhang, Jufeng; Huang, Qian

    2007-03-01

    We have applied pharmacophore generation, database searching and docking methodologies to discover new structures for the design of vascular endothelial growth factor receptors, the tyrosine kinase insert domain-containing receptor kinase inhibitors. The chemical function based pharmacophore models were built for kinase insert domain-containing receptor kinase inhibitors from a set of 10 known inhibitors using the algorithm HipHop, which is implemented in the CATALYST software. The highest scoring HipHop model consists of four features: one hydrophobic, one hydrogen bond acceptor, one hydrogen bond donor and one ring aromatic function. Using the algorithm CatShape within CATALYST, the bound conformation of 4-amino-furo [2, 3-d] pyrimidine binding to kinase insert domain-containing receptor kinase was used to generate a shape query. A merged shape and hypothesis query that is in an appropriate alignment was then built. The combined shape and hypothesis model was used as a query to search Maybridge database for other potential lead compounds. A total of 39 compounds were retrieved as hits. The hits obtained were docked into kinase insert domain-containing receptor kinase active site. One novel potential lead was proposed based on CATALYST fit value, LigandFit docking scores, and examination of how the hit retain key interactions known to be required for kinase binding. This compound inhibited vascular endothelial growth factor stimulated kinase insert domain-containing receptor phosphorylation in human umbilical vein endothelial cells. PMID:17441906

  4. Design, synthesis and biological evaluation of pyrazol-furan carboxamide analogues as novel Akt kinase inhibitors.

    PubMed

    Zhan, Wenhu; Xu, Lei; Dong, Xiaowu; Dong, Jun; Yi, Xiao; Ma, Xiaodong; Qiu, Ni; Li, Jia; Yang, Bo; Zhou, Yubo; Hu, Yongzhou

    2016-07-19

    A series of novel pyrazol-furan carboxamide analogues were designed, synthesized and biologically evaluated for their Akt1 inhibitory activities, as well as anti-proliferative efficacies against HCT116 and OVCAR-8 cell lines. Most compounds exhibited moderate to excellent Akt1 inhibitory activities, together with favorable cytotoxicities. Further kinase selectivity assay of the most promising compound 25e illustrated that it was also potent against the structurally related AGC kinases, including Akt2, Akt3, ROCK1 and PKA, but was specific over kinases from other subfamilies. In addition, the Western blot analysis indicated that 25e could significantly suppress the phosphorylation level of Akt substrate GSK3β in PC-3 cell. Moreover, 25e demonstrated a concentration-dependent inhibition of phosphorylation of PRAS40 in LNCaP cell, with IC50 value of 30.4 nM. PMID:27089211

  5. Combination of the ABL kinase inhibitor imatinib with the Janus kinase 2 inhibitor TG101348 for targeting residual BCR-ABL-positive cells

    PubMed Central

    2014-01-01

    Background The ABL kinase inhibitor imatinib is highly effective in treating most, but not all, patients with chronic myeloid leukemia (CML). This is because residual CML cells are generally present in the bone marrow microenvironment and are refractory to imatinib. Hematopoietic cytokine receptor signaling is mediated by Janus kinases (JAKs) and their downstream transcription factor, signal transducer and activator of transcription (STAT). TG101348 (SAR302503) is an oral inhibitor of JAK2. Methods We investigated the efficacy of imatinib and TG101348 using the break point cluster region-c-Abelson (BCR-ABL)-positive cell line and primary CML samples wherein leukemia cells were protected by a feeder cell line (HS-5). Results Imatinib treatment resulted in partial inhibition of cell growth in HS-5-conditioned medium. Furthermore, combined treatment with imatinib and TG101348 abrogated the protective effects of HS-5-conditioned medium on K562 cells. Phosphorylation of Crk-L, a BCR-ABL substrate, decreased considerably, while apoptosis increased. In addition, the combined treatment of CD34-positive primary samples resulted in considerably increased cytotoxicity, decreased Crk-L phosphorylation, and increased apoptosis. We also investigated TG101348 activity against feeder cells and observed that STAT5 phosphorylation, granulocyte macrophage colony-stimulating factor, and interleukin 6 levels decreased, indicating reduced cytokine production in HS-5 cells treated with TG101348. Conclusions These results showed that JAK inhibitors may enhance the cytotoxic effect of imatinib against residual CML cells and that a combined approach may be a powerful strategy against the stroma-associated drug resistance of Philadelphia chromosome-positive cells. PMID:24775308

  6. Identification of novel inhibitors of BCR-ABL tyrosine kinase via virtual screening.

    PubMed

    Peng, Hui; Huang, Niu; Qi, Jing; Xie, Ping; Xu, Chen; Wang, Jianxiang; Yang, Chunzheng

    2003-11-01

    Inhibition of BCR-ABL tyrosine kinase activity has shown to be essential for the treatment of chronic myelogenous leukemia (CML). However, drug resistance has quickly arisen in recent clinical trials for STI571 (Gleevec), which is the first approved drug of CML by inhibiting ABL tyrosine kinase. It is desirable to develop new types of ABL tyrosine kinase inhibitors that may overcome this drug resistance problem. Here we present the discovery of novel inhibitors targeted at the catalytic domain of ABL tyrosine kinase by using three-dimensional database searching techniques. From a database containing 200,000 commercially available compounds, the top 1000 compounds with the best DOCK energy score were selected and subjected to structural diversity and drug likeness analysis, 15 compounds were submitted for biological assay. Eight out of the 15 showed inhibitory activity against K562 cells with IC(50) value ranging from 10 to 200 microM. Two promising compounds showed inhibition in further ABL tyrosine phosphorylation assay. It is anticipated that those two compounds can serve as lead compounds for further drug design and optimization. PMID:14552760

  7. Optimization of a Novel Series of Ataxia-Telangiectasia Mutated Kinase Inhibitors as Potential Radiosensitizing Agents.

    PubMed

    Min, Jaeki; Guo, Kexiao; Suryadevara, Praveen K; Zhu, Fangyi; Holbrook, Gloria; Chen, Yizhe; Feau, Clementine; Young, Brandon M; Lemoff, Andrew; Connelly, Michele C; Kastan, Michael B; Guy, R Kiplin

    2016-01-28

    We previously reported a novel inhibitor of the ataxia-telangiectasia mutated (ATM) kinase, which is a target for novel radiosensitizing drugs. While our initial lead, compound 4, was relatively potent and nontoxic, it exhibited poor stability to oxidative metabolism and relatively poor selectivity against other kinases. The current study focused on balancing potency and selectivity with metabolic stability through structural modification to the metabolized site on the quinazoline core. We performed extensive structure-activity and structure-property relationship studies on this quinazoline ATM kinase inhibitor in order to identify structural variants with enhanced selectivity and metabolic stability. We show that, while the C-7-methoxy group is essential for potency, replacing the C-6-methoxy group considerably improves metabolic stability without affecting potency. Promising analogues 20, 27g, and 27n were selected based on in vitro pharmacology and evaluated in murine pharmacokinetic and tolerability studies. Compound 27g possessed significantly improve pharmacokinetics relative to that of 4. Compound 27g was also significantly more selective against other kinases than 4. Therefore, 27g is a good candidate for further development as a potential radiosensitizer. PMID:26632965

  8. Discovery of Novel Fibroblast Growth Factor Receptor 1 Kinase Inhibitors by Structure-Based Virtual Screening

    SciTech Connect

    Ravindranathan, K.; Mandiyan, V; Ekkati, A; Bae, J; Schlessinger, J; Jorgensen, W

    2010-01-01

    Fibroblast growth factors (FGFs) play important roles in embryonic development, angiogenesis, wound healing, and cell proliferation and differentiation. In search of inhibitors of FGFR1 kinase, 2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure, which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed. Following hit validation, two compounds 10 and 16, a benzylidene derivative of pseudothiohydantoin and a thienopyrimidinone derivative, respectively, were discovered that inhibit FGFR1 kinase with IC{sub 50} values of 23 and 50 {micro}M. Initial optimization of 16 led to the more unsaturated 40, which has significantly enhanced potency, 1.9 {micro}M. The core structures represent new structural motifs for FGFR1 kinase inhibitors. The study also illustrates complexities associated with the choice of protein structures for docking, possible use of multiple kinase structures to seek selectivity, and hit identification.

  9. Beyond trastuzumab: small molecule tyrosine kinase inhibitors in HER-2-positive breast cancer.

    PubMed

    Roy, Vivek; Perez, Edith A

    2009-11-01

    HER-2 is a transmembrane, tyrosine kinase (TK) receptor whose overexpression is associated with adverse prognosis in breast cancer. The biological effects of HER-2 are mediated by kinase activity causing phosphorylation of tyrosine residues in the cytoplasmic domain of the receptor molecule, leading to activation of downstream growth-promoting pathways. Antibody-mediated inhibition by trastuzumab as well as TK inhibition are clinically effective anti-HER-2 strategies. Kinase inhibitors offer some potential therapeutic advantages over antibody-based therapies. Being small molecules, TK inhibitors (TKIs) have oral bioavailability and ability to cross the blood-brain barrier. Because of their different mode of action, TKIs may be able to overcome some of the mechanisms of trastuzumab resistance. Preclinical, and limited clinical data also suggest that TKIs and trastuzumab have synergistic activity. Lapatinib is the only TKI available for clinical use at present, but several molecules with anti-HER-2 activity have been identified and are undergoing evaluation. These differ in the spectrum of kinases that they inhibit, potency of HER-2 inhibition, pharmacokinetic properties, and toxicity profiles, and are at various stages of clinical development. In this article we summarize selected HER-2 TKIs approved for clinical use or in development for which clinical data are available. PMID:19887469

  10. Phosphoinositide 3-kinase gamma (PI3Kgamma) inhibitors for the treatment of inflammation and autoimmune disease.

    PubMed

    Venable, Jennifer D; Ameriks, Michael K; Blevitt, Jonathan M; Thurmond, Robin L; Fung-Leung, Wai-Ping

    2010-01-01

    Phosphoinositide 3-kinase gamma (PI3Kgamma) is a lipid kinase in leukocytes that generates phosphatidylinositol 3,4,5-trisphosphate to recruit and activate downstream signaling molecules. Distinct from other members in the PI3K family, PI3Kgamma is activated by G-protein coupled-receptors responding to chemotactic ligands. PI3Kgamma plays an important role in migration of both myeloid and lymphoid cells. It is also required for other leukocyte functions such as neutrophil oxidative burst, T cell proliferation and mast degranulation. Mice with PI3Kgamma inactivated by genetic or pharmacological approaches are protected from disease development in a number of inflammation and autoimmune disease models. The function of PI3Kgamma depends on its kinase activity and therefore it has been suggested by many reports that small molecules inhibiting its kinase activity could be promising for the treatment of inflammation and autoimmune diseases. Over the last five years, a number of pharmaceutical companies have reported a wide variety of PI3Kgamma inhibitors, of which several x-ray crystal structures with PI3Kgamma have been elucidated. The structural characteristics and selectivity profiles of these inhibitors, in particular thiazolidinones and 2-aminoheterocycles, and those disclosed in related patent applications are summarized in this review. PMID:20017720

  11. Role of Tyrosine Kinase Inhibitors in Indolent and Other Mature B-Cell Neoplasms

    PubMed Central

    Kutsch, Nadine; Marks, Reinhard; Ratei, Richard; Held, Thomas K; Schmidt-Hieber, Martin

    2015-01-01

    Targeting tyrosine kinases represents a highly specific treatment approach for different malignancies. This also includes non-Hodgkin lymphoma since it is well known that these enzymes are frequently involved in the lymphomagenesis. Hereby, tyrosine kinases might either be dysregulated intrinsically or be activated within signal transduction pathways leading to tumor survival and growth. Among others, Bruton’s tyrosine kinase (Btk) is of particular interest as a potential therapeutic target. Btk is stimulated by B-cell receptor signaling and activates different transcription factors such as nuclear factor κB. The Btk inhibitor ibrutinib has been approved for the treatment of chronic lymphocytic leukemia and mantle-cell lymphoma recently. Numerous clinical trials evaluating this agent in different combinations (eg, with rituximab or classical chemotherapeutic agents) as a treatment option for aggressive and indolent lymphoma are under way. Here, we summarize the role of tyrosine kinase inhibitors in the treatment of indolent and other non-Hodgkin lymphomas (eg, mantle-cell lymphoma). PMID:26327780

  12. Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis

    PubMed Central

    Šalovská, Barbora; Fabrik, Ivo; Ďurišová, Kamila; Link, Marek; Vávrová, Jiřina; Řezáčová, Martina; Tichý, Aleš

    2014-01-01

    DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells. PMID:25003641

  13. Suppression of dioxins by S-N inhibitors in pilot-scale experiments.

    PubMed

    Zhan, Ming-Xiu; Fu, Jian-Ying; Chen, Tong; Lin, Xiao-Qing; Li, Xiao-Dong; Yan, Jian-Hua; Buekens, Alfons

    2016-08-01

    S-N inhibitors like thiourea and sewage sludge decomposition gases (SDG) are relatively novel dioxins suppressants and their efficiencies are proven in numerous lab-scale experiments. In this study, the suppression effects of both thiourea and SDG on the formation of dioxins are systematically tested in a pilot-scale system, situated at the bypass of a hazardous waste incinerator (HWI). Moreover, a flue gas recirculation system is used to get high dioxin suppression efficiencies. Operating experience shows that this system is capable of stable operation and to keep gaseous suppressant compounds at a high and desirable molar ratio (S + N)/Cl level in the flue gas. The suppression efficiencies of dioxins are investigated in flue gas both without and with addition of S-N inhibitors. A dioxin reduction of more than 80 % is already achieved when the (S + N)/Cl molar ratio is increased to ca. 2.20. When this (S + N)/Cl molar ratio has augmented to 4.18 by applying suppressant recirculation, the residual PCDD/Fs concentration in the flue gas shrank from 1.22 to 0.08 ng I-TEQ/Nm(3). Furthermore, the congener distribution of dioxins is analysed to find some possible explanation or suppression mechanism. In addition, a correlation analysis between (S + N)/Cl molar ratios and PCDD/Fs is also conducted to investigate the chief functional compounds for dioxin suppression. PMID:27164888

  14. Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo.

    PubMed

    Ashton, Susan; Song, Young Ho; Nolan, Jim; Cadogan, Elaine; Murray, Jim; Odedra, Rajesh; Foster, John; Hall, Peter A; Low, Susan; Taylor, Paula; Ellston, Rebecca; Polanska, Urszula M; Wilson, Joanne; Howes, Colin; Smith, Aaron; Goodwin, Richard J A; Swales, John G; Strittmatter, Nicole; Takáts, Zoltán; Nilsson, Anna; Andren, Per; Trueman, Dawn; Walker, Mike; Reimer, Corinne L; Troiano, Greg; Parsons, Donald; De Witt, David; Ashford, Marianne; Hrkach, Jeff; Zale, Stephen; Jewsbury, Philip J; Barry, Simon T

    2016-02-10

    Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans. PMID:26865565

  15. Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors.

    PubMed

    Ji, Hongbin; Zhao, Xiaojun; Yuza, Yuki; Shimamura, Takeshi; Li, Danan; Protopopov, Alexei; Jung, Boonim L; McNamara, Kate; Xia, Huili; Glatt, Karen A; Thomas, Roman K; Sasaki, Hidefumi; Horner, James W; Eck, Michael; Mitchell, Albert; Sun, Yangping; Al-Hashem, Ruqayyah; Bronson, Roderick T; Rabindran, Sridhar K; Discafani, Carolyn M; Maher, Elizabeth; Shapiro, Geoffrey I; Meyerson, Matthew; Wong, Kwok-Kin

    2006-05-16

    The tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) have shown anti-tumor activity in the treatment of non-small cell lung cancer (NSCLC). Dramatic and durable responses have occurred in NSCLC tumors with mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). In contrast, these inhibitors have shown limited efficacy in glioblastoma, where a distinct EGFR mutation, the variant III (vIII) in-frame deletion of exons 2-7, is commonly found. In this study, we determined that EGFRvIII mutation was present in 5% (3/56) of analyzed human lung squamous cell carcinoma (SCC) but was not present in human lung adenocarcinoma (0/123). We analyzed the role of the EGFRvIII mutation in lung tumorigenesis and its response to tyrosine kinase inhibition. Tissue-specific expression of EGFRvIII in the murine lung led to the development of NSCLC. Most importantly, these lung tumors depend on EGFRvIII expression for maintenance. Treatment with an irreversible EGFR inhibitor, HKI-272, dramatically reduced the size of these EGFRvIII-driven murine tumors in 1 week. Similarly, Ba/F3 cells transformed with the EGFRvIII mutant were relatively resistant to gefitinib and erlotinib in vitro but proved sensitive to HKI-272. These findings suggest a therapeutic strategy for cancers harboring the EGFRvIII mutation. PMID:16672372

  16. Novel Kinase Inhibitors Targeting the PH Domain of AKT for Preventing and Treating Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Medical Oncology Branch is seeking statements of capability or interest from parties interested in licensing and co-development collaborative research to further develop, evaluate, or commercialize novel kinase inhibitors targeting the PH domain of AKT.

  17. Biological effect of tyrosine kinase inhibitors on three canine mast cell tumor cell lines with various KIT statuses.

    PubMed

    Takeuchi, Y; Fujino, Y; Fukushima, K; Watanabe, M; Nakagawa, T; Ohno, K; Sasaki, N; Sugano, S; Tsujimoto, H

    2012-02-01

    Tyrosine kinase inhibitors (TKIs) can be important in the treatment of canine mast cell tumor (cMCT). Meanwhile, some TKIs have been identified as substrates for ABCB1. The inhibitory effect of four TKIs (axitinib, imatinib, masitinib, and vatalanib) for proliferation and phosphorylation of c-Kit receptor as well as the expression and function of ABCB1 were investigated in three cMCT cell lines (HRMC, VIMC1, and CMMC1). The IC(50) values of the TKIs in HRMC, the only cell line with wild-type KIT, were clearly higher than those in CMMC1 and VIMC1. In HRMC and CMMC1, both the growth and phosphorylation of c-Kit receptor were suppressed proportionally by the TKIs. VIMC1 required higher concentrations for the inhibition of c-Kit receptor phosphorylation than those in cell growth. The treatment with cyclosporine increased the effects of the TKIs on VIMC1 since ABCB1 was expressed in VIMC1. The results indicated that cMCT cell lines harboring wild-type KIT had lower sensitivity to TKIs. The growth of VIMC1 was seemingly reduced by TKIs through the inhibition of other tyrosine kinases than c-Kit receptor. There was little influence of ABCB1 on TKI effects to the proliferation of VIMC1. These results will be helpful to understand the different sensitivity to TKIs in cMCT patients. PMID:21480930

  18. Kinase inhibitor screening identifies CDK4 as a potential therapeutic target for melanoma

    PubMed Central

    MAHGOUB, T.; EUSTACE, A.J.; COLLINS, D.M.; WALSH, N.; O'DONOVAN, N.; CROWN, J.

    2015-01-01

    Despite recent advances in targeted therapies and immunotherapies metastatic melanoma remains only rarely curable. The objective of the present study was to identify novel therapeutic targets for metastatic melanoma. A library of 160 well-characterised and potent protein kinase inhibitors was screened in the BRAF mutant cell line Sk-Mel-28, and the NRAS mutant Sk-Mel-2, using proliferation assays. Of the 160 inhibitors tested, 20 achieved >50% growth inhibition in both cell lines. Six of the 20 were cyclin dependent kinase (CDK) inhibitors, including two CDK4 inhibitors. Fascaplysin, a synthetic CDK4 inhibitor, was further tested in 8 melanoma cell lines. The concentration of fascaplysin required to inhibit growth by 50% (IC50 value) ranged from 0.03 to 0.22 μM. Fascaplysin also inhibited clonogenic growth and induced apoptosis. Sensitivity to PD0332991, a therapeutic CDK4/6 inhibitor was also evaluated in the melanoma cell lines. PD0332991 IC50 values ranged from 0.13 to 2.29 μM. Similar to fascaplysin, PD0332991 inhibited clonogenic growth of melanoma cells and induced apoptosis. Higher levels of CDK4 protein correlated with lower sensitivity to PD0332991 in the cell lines. Combined treatment with PD0332991 and the BRAF inhibitor PLX4032, showed additive anti-proliferative effects in the BRAF mutant cell line Malme-3M. In summary, targeting CDK4 inhibits growth and induces apoptosis in melanoma cells in vitro, suggesting that CDK4 may be a rational therapeutic target for metastatic melanoma. PMID:26201960

  19. Effect of Narrow Spectrum Versus Selective Kinase Inhibitors on the Intestinal Proinflammatory Immune Response in Ulcerative Colitis

    PubMed Central

    Biancheri, Paolo; Foster, Martyn R.; Fyfe, Matthew C. T.; MacDonald, Thomas T.; Sirohi, Sameer; Solanke, Yemisi; Wood, Eleanor; Rowley, Adele; Webber, Steve

    2016-01-01

    Background: Kinases are key mediators of inflammation, highlighting the potential of kinase inhibitors as treatments for inflammatory disorders. Selective kinase inhibitors, however, have proved disappointing, particularly in the treatment of rheumatoid arthritis and inflammatory bowel disease. Consequently, to improve efficacy, attention has turned to multikinase inhibition. Methods: The activity of a narrow spectrum kinase inhibitor, TOP1210, has been compared with selective kinase inhibitors (BIRB-796, dasatinib and BAY-61-3606) in a range of kinase assays, inflammatory cell assays, and in inflamed biopsies from patients with ulcerative colitis (UC). Effects on recombinant P38α, Src, and Syk kinase activities were assessed using Z-lyte assays (Invitrogen, Paisley, United Kingdom). Anti-inflammatory effects were assessed by measurement of proinflammatory cytokine release from peripheral blood mononuclear cells, primary macrophages, HT29 cells, inflamed colonic UC biopsies, and myofibroblasts isolated from inflamed colonic UC mucosa. Results: TOP1210 potently inhibits P38α, Src, and Syk kinase activities. Similarly, TOP1210 demonstrates potent inhibitory activity against proinflammatory cytokine release in each of the cellular assays and the inflamed colonic UC biopsies and myofibroblasts isolated from inflamed colonic UC mucosa. Generally, the selective kinase inhibitors showed limited and weaker activity in the cellular assays compared with the broad inhibitory profile of TOP1210. However, combination of the selective inhibitors led to improved efficacy and potency in both cellular and UC biopsy assays. Conclusions: Targeted, multikinase inhibition with TOP1210 leads to a broad efficacy profile in both the innate and adaptive immune responses, with significant advantages over existing selective kinase approaches, and potentially offers a much improved therapeutic benefit in inflammatory bowel disease. PMID:27104822

  20. Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

    PubMed

    Cherian, Joseph; Nacro, Kassoum; Poh, Zhi Ying; Guo, Samantha; Jeyaraj, Duraiswamy A; Wong, Yun Xuan; Ho, Melvyn; Yang, Hai Yan; Joy, Joma Kanikadu; Kwek, Zekui Perlyn; Liu, Boping; Wee, John Liang Kuan; Ong, Esther H Q; Choong, Meng Ling; Poulsen, Anders; Lee, May Ann; Pendharkar, Vishal; Ding, Li Jun; Manoharan, Vithya; Chew, Yun Shan; Sangthongpitag, Kanda; Lim, Sharon; Ong, S Tiong; Hill, Jeffrey; Keller, Thomas H

    2016-04-14

    Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented. PMID:27011159

  1. Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production.

    PubMed

    Hashimoto, Shin; Yamamoto, Soh; Ogasawara, Noriko; Sato, Toyotaka; Yamamoto, Keisuke; Katoh, Hiroshi; Kubota, Toru; Shiraishi, Tsukasa; Kojima, Takashi; Himi, Tetsuo; Tsutsumi, Hiroyuki; Yokota, Shin-Ichi

    2016-01-01

    Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection. PMID:27560627

  2. Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production

    PubMed Central

    Hashimoto, Shin; Yamamoto, Soh; Ogasawara, Noriko; Sato, Toyotaka; Yamamoto, Keisuke; Katoh, Hiroshi; Kubota, Toru; Shiraishi, Tsukasa; Kojima, Takashi; Himi, Tetsuo; Tsutsumi, Hiroyuki; Yokota, Shin-ichi

    2016-01-01

    Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection. PMID:27560627

  3. Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma.

    PubMed

    Patwardhan, Parag P; Ivy, Kathryn S; Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K

    2016-01-26

    Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10-18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma. PMID:26675259

  4. Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma

    PubMed Central

    Musi, Elgilda; de Stanchina, Elisa; Schwartz, Gary K.

    2016-01-01

    Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10–18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma. PMID:26675259

  5. Echinacoside Inhibits Glutamate Release by Suppressing Voltage-Dependent Ca2+ Entry and Protein Kinase C in Rat Cerebrocortical Nerve Terminals

    PubMed Central

    Lu, Cheng Wei; Lin, Tzu Yu; Huang, Shu Kuei; Wang, Su Jane

    2016-01-01

    The glutamatergic system may be involved in the effects of neuroprotectant therapies. Echinacoside, a phenylethanoid glycoside extracted from the medicinal Chinese herb Herba Cistanche, has neuroprotective effects. This study investigated the effects of echinacoside on 4-aminopyridine-evoked glutamate release in rat cerebrocortical nerve terminals (synaptosomes). Echinacoside inhibited Ca2+-dependent, but not Ca2+-independent, 4-aminopyridine-evoked glutamate release in a concentration-dependent manner. Echinacoside also reduced the 4-aminopyridine-evoked increase in cytoplasmic free Ca2+ concentration but did not alter the synaptosomal membrane potential. The inhibitory effect of echinacoside on 4-aminopyridine-evoked glutamate release was prevented by ω-conotoxin MVIIC, a wide-spectrum blocker of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but was insensitive to the intracellular Ca2+ release-inhibitors dantrolene and 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157). Furthermore, echinacoside decreased the 4-aminopyridine-induced phosphorylation of protein kinase C, and protein kinase C inhibitors abolished the effect of echinacoside on glutamate release. According to these results, we suggest that the inhibitory effect of echinacoside on evoked glutamate release is associated with reduced voltage-dependent Ca2+ entry and subsequent suppression of protein kinase C activity. PMID:27347934

  6. Identification of ellagic acid as potent inhibitor of protein kinase CK2: a successful example of a virtual screening application.

    PubMed

    Cozza, Giorgio; Bonvini, Paolo; Zorzi, Elisa; Poletto, Giorgia; Pagano, Mario A; Sarno, Stefania; Donella-Deana, Arianna; Zagotto, Giuseppe; Rosolen, Angelo; Pinna, Lorenzo A; Meggio, Flavio; Moro, Stefano

    2006-04-20

    Casein kinase 2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase whose abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and other diseases. Using a virtual screening approach, we have identified the ellagic acid, a naturally occurring tannic acid derivative, as a novel potent CK2 inhibitor. At present, ellagic acid represents the most potent known CK2 inhibitor (K(i) = 20 nM). PMID:16610779

  7. Modulation of human basophil histamine release by protein kinase C inhibitors differs with secretagogue and with inhibitor.

    PubMed

    Bergstrand, H; Lundquist, B; Karabelas, K; Michelsen, P

    1992-03-01

    To assess possible involvement of protein kinase C (PKC) in human basophil degranulation, the present work compared effects of various purported PKC inhibitors on leukocyte histamine release triggered by different stimuli. The effects recorded varied with the inhibitor and the secretagogue used; moreover, with a given secretagogue, different inhibitors often displayed different activities. Thus, histamine release triggered by the PKC activator 4 beta-phorbol 12-myristate 13-acetate was blocked by K252a, staurosporine and the purported specific PKC inhibitor Ro 31-7549, and reduced by calphostin C, H-7, TMB-8 and W-7 but not affected by polymyxin B; it was augmented by 2.1 microM palmitoyl carnitine. The leukocyte response induced by another putative activator of PKC, 1,2-isopropylidene-3-decanoyl-sn-glycerol, was also enhanced by 2.1 microM palmitoyl carnitine, slightly increased by staurosporine, TMB-8 and W-7 but not affected by calphostin C, H-7, K252a or Ro 31-7549, whereas the hyperosmolar mannitol-induced response was reduced by H-7, calphostin C, TMB-8 and W-7 and slightly augmented by staurosporine. Anti-IgE-induced histamine release was blocked by staurosporine and K252a and reduced by calphostin C, sphingosine, TMB-8 and W-7 but not affected by H-7, polymyxin B or retinal. It was enhanced by Ro 31-7549. In contrast, leukocyte histamine release induced by calcium ionophore A23187 or by ionomycin was blocked by retinal, TMB-8 and W-7 and reduced by calphostin C and palmitoyl carnitine but enhanced by H-7, staurosporine and polymyxin B; K252a and Ro 31-7549 did not affect such responses. Formyl-methionyl-leucyl-phenylalanine-triggered histamine release was barely affected by any agent used. Thus, the specific PKC inhibitor Ro 31-7549 selectively blocked 4 beta-phorbol 12-myristate 13-acetate-triggered leukocyte histamine release. These results imply that examined secretagogues trigger human leukocyte histamine release through partly separate pathways

  8. Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

    SciTech Connect

    Zorn, Julie A.; Wang, Qi; Fujimura, Eric; Barros, Tiago; Kuriyan, John; Boggon, Titus J.

    2015-04-02

    More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain of FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.

  9. Crystal structure of the FLT3 kinase domain bound to the inhibitor quizartinib (AC220)

    DOE PAGESBeta

    Zorn, Julie A.; Wang, Qi; Fujimura, Eric; Barros, Tiago; Kuriyan, John; Boggon, Titus J.

    2015-04-02

    More than 30% of acute myeloid leukemia (AML) patients possess activating mutations in the receptor tyrosine kinase FMS-like tyrosine kinase 3 or FLT3. A small-molecule inhibitor of FLT3 (known as quizartinib or AC220) that is currently in clinical trials appears promising for the treatment of AML. Here, we report the co-crystal structure of the kinase domain of FLT3 in complex with quizartinib. FLT3 with quizartinib bound adopts an “Abl-like” inactive conformation with the activation loop stabilized in the “DFG-out” orientation and folded back onto the kinase domain. This conformation is similar to that observed for the uncomplexed intracellular domain ofmore » FLT3 as well as for related receptor tyrosine kinases, except for a localized induced fit in the activation loop. The co-crystal structure reveals the interactions between quizartinib and the active site of FLT3 that are key for achieving its high potency against both wild-type FLT3 as well as a FLT3 variant observed in many AML patients. This co-complex further provides a structural rationale for quizartinib-resistance mutations.« less

  10. Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II[S

    PubMed Central

    Cingolani, Francesca; Casasampere, Mireia; Sanllehí, Pol; Casas, Josefina; Bujons, Jordi; Fabrias, Gemma

    2014-01-01

    Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites. PMID:24875537

  11. A Novel Glycogen Synthase Kinase-3 Inhibitor Optimized for Acute Myeloid Leukemia Differentiation Activity.

    PubMed

    Hu, Sophia; Ueda, Masumi; Stetson, Lindsay; Ignatz-Hoover, James; Moreton, Stephen; Chakrabarti, Amit; Xia, Zhiqiang; Karan, Goutam; de Lima, Marcos; Agrawal, Mukesh K; Wald, David N

    2016-07-01

    Standard therapies used for the treatment of acute myeloid leukemia (AML) are cytotoxic agents that target rapidly proliferating cells. Unfortunately, this therapeutic approach has limited efficacy and significant toxicity and the majority of AML patients still die of their disease. In contrast to the poor prognosis of most AML patients, most individuals with a rare subtype of AML, acute promyelocytic leukemia, can be cured by differentiation therapy using regimens containing all-trans retinoic acid. GSK3 has been previously identified as a therapeutic target in AML where its inhibition can lead to the differentiation and growth arrest of leukemic cells. Unfortunately, existing GSK3 inhibitors lead to suboptimal differentiation activity making them less useful as clinical AML differentiation agents. Here, we describe the discovery of a novel GSK3 inhibitor, GS87. GS87 was discovered in efforts to optimize GSK3 inhibition for AML differentiation activity. Despite GS87's dramatic ability to induce AML differentiation, kinase profiling reveals its high specificity in targeting GSK3 as compared with other kinases. GS87 demonstrates high efficacy in a mouse AML model system and unlike current AML therapeutics, exhibits little effect on normal bone marrow cells. GS87 induces potent differentiation by more effectively activating GSK3-dependent signaling components including MAPK signaling as compared with other GSK3 inhibitors. GS87 is a novel GSK3 inhibitor with therapeutic potential as a differentiation agent for non-promyelocytic AML. Mol Cancer Ther; 15(7); 1485-94. ©2016 AACR. PMID:27196775

  12. JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors

    PubMed Central

    Gao, Sizhi P.; Chang, Qing; Mao, Ninghui; Daly, Laura A.; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Brewer, Monica Red; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L.; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F.

    2016-01-01

    Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non–small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells’ dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877

  13. Synthesis and Biological Evaluation of Analogues of AKT (Protein Kinase B) Inhibitor-IV

    PubMed Central

    Sun, Qi; Wu, Runzhi; Cai, Sutang; Lin, Yuan; Sellers, Llewlyn; Sakamoto, Kaori; He, Biao; Peterson, Blake R.

    2011-01-01

    Inhibitors of the PI3-kinase/AKT (protein kinase B) pathway are under investigation as anticancer and antiviral agents. The benzimidazole derivative AKT inhibitor-IV (ChemBridge 5233705) affects this pathway and exhibits potent anticancer and antiviral activity. To probe its biological activity, we synthesized AKT inhibitor-IV and 21 analogues using a novel six-step route based on ZrCl4-catalyzed cyclization of 1,2-arylenediamines with α,β-unsaturated aldehydes. We examined effects on viability of HeLa carcinoma cells, viability of normal human cells (NHBE), replication of recombinant parainfluenza virus 5 (PIV5) in HeLa cells, and replication of the intracellular bacterium Mycobacterium fortuitum in HeLa cells. Replacement of the benzimidazole N-ethyl substitutent of AKT inhibitor-IV with N-hexyl and N-dodecyl groups enhanced antiviral activity and cytotoxicity against the cancer cell line, but these compounds showed substantially lower toxicity (from 6-fold to >20-fold) against NHBE cells, and no effect on M. fortuitum, suggesting inhibition of one or more host protein(s) required for proliferation of cancer cells and PIV5. The key structural elements identified here may facilitate identification of targets of this highly biologically active scaffold. PMID:21319800

  14. Small-Molecule Inhibitors of the Receptor Tyrosine Kinases: Promising Tools for Targeted Cancer Therapies

    PubMed Central

    Hojjat-Farsangi, Mohammad

    2014-01-01

    Chemotherapeutic and cytotoxic drugs are widely used in the treatment of cancer. In spite of the improvements in the life quality of patients, their effectiveness is compromised by several disadvantages. This represents a demand for developing new effective strategies with focusing on tumor cells and minimum side effects. Targeted cancer therapies and personalized medicine have been defined as a new type of emerging treatments. Small molecule inhibitors (SMIs) are among the most effective drugs for targeted cancer therapy. The growing number of approved SMIs of receptor tyrosine kinases (RTKs) i.e., tyrosine kinase inhibitors (TKIs) in the clinical oncology imply the increasing attention and application of these therapeutic tools. Most of the current approved RTK–TKIs in preclinical and clinical settings are multi-targeted inhibitors with several side effects. Only a few specific/selective RTK–TKIs have been developed for the treatment of cancer patients. Specific/selective RTK–TKIs have shown less deleterious effects compared to multi-targeted inhibitors. This review intends to highlight the importance of specific/selective TKIs for future development with less side effects and more manageable agents. This article provides an overview of: (1) the characteristics and function of RTKs and TKIs; (2) the recent advances in the improvement of specific/selective RTK–TKIs in preclinical or clinical settings; and (3) emerging RTKs for targeted cancer therapies by TKIs. PMID:25110867

  15. JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors.

    PubMed

    Gao, Sizhi P; Chang, Qing; Mao, Ninghui; Daly, Laura A; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Red Brewer, Monica; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F

    2016-01-01

    Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non-small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells' dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877

  16. Large-Scale Computational Screening Identifies First in Class Multitarget Inhibitor of EGFR Kinase and BRD4

    PubMed Central

    Allen, Bryce K.; Mehta, Saurabh; Ember, Stewart W. J.; Schonbrunn, Ernst; Ayad, Nagi; Schürer, Stephan C.

    2015-01-01

    Inhibition of cancer-promoting kinases is an established therapeutic strategy for the treatment of many cancers, although resistance to kinase inhibitors is common. One way to overcome resistance is to target orthogonal cancer-promoting pathways. Bromo and Extra-Terminal (BET) domain proteins, which belong to the family of epigenetic readers, have recently emerged as promising therapeutic targets in multiple cancers. The development of multitarget drugs that inhibit kinase and BET proteins therefore may be a promising strategy to overcome tumor resistance and prolong therapeutic efficacy in the clinic. We developed a general computational screening approach to identify novel dual kinase/bromodomain inhibitors from millions of commercially available small molecules. Our method integrated machine learning using big datasets of kinase inhibitors and structure-based drug design. Here we describe the computational methodology, including validation and characterization of our models and their application and integration into a scalable virtual screening pipeline. We screened over 6 million commercially available compounds and selected 24 for testing in BRD4 and EGFR biochemical assays. We identified several novel BRD4 inhibitors, among them a first in class dual EGFR-BRD4 inhibitor. Our studies suggest that this computational screening approach may be broadly applicable for identifying dual kinase/BET inhibitors with potential for treating various cancers. PMID:26596901

  17. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    SciTech Connect

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-Kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-08-26

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. The interaction of PKM2 with phosphotyrosine-containing proteins inhibits enzyme activity and increases the availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small-molecule PKM2 activators inhibits the growth of xenograft tumors. Structural studies reveal that small-molecule activators bind PKM2 at the subunit interaction interface, a site that is distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. This data supports the notion that small-molecule activation of PKM2 can interfere with anabolic metabolism.

  18. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis

    PubMed Central

    Anastasiou, Dimitrios; Yu, Yimin; Israelsen, William J.; Jiang, Jian-kang; Boxer, Matthew B.; Hong, Bum Soo; Tempel, Wolfram; Dimov, Svetoslav; Shen, Min; Jha, Abhishek; Yang, Hua; Mattaini, Katherine R.; Metallo, Christian M.; Fiske, Brian P.; Courtney, Kevin D.; Malstrom, Scott; Khan, Tahsin M.; Kung, Charles; Skoumbourdis, Amanda P.; Veith, Henrike; Southall, Noel; Walsh, Martin J.; Brimacombe, Kyle R.; Leister, William; Lunt, Sophia Y.; Johnson, Zachary R.; Yen, Katharine E.; Kunii, Kaiko; Davidson, Shawn M.; Christofk, Heather R.; Austin, Christopher P.; Inglese, James; Harris, Marian H.; Asara, John M.; Stephanopoulos, Gregory; Salituro, Francesco G.; Jin, Shengfang; Dang, Lenny; Auld, Douglas S.; Park, Hee-Won; Cantley, Lewis C.; Thomas, Craig J.; Vander Heiden, Matthew G.

    2012-01-01

    Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism. PMID:22922757

  19. Analysis of pulmonary vasodilator responses to the Rho-kinase inhibitor fasudil in the anesthetized rat.

    PubMed

    Badejo, Adeleke M; Dhaliwal, Jasdeep S; Casey, David B; Gallen, Thomas B; Greco, Anthony J; Kadowitz, Philip J

    2008-11-01

    The small GTP-binding protein Rho and its downstream effector, Rho-kinase, are important regulators of vasoconstrictor tone. Rho-kinase is upregulated in experimental models of pulmonary hypertension, and Rho-kinase inhibitors decrease pulmonary arterial pressure in rodents with monocrotaline and chronic hypoxia-induced pulmonary hypertension. However, less is known about responses to fasudil when pulmonary vascular resistance is elevated on an acute basis by vasoconstrictor agents and ventilatory hypoxia. In the present study, intravenous injections of fasudil reversed pulmonary hypertensive responses to intravenous infusion of the thromboxane receptor agonist, U-46619 and ventilation with a 10% O(2) gas mixture and inhibited pulmonary vasoconstrictor responses to intravenous injections of angiotensin II, BAY K 8644, and U-46619 without prior exposure to agonists, which can upregulate Rho-kinase activity. The calcium channel blocker isradipine and fasudil had similar effects and in small doses had additive effects in blunting vasoconstrictor responses, suggesting parallel and series mechanisms in the lung. When pulmonary vascular resistance was increased with U-46619, fasudil produced similar decreases in pulmonary and systemic arterial pressure, whereas isradipine produced greater decreases in systemic arterial pressure. The hypoxic pressor response was enhanced by 5-10 mg/kg iv nitro-L-arginine methyl ester (L-NAME), and fasudil or isradipine reversed the pulmonary hypertensive response to hypoxia in control and in L-NAME-treated animals, suggesting that the response is mediated by Rho-kinase and L-type Ca(2+) channels. These results suggest that Rho-kinase is constitutively active in regulating baseline tone and vasoconstrictor responses in the lung under physiological conditions and that Rho-kinase inhibition attenuates pulmonary vasoconstrictor responses to agents that act by different mechanisms without prior exposure to the agonist. PMID:18689606

  20. Targeting mitogen-activated protein kinase kinase with the inhibitor PD0325901 decreases hepatocellular carcinoma growth in vitro and in mouse model systems.

    PubMed

    Hennig, Matthew; Yip-Schneider, Michele T; Wentz, Sabrina; Wu, Huangbing; Hekmatyar, S K; Klein, Patrick; Bansal, Navin; Schmidt, C Max

    2010-04-01

    Hepatocellular carcinoma (HCC) is a common cause of death from solid organ malignancy worldwide. Extracellular signal-regulated/mitogen-activated protein kinase kinase (MEK) signaling is a critical growth regulatory pathway in HCC. Targeting MEK with a novel small molecule inhibitor, PD0325901, may inhibit HCC tumorigenesis. PD0325901 (0.01-100 nM) inhibited growth and MEK activity in vitro in immortalized murine transforming growth factor alpha (TGF-alpha) transgenic hepatocyte (TAMH) cells, derived from the livers of TGF-alpha transgenic mice. Treatment of athymic mice bearing TAMH flank tumors with vehicle or PD0325901 (20 mg/kg) revealed a significant reduction of MEK activity ex vivo 24 hours after a single PD0325901 dose. The growth rate of TAMH flank tumors over 16 days was reduced threefold in the treatment arm (1113 +/- 269% versus 3077 +/- 483%, P < 0.01). PD0325901 exhibited similar inhibitory effects in HepG2 and Hep3B human HCC cells in vitro and in Hep3B flank tumors in vivo. To confirm this in a developmental model, MT-42 (CD-1) TGF-alpha mice were treated with vehicle or PD0325901 (20 mg/kg) for 5 weeks. Gross HCC was detected in 47% and 13.3% of the control and treatment mice, respectively. Tumor growth suppression by PD0325901 relative to vehicle was also shown by magnetic resonance imaging. These studies provide compelling preclinical evidence that targeting MEK in human clinical trials may be promising for the treatment of HCC. PMID:20112426

  1. Upstream mitogen-activated protein kinase (MAPK) pathway inhibition: MEK inhibitor followed by a BRAF inhibitor in advanced melanoma patients.

    PubMed

    Goldinger, Simone M; Zimmer, Lisa; Schulz, Carsten; Ugurel, Selma; Hoeller, Christoph; Kaehler, Katharina C; Schadendorf, Dirk; Hassel, Jessica C; Becker, Juergen; Hauschild, Axel; Dummer, Reinhard

    2014-01-01

    BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients. BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi. A total of 23 melanoma patients (six females, 17 males, aged 47-80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2-23.2) and subsequent for BRAFi 4.5 (1.2-15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed. Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies. PMID:24183461

  2. Discovery of pyrrolo[1,2-b]pyridazine-3-carboxamides as Janus kinase (JAK) inhibitors.

    PubMed

    Duan, James J-W; Lu, Zhonghui; Jiang, Bin; Yang, Bingwei V; Doweyko, Lidia M; Nirschl, David S; Haque, Lauren E; Lin, Shuqun; Brown, Gregory; Hynes, John; Tokarski, John S; Sack, John S; Khan, Javed; Lippy, Jonathan S; Zhang, Rosemary F; Pitt, Sidney; Shen, Guoxiang; Pitts, William J; Carter, Percy H; Barrish, Joel C; Nadler, Steven G; Salter-Cid, Luisa M; McKinnon, Murray; Fura, Aberra; Schieven, Gary L; Wrobleski, Stephen T

    2014-12-15

    A new class of Janus kinase (JAK) inhibitors was discovered using a rationally designed pyrrolo[1,2-b]pyridazine-3-carboxamide scaffold. Preliminary studies identified (R)-(2,2-dimethylcyclopentyl)amine as a preferred C4 substituent on the pyrrolopyridazine core (3b). Incorporation of amino group to 3-position of the cyclopentane ring resulted in a series of JAK3 inhibitors (4g-4j) that potently inhibited IFNγ production in an IL2-induced whole blood assay and displayed high functional selectivity for JAK3-JAK1 pathway relative to JAK2. Further modifications led to the discovery of an orally bioavailable (2-fluoro-2-methylcyclopentyl)amino analogue 5g which is a nanomolar inhibitor of both JAK3 and TYK2, functionally selective for the JAK3-JAK1 pathway versus JAK2, and active in a human whole blood assay. PMID:25453808

  3. Discovery of selective phosphatidylinositol 3-kinase inhibitors to treat hematological malignancies.

    PubMed

    Zhu, Jingyu; Hou, Tingjun; Mao, Xinliang

    2015-08-01

    The phosphatidylinositol 3-kinase (PI3K) signaling pathway is associated with chemoresistance and poor prognosis of many cancers, including hematological malignancies (HM), such as leukemia, lymphomas, and multiple myeloma (MM). Targeting PI3K is emerging as a promising strategy in the treatment of these blood cancers. Recent approval of idelalisib, a specific inhibitor of PI3Kδ, for the treatment of several types of HM, is likely to attract more interest in search for novel PI3K inhibitors. Here, we discuss classic and cutting-edge techniques and strategies to identify PI3K inhibitors for the treatment of HM. Each technique has its own strengths and limitations, and their combined application will accelerate the drug discovery process with fewer associated costs. PMID:25857437

  4. Design of inhibitors of thymidylate kinase from Variola virus as new selective drugs against smallpox.

    PubMed

    Guimarães, Ana P; de Souza, Felipe R; Oliveira, Aline A; Gonçalves, Arlan S; de Alencastro, Ricardo B; Ramalho, Teodorico C; França, Tanos C C

    2015-02-16

    Recently we constructed a homology model of the enzyme thymidylate kinase from Variola virus (VarTMPK) and proposed it as a new target to the drug design against smallpox. In the present work, we used the antivirals cidofovir and acyclovir as reference compounds to choose eleven compounds as leads to the drug design of inhibitors for VarTMPK. Docking and molecular dynamics (MD) studies of the interactions of these compounds inside VarTMPK and human TMPK (HssTMPK) suggest that they compete for the binding region of the substrate and were used to propose the structures of ten new inhibitors for VarTMPK. Further docking and MD simulations of these compounds, inside VarTMPK and HssTMPK, suggest that nine among ten are potential selective inhibitors of VarTMPK. PMID:25458183

  5. Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia

    PubMed Central

    Neumann, Theresa; Benajiba, Lina; Göring, Stefan; Stegmaier, Kimberly; Schmidt, Boris

    2016-01-01

    The challenge for Glycogen Synthase Kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML) may require α-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3α selectivity reported so far, but suffered from insufficient aqueous solubility. This work presents the solubility-driven optimization of our isoform-selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed high activity against GSK-3α/β with the highest GSK-3α selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3α targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation phenotype and colony formation impairment, confirming the potential of GSK-3α inhibition in AML therapy. PMID:26496242

  6. Repeated preconditioning with hyperbaric oxygen induces neuroprotection against forebrain ischemia via suppression of p38 mitogen activated protein kinase.

    PubMed

    Yamashita, Satoshi; Hirata, Takao; Mizukami, Yoichi; Cui, Ying Jun; Fukuda, Shiro; Ishida, Kazuyoshi; Matsumoto, Mishiya; Sakabe, Takefumi

    2009-12-01

    We previously reported in rats that preconditioning with hyperbaric oxygen (HBO; 100% O(2) 3.5-atomsphere absolute (ATA), 1 h/day for 5 days) provided neuroprotection against transient (8 min) forebrain ischemia possibly through protein synthesis relevant to neurotrophin receptor and inflammatory-immune system. A recent report suggested that HBO-induced neuroprotection is relevant to brain derived neurotrophic factor and its downstream event involving suppression of p38 mitogen activated protein kinase (p38) activation. In the present study, we first performed a dose comparison (1, 2, and 3.5 ATA) of HBO-induced neuroprotection and then investigated pharmacological modification by 10 mg/kg anisomycin (a protein synthesis inhibitor and potent activator for p38) and 200 microg/kg SB203580 (a p38 inhibitor), which were given intraperitoneally 60 and 30 min before every 3.5 ATA-HBO treatment, respectively. Most prominent protective effect on hippocampal CA1 neurons was observed with 3.5 ATA-HBO (survived neurons: 69% [62-73%] vs. untreated: 3.9% [2-8%], 1 ATA: 8.8% [0-26%], 2 ATA-HBO: 46% [22-62%] (median [range]) (7 days after ischemia). Anisomycin abolished a neuroprotective effect (survived neuron: 1.2% [0-7%]). SB203580, when given between administration of anisomycin and HBO treatment, resumed a neuroprotective effect (survived neuron: 52% [37-62%]). The level of phosphorylated p38 at 10-min reperfusion was significantly decreased in 3.5 ATA-HBO group (32% [12-53%] of sham). Single pretreatment with 100 and 200 microg/kg of SB203580 exerted a similar neuroprotective effect (39% [25-51%] and 59% [50-72%]) to 2 and 3.5 ATA-HBO preconditioning, respectively. It is concluded that suppression of p38 phosphorylation plays a key role in HBO-induced neuroprotection and that pretreatment with a p38 inhibitor (SB203580) can provide similar neuroprotection. PMID:19747454

  7. Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis.

    PubMed

    Hamdoun, Safae; Zhang, Chong; Gill, Manroop; Kumar, Narender; Churchman, Michelle; Larkin, John C; Kwon, Ashley; Lu, Hua

    2016-01-01

    Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions. PMID:26561564

  8. Kinase Domain Point Mutations in Ph+ Acute Lymphoblastic Leukemia (ALL) Emerge Following Therapy with BCR-ABL Kinase Inhibitors

    PubMed Central

    Jones, Dan; Thomas, Deborah; Yin, C. Cameron; O'Brien, Susan; Cortes, Jorge E.; Jabbour, Elias; Breeden, Megan; Giles, Francis J.; Zhao, Weiqiang; Kantarjian, Hagop M.

    2008-01-01

    Background BCR-ABL kinase domain (KD) mutations are detected in approximately 45% of imatinib-resistant CML. Patterns of KD mutations in Philadelphia chromosome (Ph)+ acute lymphoblastic leukemia (ALL) are less well-studied. Methods We assessed KD mutations in relapsed Ph+ ALL following treatments that included one or more kinase inhibitors (n = 24) or no prior KI therapy (n = 12). Results ABL KD mutations were detected by direct sequencing in 15 of 17 (88%) relapsed Ph+ ALL with prior imatinib (n = 16) or dasatinib (n = 1) treatment, and in 6 of 7 (86%) resistant/relapsed tumors treated with 2 or more KIs, compared with 0 of 12 relapsed Ph+ ALL never treated with KI. A restricted set of mutations was seen, mostly Y253H and T315I, detected on average 13 months following KI initiation, and mutations were not detected in the initial tumor samples prior to KI therapy in 12 patients assessed. Using a more sensitive pyrosequencing method, we did not detect mutations at codons 315 and 253 in the diagnostic samples from these 12 patients or in 30 Ph+ ALL patients who never relapsed. Conclusions ABL KD mutations, especially at codons 315 and 253, emerge upon relapse in the vast majority of patients with Ph+ ALL receiving maintenance KI therapy. Ongoing KI exposure may thus alter the patterns of relapse and favor outgrowth of clones with KI-resistant mutations. PMID:18615627

  9. Structural Requirements and Docking Analysis of Amidine-Based Sphingosine Kinase 1 Inhibitors Containing Oxadiazoles.

    PubMed

    Houck, Joseph D; Dawson, Thomas K; Kennedy, Andrew J; Kharel, Yugesh; Naimon, Niels D; Field, Saundra D; Lynch, Kevin R; Macdonald, Timothy L

    2016-05-12

    Sphingosine 1-phosphate (S1P) is a potent growth-signaling lipid that has been implicated in cancer progression, inflammation, sickle cell disease, and fibrosis. Two sphingosine kinases (SphK1 and 2) are the source of S1P; thus, inhibitors of the SphKs have potential as targeted cancer therapies and will help to clarify the roles of S1P and the SphKs in other hyperproliferative diseases. Recently, we reported a series of amidine-based inhibitors with high selectivity for SphK1 and potency in the nanomolar range. However, these inhibitors display a short half-life. With the goal of increasing metabolic stability and maintaining efficacy, we designed an analogous series of molecules containing oxadiazole moieties. Generation of a library of molecules resulted in the identification of the most selective inhibitor of SphK1 reported to date (705-fold selectivity over SphK2), and we found that potency and selectivity vary significantly depending on the particular oxadiazole isomer employed. The best inhibitors were subjected to in silico molecular dynamics docking analysis, which revealed key insights into the binding of amidine-based inhibitors by SphK1. Herein, the design, synthesis, biological evaluation, and docking analysis of these molecules are described. PMID:27190598

  10. South (S)- and North (N)-Methanocarba-7-Deazaadenosine Analogues as Inhibitors of Human Adenosine Kinase.

    PubMed

    Toti, Kiran S; Osborne, Danielle; Ciancetta, Antonella; Boison, Detlev; Jacobson, Kenneth A

    2016-07-28

    Adenosine kinase (AdK) inhibitors raise endogenous adenosine levels, particularly in disease states, and have potential for treatment of seizures, neurodegeneration, and inflammation. On the basis of the South (S) ribose conformation and molecular dynamics (MD) analysis of nucleoside inhibitors bound in AdK X-ray crystallographic structures, (S)- and North (N)-methanocarba (bicyclo[3.1.0]hexane) derivatives of known inhibitors were prepared and compared as human (h) AdK inhibitors. 5'-Hydroxy (34, MRS4202 (S); 55, MRS4380 (N)) and 5'-deoxy 38a (MRS4203 (S)) analogues, containing 7- and N(6)-NH phenyl groups in 7-deazaadenine, robustly inhibited AdK activity (IC50 ∼ 100 nM), while the 5'-hydroxy derivative 30 lacking the phenyl substituents was weak. Docking in the hAdK X-ray structure and MD simulation suggested a mode of binding similar to 5'-deoxy-5-iodotubercidin and other known inhibitors. Thus, a structure-based design approach for further potency enhancement is possible. The potent AdK inhibitors in this study are ready to be further tested in animal models of epilepsy. PMID:27410258

  11. Identifying GSK-3β kinase inhibitors of Alzheimer's disease: Virtual screening, enzyme, and cell assays.

    PubMed

    Lin, Chih-Hsin; Hsieh, Yu-Shao; Wu, Yih-Ru; Hsu, Chia-Jen; Chen, Hsuan-Chiang; Huang, Wun-Han; Chang, Kuo-Hsuan; Hsieh-Li, Hsiu Mei; Su, Ming-Tsan; Sun, Ying-Chieh; Lee, Guan-Chiun; Lee-Chen, Guey-Jen

    2016-06-30

    Glycogen synthase kinase 3β (GSK-3β) is widely known as a critical target protein for treating Alzheimer's disease (AD). We utilized virtual screening to search databases for compounds with the potential to be used in drugs targeting GSK-3β kinase, and kinase as well as cell assays to investigate top-scored, selected compounds. Virtual screening of >1.1 million compounds in the ZINC and in-house databases was conducted using an optimized computational protocol in the docking program GOLD. Of the top-ranked compounds, 16 underwent a luminescent kinase assay and a cell assay using HEK293 cells expressing DsRed-tagged ΔK280 in the repeat domain of tau (tauRD). The compounds VB-003 (a potent GSK-3β inhibitor) and VB-008 (AM404, an anandamide transport inhibitor), with determined IC50 values of 0.25 and 5.4μM, respectively, were identified as reducing tau aggregation. Both compounds increased expression of phospho-GSK-3β (Ser9) and reduced endogenous tau phosphorylation at the sites of Ser202, Thr231, and Ser396. In the ∆K280 tauRD-DsRed SH-SY5Y cells, VB-008, but not VB-003, enhanced HSPB1 and GRP78 expression, increased ∆K280 tauRD-DsRed solubility, and promoted neurite outgrowth. Thus VB-008 performed best to the end of the present study. The identified compound VB-008 may guide the identification and synthesis of potential inhibitors analogous to this compound. PMID:27094783

  12. Antitumor effects of immunotoxins are enhanced by lowering HCK or treatment with SRC kinase inhibitors.

    PubMed

    Liu, Xiu-Fen; Xiang, Laiman; FitzGerald, David J; Pastan, Ira

    2014-01-01

    Recombinant immunotoxins (RIT) are agents being developed for cancer treatment. They are composed of an Fv that binds to a cancer cell, fused to a 38-kDa fragment of Pseudomonas exotoxin A. SS1P is a RIT that targets mesothelin, a protein expressed on mesothelioma as well as pancreatic, ovarian, lung, and other cancers. Because the protein tyrosine kinase family regulates a variety of cellular processes and pathways, we hypothesized that tyrosine kinases might regulate susceptibility to immunotoxin killing. To investigate their role, we used siRNAs to lower the level of expression of the 88 known tyrosine kinases. We identified five tyrosine kinases, INSR, HCK, SRC, PDGFRβ, and BMX that enhance the activity of SS1P when their level of expression is lowered by siRNAs. We further investigated the Src family member HCK in this study. Knocking down of SRC slightly increased SS1P killing in A431/H9 cells, but knocking down HCK substantially enhanced killing by SS1P. We investigated the mechanism of enhancement and found that HCK knockdown enhanced SS1P cleavage by furin and lowered levels of Mcl-1 and raised Bax. We then found that Src inhibitors mimic the stimulatory effect of HCK knockdown; both SU6656 and SKI-606 (bosutinib) enhanced immunotoxin killing of mesothelin-expressing cells by SS1P and CD22-expressing cells by HA22 (moxetumomab pasudotox). SU6656 also enhanced the antitumor effects of SS1P and HA22 in mouse xenograft tumor models. Our data suggest that the combination of immunotoxin with tyrosine kinase inhibitors may be an effective way to treat some cancers. PMID:24145282

  13. Inhibitors of the Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

    SciTech Connect

    Sueyoshi, Noriyuki; Takao, Toshihiko; Nimura, Takaki; Sugiyama, Yasunori; Numano, Takamasa; Shigeri, Yasushi; Taniguchi, Takanobu; Kameshita, Isamu Ishida, Atsuhiko

    2007-11-23

    Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca{sup 2+}/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP family phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro.

  14. The dipeptidyl peptidase-4 inhibitor sitagliptin suppresses mouse colon tumorigenesis in type 2 diabetic mice.

    PubMed

    Yorifuji, Naoki; Inoue, Takuya; Iguchi, Munetaka; Fujiwara, Kaori; Kakimoto, Kazuki; Nouda, Sadaharu; Okada, Toshihiko; Kawakami, Ken; Abe, Yosuke; Takeuchi, Toshihisa; Higuchi, Kazuhide

    2016-02-01

    Patients with type 2 diabetes mellitus are known to have an increased risk of colorectal neoplasia. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been used as a new therapeutic tool for type 2 diabetes. Since the substrates for DPP-4 include intestinotrophic hormones and chemokines such as GLP-2 and stromal cell-derived factor-1 (SDF-1), which are associated with tumor progression, DPP-4 inhibitors may increase the risk of colorectal tumors. However, the influence of DPP-4 inhibitors on colorectal neoplasia in patients with type 2 diabetes remains unknown. In the present study, we show that long-term administration of a DPP-4 inhibitor, sitagliptin (STG), suppressed colon carcinogenesis in leptin-deficient (ob/ob) C57BL/6J mice. Colonic mucosal concentrations of glucagon‑like peptide-1 (GLP-1) and GLP-2 were significantly elevated in the ob/ob mice. However, mucosal GLP concentrations and the plasma level of SDF-1 were not affected by the administration of STG. Real‑time PCR analysis revealed that colonic mucosal IL-6 mRNA expression, which was significantly upregulated in the ob/ob mice, was significantly suppressed by the long-term administration of STG. These results suggest that a DPP-4 inhibitor may suppress colon carcinogenesis in mice with type 2 diabetes in a GLP-independent manner. Since DPP-4 has multiple biological functions, further studies analyzing other factors related to colon carcinogenesis are needed. PMID:26573958

  15. Resistance to the tyrosine kinase inhibitor axitinib is associated with increased glucose metabolism in pancreatic adenocarcinoma.

    PubMed

    Hudson, C D; Hagemann, T; Mather, S J; Avril, N

    2014-01-01

    Alterations in energy (glucose) metabolism are key events in the development and progression of cancer. In pancreatic adenocarcinoma (PDAC) cells, we investigated changes in glucose metabolism induced by resistance to the receptor tyrosine kinase inhibitor (RTKI) axitinib. Here, we show that human cell lines and mouse PDAC cell lines obtained from the spontaneous pancreatic cancer mouse model (Kras(G12D)Pdx1-cre) were sensitive to axitinib. The anti-proliferative effect was due to a G2/M block resulting in loss of 70-75% cell viability in the most sensitive PDAC cell line. However, a surviving sub-population showed a 2- to 3-fold increase in [C-14]deoxyglucose ([C-14]DG) uptake. This was sustained in axitinib-resistant cell lines, which were derived from parental PDAC. In addition to the axitinib-induced increase in [C-14]DG uptake, we observed a translocation of glucose transporter-1 (Glut-1) transporters from cytosolic pools to the cell surface membrane and a 2-fold increase in glycolysis rates measured by the extracellular acidification rate (ECAR). We demonstrated an axitinib-induced increase in phosphorylated Protein Kinase B (pAkt) and by blocking pAkt with a phosphatidylinositol-3 kinase (PI3K) inhibitor we reversed the Glut-1 translocation and restored sensitivity to axitinib treatment. Combination treatment with both axitinib and Akt inhibitor in parental pancreatic cell line resulted in a decrease in cell viability beyond that conferred by single therapy alone. Our study shows that PDAC resistance to axitinib results in increased glucose metabolism mediated by activated Akt. Combining axitinib and an Akt inhibitor may improve treatment in PDAC. PMID:24722285

  16. A Cell Biologist’s Field Guide to Aurora Kinase Inhibitors

    PubMed Central

    de Groot, Christian O.; Hsia, Judy E.; Anzola, John V.; Motamedi, Amir; Yoon, Michelle; Wong, Yao Liang; Jenkins, David; Lee, Hyun J.; Martinez, Mallory B.; Davis, Robert L.; Gahman, Timothy C.; Desai, Arshad; Shiau, Andrew K.

    2015-01-01

    Aurora kinases are essential for cell division and are frequently misregulated in human cancers. Based on their potential as cancer therapeutics, a plethora of small molecule Aurora kinase inhibitors have been developed, with a subset having been adopted as tools in cell biology. Here, we fill a gap in the characterization of Aurora kinase inhibitors by using biochemical and cell-based assays to systematically profile a panel of 10 commercially available compounds with reported selectivity for Aurora A (MLN8054, MLN8237, MK-5108, MK-8745, Genentech Aurora Inhibitor 1), Aurora B (Hesperadin, ZM447439, AZD1152-HQPA, GSK1070916), or Aurora A/B (VX-680). We quantify the in vitro effect of each inhibitor on the activity of Aurora A alone, as well as Aurora A and Aurora B bound to fragments of their activators, TPX2 and INCENP, respectively. We also report kinome profiling results for a subset of these compounds to highlight potential off-target effects. In a cellular context, we demonstrate that immunofluorescence-based detection of LATS2 and histone H3 phospho-epitopes provides a facile and reliable means to assess potency and specificity of Aurora A versus Aurora B inhibition, and that G2 duration measured in a live imaging assay is a specific readout of Aurora A activity. Our analysis also highlights variation between HeLa, U2OS, and hTERT-RPE1 cells that impacts selective Aurora A inhibition. For Aurora B, all four tested compounds exhibit excellent selectivity and do not significantly inhibit Aurora A at effective doses. For Aurora A, MK-5108 and MK-8745 are significantly more selective than the commonly used inhibitors MLN8054 and MLN8237. A crystal structure of an Aurora A/MK-5108 complex that we determined suggests the chemical basis for this higher specificity. Taken together, our quantitative biochemical and cell-based analyses indicate that AZD1152-HQPA and MK-8745 are the best current tools for selectively inhibiting Aurora B and Aurora A, respectively

  17. The cyclin-dependent kinase inhibitor butyrolactone is a potent inhibitor of p21 (WAF1/CIP1 expression).

    PubMed

    Sax, Joanna K; Dash, Bipin C; Hong, Rui; Dicker, David T; El-Deiry, Wafik S

    2002-01-01

    Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damag-ing agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21- dependent checkpoints. PMID:12429914

  18. The IκB kinase inhibitor ACHP strongly attenuates TGFβ1-induced myofibroblast formation and collagen synthesis.

    PubMed

    Mia, Masum M; Bank, Ruud A

    2015-12-01

    Excessive accumulation of a collagen-rich extracellular matrix (ECM) by myofibroblasts is a characteristic feature of fibrosis, a pathological state leading to serious organ dysfunction. Transforming growth factor beta1 (TGFβ1) is a strong inducer of myofibroblast formation and subsequent collagen production. Currently, there are no remedies for the treatment of fibrosis. Activation of the nuclear factor kappa B (NF-κB) pathway by phosphorylating IκB with the enzyme IκB kinase (IKK) plays a major role in the induction of fibrosis. ACHP {2-Amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)-3 pyridinecarbonitrile}, a selective inhibitor of IKK, prohibits the activation of the NF-κB pathway. It is not known whether ACHP has potential anti-fibrotic properties. Using adult human dermal and lung fibroblasts we have investigated whether ACHP has the ability to inhibit the TGFβ1-induced transition of fibroblasts into myofibroblasts and its excessive synthesis of ECM. The presence of ACHP strongly suppressed the induction of the myofibroblast markers alpha-smooth muscle actin (αSMA) and SM22α, as well as the deposition of the ECM components collagen type I and fibronectin. Furthermore, post-treatment with ACHP partly reversed the expression of αSMA and collagen type I production. Finally, ACHP suppressed the expression of the three collagen-modifying enzymes lysyl hydroxylase (PLOD1, PLOD2 and PLOD3) in dermal fibroblasts, but did not do so in lung fibroblasts. We conclude that the IKK inhibitor ACHP has potent antifibrotic properties, and that the NF-κB pathway plays an important role in myofibroblast biology. PMID:26337045

  19. Discovery, Synthesis and Characterization of an Orally Bioavailable, Brain Penetrant Inhibitor of Mixed Lineage Kinase 3

    PubMed Central

    Goodfellow, Val S.; Loweth, Colin J.; Ravula, Satheesh B.; Wiemann, Torsten; Nguyen, Thong; Xu, Yang; Todd, Daniel E.; Sheppard, David; Pollack, Scott; Polesskaya, Oksana; Marker, Daniel F.; Dewhurst, Stephen; Gelbard, Harris A.

    2014-01-01

    Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson’s Disease and HIV-1 Associated Neurocognitive Disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50 = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes, and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC50 = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development. PMID:24044867

  20. Identification of p38α MAP kinase inhibitors by pharmacophore based virtual screening.

    PubMed

    Gangwal, Rahul P; Das, Nihar R; Thanki, Kaushik; Damre, Mangesh V; Dhoke, Gaurao V; Sharma, Shyam S; Jain, Sanyog; Sangamwar, Abhay T

    2014-04-01

    The p38α mitogen-activated protein (MAP) kinase plays a vital role in treating many inflammatory diseases. In the present study, a combined ligand and structure based pharmacophore model was developed to identify potential DFG-in selective p38 MAP kinase inhibitors. Conformations of co-crystallised inhibitors were used in the development and validation of ligand and structure based pharmacophore modeling approached. The validated pharmacophore was utilized in database screening to identify potential hits. After Lipinski's rule of five filter and molecular docking analysis, nineteen hits were purchased and selected for in vitro analysis. The virtual hits exhibited promising activity against tumor necrosis factor-α (TNF-α) with 23-98% inhibition at 10μM concentration. Out of these seven compounds has shown potent inhibitory activity against p38 MAP kinase with IC50 values ranging from 12.97 to 223.5nM. In addition, the toxicity study against HepG2 cells was also carried out to confirm the safety profile of identified virtual hits. PMID:24473068

  1. The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

    PubMed Central

    Chang, Xiaofei; Izumchenko, Eugene; Solis, Luisa M.; Kim, Myoung Sook; Chatterjee, Aditi; Ling, Shizhang; Monitto, Constance L.; Harari, Paul M.; Hidalgo, Manuel; Goodman, Steve N.; Wistuba, Ignacio I.; Bedi, Atul; Sidransky, David

    2013-01-01

    The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01). PMID:23935914

  2. 1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazoles: i