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Sample records for inhibitor targeting kit

  1. Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells.

    PubMed

    Abbaspour Babaei, Maryam; Kamalidehghan, Behnam; Saleem, Mohammad; Huri, Hasniza Zaman; Ahmadipour, Fatemeh

    2016-01-01

    c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence. PMID:27536065

  2. Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells

    PubMed Central

    Abbaspour Babaei, Maryam; Kamalidehghan, Behnam; Saleem, Mohammad; Huri, Hasniza Zaman; Ahmadipour, Fatemeh

    2016-01-01

    c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence. PMID:27536065

  3. Combination therapy for KIT-mutant mast cells: targeting constitutive NFAT and KIT activity.

    PubMed

    Macleod, Alison C; Klug, Lillian R; Patterson, Janice; Griffith, Diana J; Beadling, Carol; Town, Ajia; Heinrich, Michael C

    2014-12-01

    Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease.

  4. Oncogenic c-kit transcript is a target for binase.

    PubMed

    Mitkevich, Vladimir A; Petrushanko, Irina Y; Kretova, Olga V; Zelenikhin, Pavel V; Prassolov, Vladimir S; Tchurikov, Nickolai A; Ilinskaya, Olga N; Makarov, Alexander A

    2010-07-01

    Mutational activation of c-Kit receptor tyrosine kinase is common in acute myelogenous leukemia (AML). One such activating point mutation is the N822K replacement in the c-Kit protein. Here we investigate the selective cytotoxic effect of binase--RNase from Bacillus intermedius--on FDC-P1-N822K cells. These cells were derived from myeloid progenitor FDC-P1 cells, in which ectopic expression of N822K c-kit gene induces interleukin-3 independent growth. In order to determine whether the sensitivity of these cells to binase is caused by the expression of c-kit oncogene, the cytotoxicity of the RNase was studied in the presence of selective inhibitor of mutated c-Kit imatinib (Gleevec). Inhibition of mutated c-Kit protein leads to the loss of cell sensitivity to the apoptotic effect of binase, while the latter still decreases the amount of cellular RNA. Using green fluorescent protein as an expression marker for the c-Kit oncoprotein, we demonstrate that the elimination of c-Kit is the key factor in selective cytotoxicity of binase. Quantitative RT-PCR with RNA samples isolated from the binase-treated FDC-P1-N822K cells shows that binase treatment results in 41% reduction in the amount of с-kit mRNA. This indicates that the transcript of the activated mutant c-kit is the target for toxic action of binase. Thus, the combination of inhibition of oncogenic protein with the destruction of its mRNA is a promising approach to eliminating malignant cells.

  5. Targeting KIT in Melanoma: A Paradigm of Molecular Medicine and Targeted Therapeutics

    PubMed Central

    Woodman, Scott E.; Davies, Michael A.

    2014-01-01

    Despite multiple clinical trials utilizing a spectrum of therapeutic modalities, melanoma remains a disease with dismal outcomes in patients with advanced disease. However, it is now clear that melanoma is not a single entity, but can be molecularly divided into subtypes that generally correspond to the anatomical location of the primary melanoma. Melanomas from acral lentiginous, mucosal, and chronic sun-damaged sites frequently harbor activating mutations and/or increased copy number in the KIT tyrosine kinase receptor gene, which are very rare in the more common cutaneous tumors. Multiple case reports and early observations from clinical trials suggest that targeting mutant KIT with tyrosine kinase inhibitors is efficacious in KIT mutant melanoma. This review recounts what is known about the role of KIT in melanocyte maturation, our current understanding of KIT genetic aberrations in melanoma, and how this knowledge is being translated into clinical oncology. PMID:20457136

  6. Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

    PubMed

    Choudhary, Swati; Pardo, Alessa; Rosinke, Reinhard; Batra, Janendra K; Barth, Stefan; Verma, Rama S

    2016-01-01

    Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system.

  7. Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

    PubMed

    Choudhary, Swati; Pardo, Alessa; Rosinke, Reinhard; Batra, Janendra K; Barth, Stefan; Verma, Rama S

    2016-01-01

    Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system. PMID:26428235

  8. PCR inhibitor removal using the NucleoSpin® DNA Clean-Up XS kit.

    PubMed

    Faber, Korie L; Person, Eric C; Hudlow, William R

    2013-01-01

    Forensic evidence samples are collected from an unlimited variety of substrates, which may contain compounds known to inhibit the polymerase chain reaction (PCR). These PCR inhibitors are co-extracted with the DNA sample and can negatively affect the DNA typing results, which can range from partial to complete inhibition of the short tandem repeat (STR) PCR. One potential solution is to remove the PCR inhibitors from the extracts prior to the STR PCR with the NucleoSpin(®) DNA Clean-Up XS kit. The kit contains a NucleoSpin(®) XS silica column that has a special funnel design of thrust rings along with a very small silica membrane, which allows for sample elution in a small volume that is appropriate for use with current STR typing kits. The NucleoSpin(®) DNA Clean-Up XS kit was optimized for the best possible DNA recovery and then evaluated for its ability to remove eight commonly encountered PCR inhibitors including: bile salt, collagen, hematin, humic acid, indigo, melanin, tannic acid and urea. Each of these PCR inhibitors was effectively removed by the NucleoSpin(®) DNA Clean-Up XS kit as demonstrated by generating more complete STR profiles from the cleaned up inhibitor samples than from the raw inhibitor samples.

  9. Synthesis and biological evaluation of di-aryl urea derivatives as c-Kit inhibitors.

    PubMed

    Ravez, Séverine; Arsenlis, Stéphane; Barczyk, Amélie; Dupont, Anthony; Frédérick, Raphaël; Hesse, Stéphanie; Kirsch, Gilbert; Depreux, Patrick; Goossens, Laurence

    2015-11-15

    Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of many types of human cancers and many potent small molecules kinase inhibitors have been discovered the last decade. In the present study, we describe the synthesis of thienopyrimidine derivatives and their pharmacological evaluation against nine kinases (EGFR, PDGFR-ß, c-Kit, c-Met, Src, Raf, VEGFR-1, -2 and -3). Most of the synthesized compounds showed from moderate to potent activities against c-Kit with IC50 values in the nanomolar range. Among them, 4-anilino(urea)thienopyrimidine analogs showed selectivity and potent c-Kit inhibition with IC50 values less than 6 nM. Docking simulation was performed for the most promising compound 9 into the c-Kit active site to determine the potential binding mode. This study reveal that the 4-anilino(urea)thienopyrimidine is an interesting scaffold to design novel potent and selective c-Kit inhibitors which may make promising candidates for cancers where c-Kit receptors are overexpressed.

  10. Discovery of N-(3-((1-Isonicotinoylpiperidin-4-yl)oxy)-4-methylphenyl)-3-(trifluoromethyl)benzamide (CHMFL-KIT-110) as a Selective, Potent, and Orally Available Type II c-KIT Kinase Inhibitor for Gastrointestinal Stromal Tumors (GISTs).

    PubMed

    Wang, Qiang; Liu, Feiyang; Wang, Beilei; Zou, Fengming; Chen, Cheng; Liu, Xiaochuan; Wang, Aoli; Qi, Shuang; Wang, Wenchao; Qi, Ziping; Zhao, Zheng; Hu, Zhenquan; Wang, Wei; Wang, Li; Zhang, Shanchun; Wang, Yuexiang; Liu, Jing; Liu, Qingsong

    2016-04-28

    c-KIT kinase is a validated drug discovery target for gastrointestinal stromal tumors (GISTs). Clinically used c-KIT kinase inhibitors, i.e., Imatinib and Sunitinib, bear other important targets such as ABL or FLT3 kinases. Here we report our discovery of a more selective c-KIT inhibitor, compound 13 (CHMFL-KIT-110), which completely abolished ABL and FLT3 kinase activity. KinomeScan selectivity profiling (468 kinases) of 13 exhibited a high selectivity (S score (1) = 0.01). 13 displayed great antiproliferative efficacy against GISTs cell lines GIST-T1 and GIST-882 (GI50: 0.021 and 0.043 μM, respectively). In the cellular context, it effectively affected c-KIT-mediated signaling pathways and induced apoptosis as well as cell cycle arrest. In addition, 13 possessed acceptable bioavailability (36%) and effectively suppressed the tumor growth in GIST-T1 cell inoculated xenograft model without apparent toxicity. 13 currently is undergoing extensive preclinical evaluation and might be a potential drug candidate for GISTs. PMID:27077705

  11. Synthesis and biological evaluation of analogues of the kinase inhibitor nilotinib as Abl and Kit inhibitors

    PubMed Central

    Duveau, Damien Y.; Hu, Xin; Walsh, Martin J.; Shukla, Suneet; Skoumbourdis, Amanda P.; Boxer, Matthew B.; Ambudkar, Suresh V.; Shen, Min; Thomas, Craig J.

    2013-01-01

    The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these analogues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a–c to the binding pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the stabilizing effect from its juxtamembrane region. PMID:23273517

  12. The effects of metal ion PCR inhibitors on results obtained with the Quantifiler(®) Human DNA Quantification Kit.

    PubMed

    Combs, Laura Gaydosh; Warren, Joseph E; Huynh, Vivian; Castaneda, Joanna; Golden, Teresa D; Roby, Rhonda K

    2015-11-01

    targets human DNA in the Quantifiler(®) kit. Evidence of inhibition was observed for the human-specific assay at a lower metal concentration than detected by the IPC, for all metals examined except calcium. These results strongly suggest that determination of a "true negative" sample should not be based solely on the failure of the IPC to indicate the presence of a PCR inhibitor and indicate that amplification of all samples should be attempted, regardless of the quantification results.

  13. Efficient c-kit Receptor-Targeted Gene Transfer to Primary Human CD34-Selected Hematopoietic Stem Cells

    PubMed Central

    Zhong, Qiu; Oliver, Peter; Huang, Weitao; Good, David; La Russa, Vincent; Zhang, Zili; Cork, John R.; Veith, Robert Woody; Theodossiou, Chris; Kolls, Jay K.; Schwarzenberger, Paul

    2001-01-01

    We have previously reported effective gene transfer with a targeted molecular conjugate adenovirus vector through the c-kit receptor in hematopoietic progenitor cell lines. However, a c-kit-targeted recombinant retroviral vector failed to transduce cells, indicating the existence of significant differences for c-kit target gene transfer between these two viruses. Here we demonstrate that conjugation of an adenovirus to a c-kit-retargeted retrovirus vector enables retroviral transduction. This finding suggests the requirement of endosomalysis for successful c-kit-targeted gene transfer. Furthermore, we show efficient gene transfer to, and high transgene expression (66%) in, CD34-selected, c-kit+ human peripheral blood stem cells using a c-kit-targeted adenovirus vector. These findings may have important implications for future vector development in c-kit-targeted stem cell gene transfer. PMID:11581407

  14. Intranasal sirna targeting c-kit reduces airway inflammation in experimental allergic asthma.

    PubMed

    Wu, Wei; Chen, Hui; Li, Ya-Ming; Wang, Sheng-Yu; Diao, Xin; Liu, Kai-Ge

    2014-01-01

    Allergic asthma is characterized by airway inflammation caused by infiltration and activation of inflammatory cells that produce cytokines. Many studies have revealed that c-kit, a proto-oncogene, and its ligand, stem cell factor (SCF), play an important role in the development of asthmatic inflammation. Intranasal small interference RNA (siRNA) nanoparticles targeting specific viral gene could inhibit airway inflammation. In this study, we assessed whether silencing of c-kit with intranasal small interference RNA could reduce inflammation in allergic asthma. A mouse model of experimental asthma was treated with intranasal administration of anti-c-kit siRNA to inhibit the expression of the c-kit gene. We assessed the inflammatory response in both anti-c-kit siRNA-treated and control mice. Local administration of siRNA effectively inhibited the expression of the c-kit gene and reduced airway mucus secretion and the infiltration of eosinophils in bronchoalveolar lavage fluid. Moreover, c-kit siRNA reduced the production of SCF, interleukin-4 (IL-4), and IL-5, but had no effect on interferon-γ (IFN-γ) generation. These results show that intranasal siRNA nanoparticles targeting c-kit can decrease the inflammatory response in experimental allergic asthma.

  15. Discovery of Aryl Aminoquinazoline Pyridones as Potent, Selective, and Orally Efficacious Inhibitors of Receptor Tyrosine Kinase c-Kit

    SciTech Connect

    Hu, Essa; Tasker, Andrew; White, Ryan D.; Kunz, Roxanne K.; Human, Jason; Chen, Ning; Bürli, Roland; Hungate, Randall; Novak, Perry; Itano, Andrea; Zhang, Xuxia; Yu, Violeta; Nguyen, Yen; Tudor, Yanyan; Plant, Matthew; Flynn, Shaun; Xu, Yang; Meagher, Kristin L.; Whittington, Douglas A.; Ng, Gordon Y.

    2008-12-09

    Inhibition of c-Kit has the potential to treat mast cell associated fibrotic diseases. We report the discovery of several aminoquinazoline pyridones that are potent inhibitors of c-Kit with greater than 200-fold selectivity against KDR, p38, Lck, and Src. In vivo efficacy of pyridone 16 by dose-dependent inhibition of histamine release was demonstrated in a rodent pharmacodynamic model of mast cell activation.

  16. 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

  17. [KIT and KIT: from biology to clinical use].

    PubMed

    Curtit, Elsa; Mansi, Laura; Viel, Erika; Dobi, Erion; Chaigneau, Loïc; Nguyen, Thierry; Pivot, Xavier; Blay, Jean-Yves; Kalbacher, Elsa

    2012-02-01

    Scientific knowledge on gastrointestinal stromal tumors (GIST) has highly progressed over the last 10 years. The molecular bases of oncogenic transformation, KIT activating mutations, were identified in 1998 by Hirota et al. The product of KIT proto-oncogene, KIT protein, is a transmembrane receptor with tyrosine kinase activity. Tyrosine kinase inhibitors targeting these mutated activated kinases, namely imatinib and more recently sunitinib, nilotinib, masitinib or sorafenib, have deeply modified GIST prognosis. Molecular biology in GIST is now becoming a routine tool for treatment selection. In patients with advanced GIST, imatinib should be given until progression, and then, other tyrosine kinase inhibitors targeting KIT should be used. In the adjuvant setting, the optimal duration of imatinib treatment remains unknown.

  18. In silico exploration of c-KIT inhibitors by pharmaco-informatics methodology: pharmacophore modeling, 3D QSAR, docking studies, and virtual screening.

    PubMed

    Chaudhari, Prashant; Bari, Sanjay

    2016-02-01

    c-KIT is a component of the platelet-derived growth factor receptor family, classified as type-III receptor tyrosine kinase. c-KIT has been reported to be involved in, small cell lung cancer, other malignant human cancers, and inflammatory and autoimmune diseases associated with mast cells. Available c-KIT inhibitors suffer from tribulations of growing resistance or cardiac toxicity. A combined in silico pharmacophore and structure-based virtual screening was performed to identify novel potential c-KIT inhibitors. In the present study, five molecules from the ZINC database were retrieved as new potential c-KIT inhibitors, using Schrödinger's Maestro 9.0 molecular modeling suite. An atom-featured 3D QSAR model was built using previously reported c-KIT inhibitors containing the indolin-2-one scaffold. The developed 3D QSAR model ADHRR.24 was found to be significant (R2 = 0.9378, Q2 = 0.7832) and instituted to be sufficiently robust with good predictive accuracy, as confirmed through external validation approaches, Y-randomization and GH approach [GH score 0.84 and Enrichment factor (E) 4.964]. The present QSAR model was further validated for the OECD principle 3, in that the applicability domain was calculated using a "standardization approach." Molecular docking of the QSAR dataset molecules and final ZINC hits were performed on the c-KIT receptor (PDB ID: 3G0E). Docking interactions were in agreement with the developed 3D QSAR model. Model ADHRR.24 was explored for ligand-based virtual screening followed by in silico ADME prediction studies. Five molecules from the ZINC database were obtained as potential c-KIT inhibitors with high in -silico predicted activity and strong key binding interactions with the c-KIT receptor.

  19. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    PubMed Central

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess, whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  20. Discovery of N-((1-(4-(3-(3-((6,7-Dimethoxyquinolin-3-yl)oxy)phenyl)ureido)-2-(trifluoromethyl)phenyl)piperidin-4-yl)methyl)propionamide (CHMFL-KIT-8140) as a Highly Potent Type II Inhibitor Capable of Inhibiting the T670I "Gatekeeper" Mutant of cKIT Kinase.

    PubMed

    Li, Binhua; Wang, Aoli; Liu, Juan; Qi, Ziping; Liu, Xiaochuan; Yu, Kailin; Wu, Hong; Chen, Cheng; Hu, Chen; Wang, Wenchao; Wu, Jiaxin; Hu, Zhenquan; Ye, Ling; Zou, Fengming; Liu, Feiyang; Wang, Beilei; Wang, Li; Ren, Tao; Zhang, Shaojuan; Bai, Mingfeng; Zhang, Shanchun; Liu, Jing; Liu, Qingsong

    2016-09-22

    cKIT kinase inhibitors, e.g., imatinib, could induce drug-acquired mutations such as cKIT T670I that rendered drug resistance after chronic treatment. Through a type II kinase inhibitor design approach we discovered a highly potent type II cKIT kinase inhibitor compound 35 (CHMFL-KIT-8140), which potently inhibited both cKIT wt (IC50 = 33 nM) and cKIT gatekeeper T670I mutant (IC50 = 99 nM). Compound 35 displayed strong antiproliferative effect against GISTs cancer cell lines GIST-T1 (cKIT wt, GI50 = 4 nM) and GIST-5R (cKIT T670I, GI50 = 26 nM). In the cellular context it strongly inhibited c-KIT mediated signaling pathways and induced apoptosis. In the BaF3-TEL-cKIT-T670I isogenic cell inoculated xenograft mouse model, 35 exhibited dose dependent tumor growth suppression efficacy and 100 mg/kg dosage provided 47.7% tumor growth inhibition (TGI) without obvious toxicity. We believe compound 35 would be a good pharmacological tool for exploration of the cKIT-T670I mutant mediated pathology in GISTs. PMID:27545040

  1. Development and performance of a targeted whole exome sequencing enrichment kit for the dog (Canis Familiaris Build 3.1).

    PubMed

    Broeckx, Bart J G; Coopman, Frank; Verhoeven, Geert E C; Bavegems, Valérie; De Keulenaer, Sarah; De Meester, Ellen; Van Niewerburgh, Filip; Deforce, Dieter

    2014-01-01

    Whole exome sequencing is a technique that aims to selectively sequence all exons of protein-coding genes. A canine whole exome sequencing enrichment kit was designed based on the latest canine reference genome (build 3.1.72). Its performance was tested by sequencing 2 exome captures, each consisting of 4 pre-capture pooled, barcoded Illumina libraries on an Illumina HiSeq 2500. At an average sequencing depth of 102x, 83 to 86% of the target regions were completely sequenced with a minimum coverage of five and 90% of the reads mapped on the target regions. Additionally, it is shown that the reproducibility within and between captures is high and that pooling four samples per capture is a valid option. Overall, we have demonstrated the strong performance of this WES enrichment kit and are confident it will be a valuable tool in future disease association studies.

  2. Female Adnexal Tumor of Probable Wolffian Origin (FATWO) With Recurrence 3 Years Postsurgery: C-kit Gene Analysis and a Possibility of a New Molecular Targeted Therapy

    PubMed Central

    Syriac, Susanna; Durie, Nicole; Kesterson, Joshua; Lele, Shashrikant; Mhawech-Fauceglia, Paulette

    2016-01-01

    Summary This is the case report of a 38-year-old woman who presented with a mass of the right broad ligament that was diagnosed as a female adnexal tumor of probable Wollfian origin (FATWO). The patient was treated with a simple mass excision. Three years after the excision, the patient presented with uterine bleeding. A total abdominal hysterectomy was advised. Intraoperative histologic consultation showed a poorly differentiated tumor on the surface of the left ovary. After extensive immunohistochemistry analysis and after reviewing the histology slides from the primary tumor, the final diagnosis was concluded to be recurrent FATWO on the surface of the ovary. C-kit immunohistochemistry was found to be strongly positive. Polymerase chain reaction amplification of C-kit genes on exons 9, 11, 13, and 17 and of PDGFR gene on exons 12 and 18 showed no mutational changes. Owing to the limited options in treating recurrent disease and the lack of prognostic factors for recurrence or metastasis, the patient was started on 400 mg of imatinib mesylate therapy for 6 months. In addition, the patient is undergoing continuous follow-up by computed tomographic imaging every 6 months. As chemotherapy and radiation therapy for recurrent or metastatic FATWO are most often unsuccessful, a molecular targeted therapy, such as tyrosine kinase inhibitor, could be considered. However, collective data are needed from multiple centers to determine its effectiveness in these patients. PMID:21464731

  3. Targeting cancer with small-molecular-weight kinase inhibitors.

    PubMed

    Fabbro, Doriano; Cowan-Jacob, Sandra W; Möbitz, Henrik; Martiny-Baron, Georg

    2012-01-01

    Protein and lipid kinases fulfill essential roles in many signaling pathways that regulate normal cell functions. Deregulation of these kinase activities lead to a variety of pathologies ranging from cancer to inflammatory diseases, diabetes, infectious diseases, cardiovascular disorders, cell growth and survival. 518 protein kinases and about 20 lipid-modifying kinases are encoded by the human genome, and a much larger proportion of additional kinases are present in parasite, bacterial, fungal, and viral genomes that are susceptible to exploitation as drug targets. Since many human diseases result from overactivation of protein and lipid kinases due to mutations and/or overexpression, this enzyme class represents an important target for the pharmaceutical industry. Approximately one third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases.The kinase inhibitors that have been launched, thus far, are mainly in oncology indications and are directed against a handful of protein and lipid kinases. With one exception, all of these registered kinase inhibitors are directed toward the ATP-site and display different selectivities, potencies, and pharmacokinetic properties. At present, about 150 kinase-targeted drugs are in clinical development and many more in various stages of preclinical development. Kinase inhibitor drugs that are in clinical trials target all stages of signal transduction from the receptor protein tyrosine kinases that initiate intracellular signaling, through second-messenger-dependent lipid and protein kinases, and protein kinases that regulate the cell cycle.This review provides an insight into protein and lipid kinase drug discovery with respect to achievements, binding modes of inhibitors, and novel avenues for the generation of second-generation kinase inhibitors to treat cancers.

  4. Novel bone-targeted Src tyrosine kinase inhibitor drug discovery.

    PubMed

    Shakespeare, William C; Metcalf, Chester A; Wang, Yihan; Sundaramoorthi, Raji; Keenan, Terence; Weigele, Manfred; Bohacek, Regine S; Dalgarno, David C; Sawyer, Tomi K

    2003-09-01

    Bone-targeted Src tyrosine kinase (STK) inhibitors have recently been developed for the treatment of osteoporosis and cancer-related bone diseases. The concept of bone targeting derives from bisphosphonates, and from the evolution of such molecules in terms of therapeutic efficacy for the treatment of bone disorders. Interestingly, some of the earliest bisphosphonates were recognized for their ability to inhibit calcium carbonate precipitation (scaling) by virtue of their affinity to chelate calcium. This chelating property was subsequently exploited in the development of bisphosphonate analogs as inhibitors of the bone-resorbing cells known as osteoclasts, giving rise to breakthrough medicines, such as Fosamax (for the treatment of osteoporosis) and Zometa (for the treatment of osteoporosis and bone metastases). Relative to these milestone achievements, there is a tremendous opportunity to explore beyond the limited chemical space (functional group diversity) of such bisphosphonates to design novel bone-targeting moieties, which may be used to develop other classes of promising small-molecule drugs affecting different biological pathways. Here, we review studies focused on bone-targeted inhibitors of STK, a key enzyme in osteoclast-dependent bone resorption. Two strategies are described relative to bone-targeted STK inhibitor drug discovery: (i) the development of novel Src homology (SH)-2 inhibitors incorporating non-hydrolyzable phosphotyrosine mimics and exhibiting molecular recognition and bone-targeting properties, leading to the in vivo-effective lead compound AP-22408; and (ii) the development of novel ATP-based Src kinase inhibitors incorporating bone-targeting moieties, leading to the in vivo-effective lead compound AP-23236. In summary, AP-22408 and AP-23236, which differ mechanistically by virtue of blocking Src-dependent non-catalytic or catalytic activities in osteoclasts, exemplify ARIAD Pharmaceuticals' structure-based design of novel bone-targeted

  5. Targeting the inhibitor of Apoptosis Protein BIR3 binding domains.

    PubMed

    Jaquith, James B

    2014-05-01

    The Inhibitor of Apoptosis Proteins (IAPs) play a critical role in the regulation of cellular apoptosis and cytokine signaling. IAP family members include XIAP, cIAP1, cIAP2, NAIP, survivin, Apollon/Bruce, ML-IAP/livin and TIAP. The IAPs have been targeted using both antisense oligonucleotides and small molecule inhibitors. Several research teams have advanced compounds that bind the highly conserved BIR3 domains of the IAPs into clinical trials, as single agents and in combination with standard of care. This patent review highlights the medicinal chemistry strategies that have been applied to the development of clinical compounds. PMID:24998289

  6. A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth.

    PubMed

    Lebron, Maria B; Brennan, Laura; Damoci, Christopher B; Prewett, Marie C; O'Mahony, Marguerita; Duignan, Inga J; Credille, Kelly M; DeLigio, James T; Starodubtseva, Marina; Amatulli, Michael; Zhang, Yiwei; Schwartz, Kaben D; Burtrum, Douglas; Balderes, Paul; Persaud, Kris; Surguladze, David; Loizos, Nick; Paz, Keren; Kotanides, Helen

    2014-09-01

    Stem cell factor receptor (c-Kit) exerts multiple biological effects on target cells upon binding its ligand stem cell factor (SCF). Aberrant activation of c-Kit results in dysregulated signaling and is implicated in the pathogenesis of numerous cancers. The development of more specific and effective c-Kit therapies is warranted given its essential role in tumorigenesis. In this study, we describe the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds c-Kit receptor with high affinity (EC 50 = 0.06 nM) and strongly blocks its interaction with SCF (IC 50 = 0.41 nM) in solid phase assays. Flow cytometry shows CK6 binding to c-Kit on the cell surface of human small cell lung carcinoma (SCLC), melanoma, and leukemia tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways such as mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), in addition to reducing tumor cell line growth in vitro. CK6 treatment significantly decreases human xenograft tumor growth in NCI-H526 SCLC (T/C% = 57) and Malme-3M melanoma (T/C% = 58) models in vivo. The combination of CK6 with standard of care chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, more potently reduces tumor growth (SCLC T/C% = 24, melanoma T/C% = 38) compared with CK6 or chemotherapy alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human malignancies harboring c-Kit receptor. PMID:24921944

  7. PLK-1 Targeted Inhibitors and Their Potential against Tumorigenesis

    PubMed Central

    Kumar, Shiv; Kim, Jaebong

    2015-01-01

    Mitotic kinases are the key components of the cell cycle machinery and play vital roles in cell cycle progression. PLK-1 (Polo-like kinase-1) is a crucial mitotic protein kinase that plays an essential role in both the onset of G2/M transition and cytokinesis. The overexpression of PLK-1 is strongly correlated with a wide spectrum of human cancers and poor prognosis. The (si)RNA-mediated depletion of PLK-1 arrests tumor growth and triggers apoptosis in cancer cells without affecting normal cells. Therefore, PLK-1 has been selected as an attractive anticancer therapeutic drug target. Some small molecules have been discovered to target the catalytic and noncatalytic domains of PLK-1. These domains regulate the catalytic activation and subcellular localization of PLK-1. However, while PLK-1 inhibitors block tumor growth, they have been shown to cause severe adverse complications, such as toxicity, neutropenia, and bone marrow suppression during clinical trials, due to a lack of selectivity and specificity within the human kinome. To minimize these toxicities, inhibitors should be tested against all protein kinases in vivo and in vitro to enhance selectivity and specificity against targets. Here, we discuss the potency and selectivity of PLK-1-targeted inhibitors and their molecular interactions with PLK-1 domains. PMID:26557691

  8. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  9. Inhibitors of apoptotic proteins: new targets for anticancer therapy.

    PubMed

    Saleem, Mohammad; Qadir, Muhammad Imran; Perveen, Nadia; Ahmad, Bashir; Saleem, Uzma; Irshad, Tehseen; Ahmad, Bashir

    2013-09-01

    Inhibitors of apoptotic proteins (IAPs) can play an important role in inhibiting apoptosis by exerting their negative action on caspases (apoptotic proteins). There are eight proteins in this family: NAIP/BIRC1/NLRB, cellular IAP1 (cIAP1)/human IAP2/BIRC2, cellular IAP2 (cIAP2)/human IAP1/BIRC3, X-linked IAP (XIAP)/BIRC4, survivin/BIRC5, baculoviral IAP repeat (BIR)-containing ubiquitin-conjugating enzyme/apollon/BIRC6, livin/melanoma-IAP (ML-IAP)/BIRC7/KIAP, and testis-specific IAP (Ts-IAP)/hILP-2/BIRC8. Deregulation of these inhibitors of apoptotic proteins (IAPs) may push cell toward cancer and neurodegenerative disorders. Inhibitors of apoptotic proteins (IAPs) may provide new target for anticancer therapy. Drugs may be developed that are inhibiting these IAPs to induce apoptosis in cancerous cells.

  10. Inhibitors of apoptotic proteins: new targets for anticancer therapy.

    PubMed

    Saleem, Mohammad; Qadir, Muhammad Imran; Perveen, Nadia; Ahmad, Bashir; Saleem, Uzma; Irshad, Tehseen; Ahmad, Bashir

    2013-09-01

    Inhibitors of apoptotic proteins (IAPs) can play an important role in inhibiting apoptosis by exerting their negative action on caspases (apoptotic proteins). There are eight proteins in this family: NAIP/BIRC1/NLRB, cellular IAP1 (cIAP1)/human IAP2/BIRC2, cellular IAP2 (cIAP2)/human IAP1/BIRC3, X-linked IAP (XIAP)/BIRC4, survivin/BIRC5, baculoviral IAP repeat (BIR)-containing ubiquitin-conjugating enzyme/apollon/BIRC6, livin/melanoma-IAP (ML-IAP)/BIRC7/KIAP, and testis-specific IAP (Ts-IAP)/hILP-2/BIRC8. Deregulation of these inhibitors of apoptotic proteins (IAPs) may push cell toward cancer and neurodegenerative disorders. Inhibitors of apoptotic proteins (IAPs) may provide new target for anticancer therapy. Drugs may be developed that are inhibiting these IAPs to induce apoptosis in cancerous cells. PMID:23790005

  11. From polypharmacology to target specificity: the case of PARP inhibitors.

    PubMed

    Liscio, Paride; Camaioni, Emidio; Carotti, Andrea; Pellicciari, Roberto; Macchiarulo, Antonio

    2013-01-01

    Poly(ADP-ribose)polymerases (PARPs) catalyze a post-transcriptional modification of proteins, consisting in the attachment of mono, oligo or poly ADP-ribose units from NAD+ to specific polar residues of target proteins. The scientific interest in members of this superfamily of enzymes is continuously growing since they have been implicated in a range of diseases including stroke, cardiac ischemia, cancer, inflammation and diabetes. Despite some inhibitors of PARP-1, the founder member of the superfamily, have advanced in clinical trials for cancer therapy, and other members of PARPs have recently been proposed as interesting drug targets, challenges exist in understanding the polypharmacology of current PARP inhibitors as well as developing highly selective chemical tools to unravel specific functions of each member of the superfamily. Beginning with an overview on the molecular aspects that affect polypharmacology, in this article we discuss how these may have an impact on PARP research and drug discovery. Then, we review the most selective PARP inhibitors hitherto reported in literature, giving an update on the molecular aspects at the basis of selective PARP inhibitor design. Finally, some outlooks on current issues and future directions in this field of research are also provided.

  12. From polypharmacology to target specificity: the case of PARP inhibitors.

    PubMed

    Liscio, Paride; Camaioni, Emidio; Carotti, Andrea; Pellicciari, Roberto; Macchiarulo, Antonio

    2013-01-01

    Poly(ADP-ribose)polymerases (PARPs) catalyze a post-transcriptional modification of proteins, consisting in the attachment of mono, oligo or poly ADP-ribose units from NAD+ to specific polar residues of target proteins. The scientific interest in members of this superfamily of enzymes is continuously growing since they have been implicated in a range of diseases including stroke, cardiac ischemia, cancer, inflammation and diabetes. Despite some inhibitors of PARP-1, the founder member of the superfamily, have advanced in clinical trials for cancer therapy, and other members of PARPs have recently been proposed as interesting drug targets, challenges exist in understanding the polypharmacology of current PARP inhibitors as well as developing highly selective chemical tools to unravel specific functions of each member of the superfamily. Beginning with an overview on the molecular aspects that affect polypharmacology, in this article we discuss how these may have an impact on PARP research and drug discovery. Then, we review the most selective PARP inhibitors hitherto reported in literature, giving an update on the molecular aspects at the basis of selective PARP inhibitor design. Finally, some outlooks on current issues and future directions in this field of research are also provided. PMID:24171773

  13. Targeting histone deacetylase inhibitors for anti-malarial therapy.

    PubMed

    Andrews, Katherine T; Tran, Thanh N; Wheatley, Nicole C; Fairlie, David P

    2009-01-01

    It is now clear that histone acetylation plays key roles in regulating gene transcription in both eukaryotes and prokaryotes, the acetylated form inducing gene expression while deacetylation silences genes. Recent studies have identified roles for histone acetyltransferases (HATs) and/or histone deacetylases (HDACs) in a number of parasites including Entamoeba histolytica, Toxoplasma gondii, Schistosoma mansoni, Cryptosporidium sp., Leishmania donovani, Neospora caninum, and Plasmodium falciparum. Here we survey fairly limited efforts to date in profiling antimalarial activities of HDAC inhibitors, showing that such compounds are potent inhibitors of the growth of P. falciparum in vitro and in vivo. Most of the compounds evaluated so far have borne a zinc-binding hydroxamate group that tends to be metabolized in vivo, and thus new zinc-binding groups need to be incorporated into second generation inhibitors in order to mask the catalytic zinc in the active site of HDACs. Also the development of compounds that are selective for parasitic HDACs over mammalian HDACs is still in relative infancy and it will take some time to derive antiparasitic HDAC inhibitor compounds with minimal toxicity for the host and acceptable pharmacokinetic and pharmacodynamic profiles for human treatment. Nevertheless, results to date suggest that HDAC inhibitor development represents a promising new approach to the potential treatment of parasitic infections, including those induced by malaria protozoa, and may offer new therapeutic targets within increasingly drug-resistant malarial parasites. PMID:19355992

  14. New Targets and Inhibitors of Mycobacterial Sulfur Metabolism§

    PubMed Central

    Paritala, Hanumantharao; Carroll, Kate S.

    2015-01-01

    The identification of new antibacterial targets is urgently needed to address multidrug resistant and latent tuberculosis infection. Sulfur metabolic pathways are essential for survival and the expression of virulence in many pathogenic bacteria, including Mycobacterium tuberculosis. In addition, microbial sulfur metabolic pathways are largely absent in humans and therefore, represent unique targets for therapeutic intervention. In this review, we summarize our current understanding of the enzymes associated with the production of sulfated and reduced sulfur-containing metabolites in Mycobacteria. Small molecule inhibitors of these catalysts represent valuable chemical tools that can be used to investigate the role of sulfur metabolism throughout the Mycobacterial lifecycle and may also represent new leads for drug development. In this light, we also summarize recent progress made in the development of inhibitors of sulfur metabolism enzymes. PMID:23808874

  15. 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

  16. A New Staple: Peptide-Targeted Covalent Inhibitors.

    PubMed

    Leverson, Joel D

    2016-09-22

    In this issue of Cell Chemical Biology, Huhn et al. (2016) unveil a clever strategy for selectively and irreversibly inhibiting an anti-apoptotic protein, BFL-1. The authors describe stapled peptides bearing carefully placed electrophiles that target a unique cysteine residue in BFL-1 via covalent modification, thus representing an extension of the stapled peptide concept into the covalent inhibitor space. PMID:27662249

  17. Slip Kits.

    ERIC Educational Resources Information Center

    Coombes, S. D.

    1979-01-01

    Discusses the process of developing the Science Lessons from Industrial Processes (SLIP) kits by 16 British science teachers. The content, applicability, and components of these kits (based upon local industries) are also included. (HM)

  18. Mammalian Target of Rapamycin Inhibitors and Nephrotoxicity: Fact or Fiction.

    PubMed

    Barbari, Antoine; Maawad, Maria; Kfoury Kassouf, Hala; Kamel, Gaby

    2015-10-01

    Mammalian target of rapamycin inhibitors, such as rapamycin and more recently everolimus, have substituted calcineurin inhibitors in many minimization strategies. Despite their acclaimed renal safety profile, several lines of evidence are emerging on their potential nephrotoxic effect. Predisposing conditions for nephrotoxicity involve a complex interplay between several environmental and genetic factors in the donor-recipient pair. Renal injury may be enhanced by pharmacodynamic interactions when combined with other drugs such as calcineurin inhibitors or nutrients that are predominantly related to an increase in local tissue exposure. These toxic interactions may occur within adequate doses and therapeutic blood levels. This explains the occurrence of nephrotoxicity in some but not all cases. Here, we postulated that activity of a low permeability glycoprotein efflux pump related to low protein expression and/or inhibition enhanced immunosuppressive drug entry in different cells. A rise in intracellular drug concentration increases bioactivity, leading to greater immunosuppression and more immune-related, nonrenal adverse events in the recipient and increased nephrotoxicity in the kidney graft. Under specific isolated or combined environmental and/or genetic conditions in both the recipient and donor affecting the glycoprotein efflux pump and/or the mammalian target of rapamycin pathway, these renal injuries may be aggravated by heightened drug tissue concentrations despite adherence to therapeutic drug and blood levels. Mammalian target of rapamycin inhibitors may induce predominantly a dose-dependent renal epithelial cell injury affecting either the glomerular or the renal tubular epithelial cells, leading to cell death and apoptosis. Epithelial mesenchymal transition mediated interstitial fibrosis and tubular atrophy observed with these drugs may be the result of a cumulative toxic renal tubular injury induced by the direct insult of the drug itself and

  19. Targeting inhibitor of apoptosis proteins (IAPs) for cancer therapy.

    PubMed

    Fulda, Simone

    2008-06-01

    Since cell death by apoptosis plays a key role in the regulation of tissue homeostasis, dysregulation of the cell's intrinsic death program may foster tumor formation and progression. "Inhibitor of apoptosis proteins" (IAPs) block apoptosis at the core of the apoptotic machinery by inhibiting effector caspases. Aberrant expression and/or function of IAPs are found in many human cancers and have been implied in resistance to current treatment approaches. Recent insights into the role of IAPs have provided the basis for various exciting discoveries that aim at modulating expression or function of IAPs. Thus, targeting IAPs, e.g. by antisense approaches or small molecule inhibitors, presents a promising novel approach for future drug development and may proof to be a successful strategy to overcome apoptosis resistance of human cancers.

  20. Targeting mitochondrial biogenesis to overcome drug resistance to MAPK inhibitors

    PubMed Central

    Zhang, Gao; Frederick, Dennie T.; Wu, Lawrence; Wei, Zhi; Krepler, Clemens; Srinivasan, Satish; Chae, Young Chan; Xu, Xiaowei; Choi, Harry; Dimwamwa, Elaida; Shannan, Batool; Basu, Devraj; Zhang, Dongmei; Guha, Manti; Xiao, Min; Randell, Sergio; Sproesser, Katrin; Xu, Wei; Liu, Jephrey; Karakousis, Giorgos C.; Schuchter, Lynn M.; Gangadhar, Tara C.; Amaravadi, Ravi K.; Gu, Mengnan; Xu, Caiyue; Ghosh, Abheek; Xu, Weiting; Tian, Tian; Zhang, Jie; Zha, Shijie; Brafford, Patricia; Weeraratna, Ashani; Davies, Michael A.; Wargo, Jennifer A.; Avadhani, Narayan G.; Lu, Yiling; Mills, Gordon B.; Altieri, Dario C.; Flaherty, Keith T.

    2016-01-01

    Targeting multiple components of the MAPK pathway can prolong the survival of patients with BRAFV600E melanoma. This approach is not curative, as some BRAF-mutated melanoma cells are intrinsically resistant to MAPK inhibitors (MAPKi). At the systemic level, our knowledge of how signaling pathways underlie drug resistance needs to be further expanded. Here, we have shown that intrinsically resistant BRAF-mutated melanoma cells with a low basal level of mitochondrial biogenesis depend on this process to survive MAPKi. Intrinsically resistant cells exploited an integrated stress response, exhibited an increase in mitochondrial DNA content, and required oxidative phosphorylation to meet their bioenergetic needs. We determined that intrinsically resistant cells rely on the genes encoding TFAM, which controls mitochondrial genome replication and transcription, and TRAP1, which regulates mitochondrial protein folding. Therefore, we targeted mitochondrial biogenesis with a mitochondrium-targeted, small-molecule HSP90 inhibitor (Gamitrinib), which eradicated intrinsically resistant cells and augmented the efficacy of MAPKi by inducing mitochondrial dysfunction and inhibiting tumor bioenergetics. A subset of tumor biopsies from patients with disease progression despite MAPKi treatment showed increased mitochondrial biogenesis and tumor bioenergetics. A subset of acquired drug-resistant melanoma cell lines was sensitive to Gamitrinib. Our study establishes mitochondrial biogenesis, coupled with aberrant tumor bioenergetics, as a potential therapy escape mechanism and paves the way for a rationale-based combinatorial strategy to improve the efficacy of MAPKi. PMID:27043285

  1. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers

    PubMed Central

    Goda, Jayant S.; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-01-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies. PMID:27121513

  2. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

    PubMed

    Goda, Jayant S; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-02-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  3. Targeting PTPs with small molecule inhibitors in cancer treatment

    PubMed Central

    Jiang, Zhong-Xing; Zhang, Zhong-Yin

    2009-01-01

    Protein tyrosine phosphorylation plays a major role in cellular signaling. The level of tyrosine phosphorylation is controlled by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Disturbance of the normal balance between PTK and PTP activity results in aberrant tyrosine phosphorylation, which has been linked to the etiology of several human diseases, including cancer. A number of PTPs have been implicated in oncogenesis and tumor progression and therefore are potential drug targets for cancer chemotherapy. These include PTP1B, which may augment signaling downstream of HER2/Neu; SHP2, which is the first oncogene in the PTP superfamily and is essential for growth factor-mediated signaling; the Cdc25 phosphatases, which are positive regulators of cell cycle progression; and the PRL phosphatases, which promote tumor metastases. As PTPs have emerged as drug targets for cancer, a number of strategies are currently been explored for the identification of various classes of PTP inhibitors. These efforts have resulted many potent, and in some cases selective, inhibitors for PTP1B, SHP2, Cdc25 and PRL phosphatases. Structural information derived from these compounds serves as a solid foundation upon which novel anti-cancer agents targeted to these PTPs can be developed. PMID:18259840

  4. Molecular drug targets in myeloproliferative neoplasms: mutant ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB and FGFR1

    PubMed Central

    Tefferi, Ayalew

    2009-01-01

    Abstract Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPN) include BCR-ABL1 and rearranged PDGFR proteins. The latter are products of intra- (e.g. FIP1L1-PDGFRA) or inter-chromosomal (e.g.ETV6-PDGFRB) gene fusions. BCR-ABL1 is associated with chronic myelogenous leukaemia (CML) and mutant PDGFR with an MPN phenotype characterized by eosinophilia and in addition, in case of FIP1L1-PDGFRA, bone marrow mastocytosis. These genotype-phenotype associations have been effectively exploited in the development of highly accurate diagnostic assays and molecular targeted therapy. It is hoped that the same will happen in other MPN with specific genetic alterations: polycythemia vera (JAK2V617F and other JAK2 mutations), essential thrombocythemia (JAK2V617F and MPL515 mutations), primary myelofibrosis (JAK2V617F and MPL515 mutations), systemic mastocytosis (KITD816V and other KIT mutations) and stem cell leukaemia/lymphoma (ZNF198-FGFR1 and other FGFR1 fusion genes). The current review discusses the above-listed mutant molecules in the context of their value as drug targets. PMID:19175693

  5. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    NASA Astrophysics Data System (ADS)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  6. TAK1 and IKK2, novel mediators of SCF-induced signaling and potential targets for c-Kit-driven diseases.

    PubMed

    Drube, Sebastian; Weber, Franziska; Göpfert, Christiane; Loschinski, Romy; Rothe, Mandy; Boelke, Franziska; Diamanti, Michaela A; Löhn, Tobias; Ruth, Julia; Schütz, Dagmar; Häfner, Norman; Greten, Florian R; Stumm, Ralf; Hartmann, Karin; Krämer, Oliver H; Dudeck, Anne; Kamradt, Thomas

    2015-10-01

    NF-κB activation depends on the IKK complex consisting of the catalytically active IKK1 and 2 subunits and the scaffold protein NEMO. Hitherto, IKK2 activation has always been associated with IκBα degradation, NF-κB activation, and cytokine production. In contrast, we found that in SCF-stimulated primary bone marrow-derived mast cells (BMMCs), IKK2 is alternatively activated. Mechanistically, activated TAK1 mediates the association between c-Kit and IKK2 and therefore facilitates the Lyn-dependent IKK2 activation which suffices to mediate mitogenic signaling but, surprisingly, does not result in NF-κB activation. Moreover, the c-Kit-mediated and Lyn-dependent IKK2 activation is targeted by MyD88-dependent pathways leading to enhanced IKK2 activation and therefore to potentiated effector functions. In neoplastic cells, expressing constitutively active c-Kit mutants, activated TAK1 and IKKs do also not induce NF-κB activation but mediate uncontrolled proliferation, resistance to apoptosis and enables IL-33 to mediate c-Kit-dependent signaling. Together, we identified the formation of the c-Kit-Lyn-TAK1 signalosome which mediates IKK2 activation. Unexpectedly, this IKK activation is uncoupled from the NF-κB-machinery but is critical to modulate functional cell responses in primary-, and mediates uncontrolled proliferation and survival of tumor-mast cells. Therefore, targeting TAK1 and IKKs might be a novel approach to treat c-Kit-driven diseases. PMID:26353931

  7. Benzothiazole and Pyrrolone Flavivirus Inhibitors Targeting the Viral Helicase

    PubMed Central

    Sweeney, Noreena L.; Hanson, Alicia M.; Mukherjee, Sourav; Ndjomou, Jean; Geiss, Brian J.; Steel, J. Jordan; Frankowski, Kevin J.; Li, Kelin; Schoenen, Frank J.; Frick, David N.

    2015-01-01

    The flavivirus nonstructural protein 3 (NS3) is a protease and helicase, and on the basis of its similarity to its homologue encoded by the hepatitis C virus (HCV), the flavivirus NS3 might be a promising drug target. Few flavivirus helicase inhibitors have been reported, in part, because few specific inhibitors have been identified when nucleic acid unwinding assays have been used to screen for helicase inhibitors. To explore the possibility that compounds inhibiting NS3-catalyzed ATP hydrolysis might function as antivirals even if they do not inhibit RNA unwinding in vitro, we designed a robust dengue virus (DENV) NS3 ATPase assay suitable for high-throughput screening. Members of two classes of inhibitory compounds were further tested in DENV helicase-catalyzed RNA unwinding assays, assays monitoring HCV helicase action, subgenomic DENV replicon assays, and cell viability assays and for their ability to inhibit West Nile virus (Kunjin subtype) replication in cells. The first class contained analogues of NIH molecular probe ML283, a benzothiazole oligomer derived from the dye primuline, and they also inhibited HCV helicase and DENV NS3-catalyzed RNA unwinding. The most intriguing ML283 analogue inhibited DENV NS3 with an IC50 value of 500 nM and was active against the DENV replicon. The second class contained specific DENV ATPase inhibitors that did not inhibit DENV RNA unwinding or reactions catalyzed by HCV helicase. Members of this class contained a 4-hydroxy-3-(5-methylfuran-2-carbonyl)-2H-pyrrol-5-one scaffold, and about 20 μM of the most potent pyrrolone inhibited both DENV replicons and West Nile virus replication in cells by 50%. PMID:26029739

  8. 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

  9. Methionine AminoPeptidase Type-2 Inhibitors Targeting Angiogenesis.

    PubMed

    Ehlers, Tedman; Furness, Scott; Robinson, Thomas Philip; Zhong, Haizhen A; Goldsmith, David; Aribser, Jack; Bowen, J Phillip

    2016-01-01

    Angiogenesis has been identified as a crucial process in the development and spread of cancers. There are many regulators of angiogenesis which are not yet fully understood. Methionine aminiopeptidase is a metalloenzyme with two structurally distinct forms in humans, Type-1 (MetAP-1) and Type-2 (MetAP-2). It has been shown that small molecule inhibitors of MetAP-2 suppress endothelial cell proliferation. The initial discovery by Donald Ingber of MetAP-2 inhibition as a potential target in angiogenesis began with a fortuitous observation similar to the discovery of penicillin activity by Sir Alexander Fleming. From a drug design perspective, MetAP-2 is an attractive target. Fumagillin and ovalicin, known natural products, bind with IC50 values in low nanomolar concentrations. Crystal structures of the bound complexes provide 3-dimensional coordinates for advanced computational studies. More recent discoveries have shown other biological activities for MetAP-2 inhibition, which has generated new interests in the design of novel inhibitors. Semisynthetic fumagillin derivatives such as AGM-1470 (TNP-470) have been shown to have better drug properties, but have not been very successful in clinical trials. The rationale and development of novel multicyclic analogs of fumagillin are reviewed.

  10. Methionine AminoPeptidase Type-2 Inhibitors Targeting Angiogenesis.

    PubMed

    Ehlers, Tedman; Furness, Scott; Robinson, Thomas Philip; Zhong, Haizhen A; Goldsmith, David; Aribser, Jack; Bowen, J Phillip

    2016-01-01

    Angiogenesis has been identified as a crucial process in the development and spread of cancers. There are many regulators of angiogenesis which are not yet fully understood. Methionine aminiopeptidase is a metalloenzyme with two structurally distinct forms in humans, Type-1 (MetAP-1) and Type-2 (MetAP-2). It has been shown that small molecule inhibitors of MetAP-2 suppress endothelial cell proliferation. The initial discovery by Donald Ingber of MetAP-2 inhibition as a potential target in angiogenesis began with a fortuitous observation similar to the discovery of penicillin activity by Sir Alexander Fleming. From a drug design perspective, MetAP-2 is an attractive target. Fumagillin and ovalicin, known natural products, bind with IC50 values in low nanomolar concentrations. Crystal structures of the bound complexes provide 3-dimensional coordinates for advanced computational studies. More recent discoveries have shown other biological activities for MetAP-2 inhibition, which has generated new interests in the design of novel inhibitors. Semisynthetic fumagillin derivatives such as AGM-1470 (TNP-470) have been shown to have better drug properties, but have not been very successful in clinical trials. The rationale and development of novel multicyclic analogs of fumagillin are reviewed. PMID:26369821

  11. Discovery of Dual-Stage Malaria Inhibitors with New Targets

    PubMed Central

    Raphemot, Rene; Lafuente-Monasterio, Maria J.; Gamo-Benito, Francisco Javier; Clardy, Jon

    2015-01-01

    Malaria remains a major global health problem, with more than half of the world population at risk of contracting the disease and nearly a million deaths each year. Here, we report the discovery of inhibitors that target multiple stages of malaria parasite growth. To identify these inhibitors, we took advantage of the Tres Cantos Antimalarial Compound Set (TCAMS) small-molecule library, which is comprised of diverse and potent chemical scaffolds with activities against the blood stage of the malaria parasite, and investigated their effects against the elusive liver stage of the malaria parasite using a forward chemical screen. From a screen of nearly 14,000 compounds, we identified and confirmed 103 compounds as dual-stage malaria inhibitors. Interestingly, these compounds show preferential inhibition of parasite growth in liver- versus blood-stage malaria parasite assays, highlighting the drug susceptibility of this parasite form. Mode-of-action studies were completed using genetically modified and drug-resistant Plasmodium parasite strains. While we identified some compound targets as classical antimalarial pathways, such as the mitochondrial electron transport chain through cytochrome bc1 complex inhibition or the folate biosynthesis pathway, most compounds induced parasite death through as yet unknown mechanisms of action. Importantly, the identification of new chemotypes with different modes of action in killing Plasmodium parasites represents a promising opportunity for probing essential and novel molecular processes that remain to be discovered. The chemical scaffolds identified with activity against drug-resistant Plasmodium parasites represent starting points for dual-stage antimalarial development to surmount the threat of malaria parasite drug resistance. PMID:26666931

  12. Discovery of Dual-Stage Malaria Inhibitors with New Targets.

    PubMed

    Raphemot, Rene; Lafuente-Monasterio, Maria J; Gamo-Benito, Francisco Javier; Clardy, Jon; Derbyshire, Emily R

    2016-03-01

    Malaria remains a major global health problem, with more than half of the world population at risk of contracting the disease and nearly a million deaths each year. Here, we report the discovery of inhibitors that target multiple stages of malaria parasite growth. To identify these inhibitors, we took advantage of the Tres Cantos Antimalarial Compound Set (TCAMS) small-molecule library, which is comprised of diverse and potent chemical scaffolds with activities against the blood stage of the malaria parasite, and investigated their effects against the elusive liver stage of the malaria parasite using a forward chemical screen. From a screen of nearly 14,000 compounds, we identified and confirmed 103 compounds as dual-stage malaria inhibitors. Interestingly, these compounds show preferential inhibition of parasite growth in liver- versus blood-stage malaria parasite assays, highlighting the drug susceptibility of this parasite form. Mode-of-action studies were completed using genetically modified and drug-resistant Plasmodium parasite strains. While we identified some compound targets as classical antimalarial pathways, such as the mitochondrial electron transport chain through cytochrome bc1 complex inhibition or the folate biosynthesis pathway, most compounds induced parasite death through as yet unknown mechanisms of action. Importantly, the identification of new chemotypes with different modes of action in killing Plasmodium parasites represents a promising opportunity for probing essential and novel molecular processes that remain to be discovered. The chemical scaffolds identified with activity against drug-resistant Plasmodium parasites represent starting points for dual-stage antimalarial development to surmount the threat of malaria parasite drug resistance. PMID:26666931

  13. Brainstorming: weighted voting prediction of inhibitors for protein targets.

    PubMed

    Plewczynski, Dariusz

    2011-09-01

    The "Brainstorming" approach presented in this paper is a weighted voting method that can improve the quality of predictions generated by several machine learning (ML) methods. First, an ensemble of heterogeneous ML algorithms is trained on available experimental data, then all solutions are gathered and a consensus is built between them. The final prediction is performed using a voting procedure, whereby the vote of each method is weighted according to a quality coefficient calculated using multivariable linear regression (MLR). The MLR optimization procedure is very fast, therefore no additional computational cost is introduced by using this jury approach. Here, brainstorming is applied to selecting actives from large collections of compounds relating to five diverse biological targets of medicinal interest, namely HIV-reverse transcriptase, cyclooxygenase-2, dihydrofolate reductase, estrogen receptor, and thrombin. The MDL Drug Data Report (MDDR) database was used for selecting known inhibitors for these protein targets, and experimental data was then used to train a set of machine learning methods. The benchmark dataset (available at http://bio.icm.edu.pl/∼darman/chemoinfo/benchmark.tar.gz ) can be used for further testing of various clustering and machine learning methods when predicting the biological activity of compounds. Depending on the protein target, the overall recall value is raised by at least 20% in comparison to any single machine learning method (including ensemble methods like random forest) and unweighted simple majority voting procedures.

  14. Pyrazolopyrimidines: Potent Inhibitors Targeting the Capsid of Rhino- and Enteroviruses.

    PubMed

    Makarov, Vadim A; Braun, Heike; Richter, Martina; Riabova, Olga B; Kirchmair, Johannes; Kazakova, Elena S; Seidel, Nora; Wutzler, Peter; Schmidtke, Michaela

    2015-10-01

    There are currently no drugs available for the treatment of enterovirus (EV)-induced acute and chronic diseases such as the common cold, meningitis, encephalitis, pneumonia, and myocarditis with or without consecutive dilated cardiomyopathy. Here, we report the discovery and characterization of pyrazolopyrimidines, a well-tolerated and potent class of novel EV inhibitors. The compounds inhibit the replication of a broad spectrum of EV in vitro with IC50 values between 0.04 and 0.64 μM for viruses resistant to pleconaril, a known capsid-binding inhibitor, without affecting cytochrome P450 enzyme activity. Using virological and genetics methods, the viral capsid was identified as the target of the most promising, orally bioavailable compound 3-(4-trifluoromethylphenyl)amino-6-phenylpyrazolo[3,4-d]pyrimidine-4-amine (OBR-5-340). Its prophylactic as well as therapeutic application was proved for coxsackievirus B3-induced chronic myocarditis in mice. The favorable pharmacokinetic, toxicological, and pharmacodynamics profile in mice renders OBR-5-340 a highly promising drug candidate, and the regulatory nonclinical program is ongoing.

  15. Influenza A Virus Entry Inhibitors Targeting the Hemagglutinin

    PubMed Central

    Yang, Jie; Li, Minmin; Shen, Xintian; Liu, Shuwen

    2013-01-01

    Influenza A virus (IAV) has caused seasonal influenza epidemics and influenza pandemics, which resulted in serious threat to public health and socioeconomic impacts. Until now, only 5 drugs belong to two categories are used for prophylaxis and treatment of IAV infection. Hemagglutinin (HA), the envelope glycoprotein of IAV, plays a critical role in viral binding, fusion and entry. Therefore, HA is an attractive target for developing anti‑IAV drugs to block the entry step of IAV infection. Here we reviewed the recent progress in the study of conformational changes of HA during viral fusion process and the development of HA-based IAV entry inhibitors, which may provide a new choice for controlling future influenza pandemics. PMID:23340380

  16. Targeting cancer using KAT inhibitors to mimic lethal knockouts.

    PubMed

    Brown, James A L; Bourke, Emer; Eriksson, Leif A; Kerin, Michael J

    2016-08-15

    Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies. PMID:27528742

  17. Targeting cancer using KAT inhibitors to mimic lethal knockouts

    PubMed Central

    Brown, James A.L.; Bourke, Emer; Eriksson, Leif A.; Kerin, Michael J.

    2016-01-01

    Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies. PMID:27528742

  18. Diverse Inhibitor Chemotypes Targeting Trypanosoma cruzi CYP51

    PubMed Central

    Johnston, Jonathan B.; Chen, Chiung-Kuang; Erenburg, Grigori; Gut, Jiri; Engel, Juan C.; Ang, Kenny K. H.; Mulvaney, Joseph; Chen, Steven; Arkin, Michelle R.; McKerrow, James H.; Podust, Larissa M.

    2012-01-01

    Background Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority. Methodology/Principal Findings The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar KD values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC50 <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC50 of 17 nM and was trypanocidal at 40 nM. Conclusions/Significance The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or

  19. Analytical specificity and sensitivity of the novel dual-target GeneProof Neisseria gonorrhoeae PCR kit for detection of N. gonorrhoeae.

    PubMed

    Golparian, Daniel; Hellmark, Bengt; Unemo, Magnus

    2015-11-01

    Detection of Neisseria gonorrhoeae relies increasingly on nucleic acid amplification tests (NAATs). The specificity of many gonococcal NAATs has been suboptimal and supplementary testing remains recommended in Europe and several additional countries. The novel dual-target GeneProof Neisseria gonorrhoeae PCR kit, targeting porA pseudogene and 16S rRNA gene, showed a high specificity and sensitivity when isolates of non-gonococcal Neisseria and related species (n = 144), and gonococci (n = 104) were tested. However, rare gonococcal porA mutants were only detected in the 16S rRNA gene target and two non-gonococcal isolates showed a low-level cross-reactivity in the 16S rRNA gene target. The detection limit for both targets was 1.5 copies per reaction.

  20. Centrin: Another target of monastrol, an inhibitor of mitotic spindle

    NASA Astrophysics Data System (ADS)

    Duan, Lian; Wang, Tong-Qing; Bian, Wei; Liu, Wen; Sun, Yue; Yang, Bin-Sheng

    2015-02-01

    Monastrol, a cell-permeable inhibitor, considered to specifically inhibit kinesin Eg5, can cause mitotic arrest and monopolar spindle formation, thus exhibiting antitumor properties. Centrin, a ubiquitous protein associated with centrosome, plays a critical role in centrosome duplication. Moreover, a correlation between centrosome amplification and cancer has been reported. In this study, it is proposed for the first time that centrin may be another target of the anticancer drug monastrol since monastrol can effectively inhibit not only the growth of the transformed Escherichia coli cells in vivo, but also the Lu3+-dependent self-assembly of EoCen in vitro. The two closely related compounds (Compounds 1 and 2) could not take the same effect. Fluorescence titration experiments suggest that four monastrols per protein is the optimum binding pattern, and the binding constants at different temperatures were obtained. Detailed thermodynamic analysis indicates that hydrophobic force is the main acting force between monastrol and centrin, and the extent of monastrol inhibition of centrin self-assembly is highly dependent upon the hydrophobic region of the protein, which is largely exposed by the binding of metal ions.

  1. Crosstalk between KIT and FGFR3 Promotes Gastrointestinal Stromal Tumor Cell Growth and Drug Resistance

    PubMed Central

    Javidi-Sharifi, Nathalie; Traer, Elie; Martinez, Jacqueline; Gupta, Anu; Taguchi, Takehiro; Dunlap, Jennifer; Heinrich, Michael C.; Corless, Christopher L.; Rubin, Brian P.; Druker, Brian J.; Tyner, Jeffrey W.

    2014-01-01

    Kinase inhibitors such as imatinib have dramatically improved outcomes for GIST patients, but many patients develop resistance to these treatments. While in some patients this event corresponds with mutations in the GIST driver oncogenic kinase KIT, other patients development resistance without KIT mutations. In this study, we address this patient subset in reporting a functional dependence of GIST on the FGF receptor FGFR3 and its crosstalk with KIT in GIST cells. Addition of the FGFR3 ligand FGF2 to GIST cells restored KIT phosphorylation during imatinib treatment, allowing sensitive cells to proliferate in the presence of the drug. FGF2 expression was increased in imatinib-resistant GIST cells, the growth of which was blocked by RNAi-mediated silencing of FGFR3. Moreover, combining KIT and FGFR3 inhibitors synergized to block the growth of imatinib-resistant cells. Signaling crosstalk between KIT and FGFR3 activated the MAPK pathway to promote resistance to imatinib. Clinically, an immunohistochemical analysis of tumor specimens from imatinib-resistant GIST patients revealed a relative increase in FGF2 levels, with a trend towards increased expression in imatinib-naïve samples consistent with possible involvement in drug resistance. Our findings provide a mechanistic rationale to evaluate existing FGFR inhibitors and multi-kinase inhibitors that target FGFR3 as promising strategies to improve treatment of GIST patients with de novo or acquired resistance to imatinib. PMID:25432174

  2. Next generation sequencing analysis of platinum refractory advanced germ cell tumor sensitive to Sunitinib (Sutent®) a VEGFR2/PDGFRβ/c-kit/ FLT3/RET/CSF1R inhibitor in a phase II trial

    PubMed Central

    2014-01-01

    Background Germ cell tumors (GCT) are the most common solid tumors in adolescent and young adult males (age 15 and 35 years) and remain one of the most curable of all solid malignancies. However a subset of patients will have tumors that are refractory to standard chemotherapy agents. The management of this refractory population remains challenging and approximately 400 patients continue to die every year of this refractory disease in the United States. Methods Given the preclinical evidence implicating vascular endothelial growth factor (VEGF) signaling in the biology of germ cell tumors, we hypothesized that the vascular endothelial growth factor receptor (VEGFR) inhibitor sunitinib (Sutent) may possess important clinical activity in the treatment of this refractory disease. We proposed a Phase II efficacy study of sunitinib in seminomatous and non-seminomatous metastatic GCT’s refractory to first line chemotherapy treatment (ClinicalTrials.gov Identifier: NCT00912912). Next generation targeted exome sequencing using HiSeq 2000 (Illumina Inc., San Diego, CA, USA) was performed on the tumor sample of the unusual responder. Results Five patients are enrolled into this Phase II study. Among them we report here the clinical course of a patient (Patient # 5) who had an exceptional response to sunitinib. Next generation sequencing to understand this patient’s response to sunitinib revealed RET amplification, EGFR and KRAS amplification as relevant aberrations. Oncoscan MIP array were employed to validate the copy number analysis that confirmed RET gene amplification. Conclusion Sunitinib conferred clinical benefit to this heavily pre-treated patient. Next generation sequencing of this ‘exceptional responder’ identified the first reported case of a RET amplification as a potential basis of sensitivity to sunitinib (VEGFR2/PDGFRβ/c-kit/ FLT3/RET/CSF1R inhibitor) in a patient with refractory germ cell tumor. Further characterization of GCT patients using

  3. Increased KIT inhibition enhances therapeutic efficacy in gastrointestinal stromal tumor

    PubMed Central

    Kim, Teresa S.; Cavnar, Michael J.; Cohen, Noah A.; Sorenson, Eric C.; Greer, Jonathan B.; Seifert, Adrian M.; Crawley, Megan H.; Green, Benjamin L.; Popow, Rachel; Pillarsetty, Nagavarakishore; Veach, Darren R.; Ku, Anson T.; Rossi, Ferdinand; Besmer, Peter; Antonescu, Cristina R.; Zeng, Shan; DeMatteo, Ronald P.

    2014-01-01

    Purpose Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and a model of targeted molecular therapy. GIST depends on oncogenic KIT signaling and responds to the tyrosine kinase inhibitor imatinib. However, imatinib is rarely curative. We hypothesized that PLX3397, which inhibits KIT and CSF1R, would be more efficacious than imatinib in GIST by also depleting tumor-associated macrophages, which are generally thought to support tumor growth. Experimental Design We treated KitV558del/+ mice that develop GIST or mice with subcutaneous human GIST xenografts with imatinib or PLX3397 and analyzed tumor weight, cellular composition, histology, molecular signaling, and fibrosis. In vitro assays on human GIST cell lines were also performed. Results PLX3397 was more effective than imatinib in reducing tumor weight and cellularity in both KitV558del/+ murine GIST and human GIST xenografts. The superiority of PLX3397 did not depend on depletion of tumor-associated macrophages, since adding CSF1R inhibition did not improve the effects of imatinib. Instead, PLX3397 was a more potent KIT inhibitor than imatinib in vitro. PLX3397 therapy also induced substantial intratumoral fibrosis, which impaired the subsequent delivery of small molecules. Conclusions PLX3397 therapy has greater efficacy than imatinib in pre-clinical GIST models and warrants study in GIST patients. The resultant intratumoral fibrosis may represent one of the barriers to achieving complete tumor eradication. PMID:24583793

  4. Discovery – Targeted Treatments and mTOR Inhibitors

    Cancer.gov

    Thanks to discovering the anticancer effects of mTOR inhibitors, cancer treatment for pNet, a rare type of pancreatic cancer, were revolutionized. Through clinical trials, NCI continues to investigate the life-saving potential of mTOR inhibitors.

  5. Stopping cancer in its tracks: using small molecular inhibitors to target glioblastoma migrating cells.

    PubMed

    Mattox, Austin K; Li, Jing; Adamson, David C

    2012-12-01

    Glioblastoma multiforme (GBM) represents one of the most common aggressive types of primary brain tumors. Despite advances in surgical resection, novel neuroimaging procedures, and the most recent adjuvant radiotherapy and chemotherapy, the median survival after diagnosis is about 12-14 months. Targeting migrating GBM cells is a key research strategy in the fight against this devastating cancer. Though the vast majority of the primary tumor focus can be surgically resected, these migrating cells are responsible for its universal recurrence. Numerous strategies and technologies are being explored to target migrating glioma cells, with small molecular inhibitors as one of the most commonly studied. Small molecule inhibitors, such as protein kinase inhibitors, phosphorylation site inhibitors, protease inhibitors, and antisense oligonucleotides show promise in slowing the progression of this disease. A better understanding of these small molecule inhibitors and how they target various extra- and intracellular signaling pathways may eventually lead to a cure for GBM.

  6. Feasibility of using molecular docking-based virtual screening for searching dual target kinase inhibitors.

    PubMed

    Zhou, Shunye; Li, Youyong; Hou, Tingjun

    2013-04-22

    Multitarget agents have been extensively explored for solving limited efficacies, poor safety, and resistant profiles of an individual target. Theoretical approaches for searching and designing multitarget agents are critically useful. Here, the performance of molecular docking to search dual-target inhibitors for four kinase pairs (CDK2-GSK3B, EGFR-Src, Lck-Src, and Lck-VEGFR2) was assessed. First, the representative structures for each kinase target were chosen by structural clustering of available crystal structures. Next, the performance of molecular docking to distinguish inhibitors from noninhibitors for each individual kinase target was evaluated. The results show that molecular docking-based virtual screening illustrates good capability to find known inhibitors for individual targets, but the prediction accuracy is structurally dependent. Finally, the performance of molecular docking to identify the dual-target kinase inhibitors for four kinase pairs was evaluated. The analyses show that molecular docking successfully filters out most noninhibitors and achieves promising performance for identifying dual-kinase inhibitors for CDK2-GSK3B and Lck-VEGFR2. But a high false-positive rate leads to low enrichment of true dual-target inhibitors in the final list. This study suggests that molecular docking serves as a useful tool in searching inhibitors against dual or even multiple kinase targets, but integration with other virtual screening tools is necessary for achieving better predictions.

  7. 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

  8. Next Generation of Targeted Molecules for Non-Hodgkin Lymphomas: Small-Molecule Inhibitors of Intracellular Targets and Signaling Pathways.

    PubMed

    Choe, Hannah; Ruan, Jia

    2016-09-15

    Advances in our understanding of the molecular pathogenesis of B-cell lymphoma have guided the development of targeted therapies that disrupt aberrant signaling pathways important for communication within lymphoma cells and for their interactions with the tumor microenvironment. This has led to unprecedented therapeutic progress, with biologic agents that have begun to transform the care of patients with lymphoma and chronic lymphocytic leukemia. This review discusses the mechanisms of action, clinical development, and emerging applications of small-molecule inhibitors that target B-cell receptor signaling pathways, B-cell lymphoma-2 inhibitors, selective inhibitors of nuclear export, and epigenetic modifiers. PMID:27633417

  9. Finding off-targets, biological pathways, and target diseases for chymase inhibitors via structure-based systems biology approach.

    PubMed

    Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang Ping; Kim, Songmi; Arulalapperumal, Venkatesh; Lee, Keun Woo

    2015-07-01

    Off-target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off-targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off-target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off-targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off-targets into biological pathways and to establish links between pathways and particular adverse effects. Off-targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off-targets. These off-targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors.

  10. Pathophysiology of cardiotoxicity from target therapy and angiogenesis inhibitors.

    PubMed

    Maurea, Nicola; Coppola, Carmela; Piscopo, Giovanna; Galletta, Francesca; Riccio, Gennaro; Esposito, Emanuela; De Lorenzo, Claudia; De Laurentiis, Michelino; Spallarossa, Paolo; Mercuro, Giuseppe

    2016-05-01

    The progress in cancer therapy and the increase in number of long-term survivors reveal the issue of cardiovascular side-effects of anticancer drugs. Cardiotoxicity has become a significant problem, and the risks of adverse cardiac events induced by systemic drugs need to be seriously considered. Potential cardiovascular toxicities linked to anticancer agents include arrhythmias, myocardial ischemia and infarction, hypertension, thromboembolism, left ventricular dysfunction, and heart failure. It has been shown that several anticancer drugs seriously affect the cardiovascular system, such as ErbB2 inhibitors, vascular endothelial growth factor (VEGF) inhibitors, multitargeted kinase inhibitors, Abelson murine leukemia viral oncogene homolog inhibitors, and others. Each of these agents has a different mechanism through which it affects the cardiovascular system. ErbB2 inhibitors block the ErbB4/ErbB2 heterodimerization pathway triggered by Neuregulin-1, which is essential for cardiomyocyte survival. VEGF signaling is crucial for vascular growth, but it also has a major impact on myocardial function, and the VEGF pathway is also essential for maintenance of cardiovascular homeostasis. Drugs that inhibit the VEGF signaling pathway lead to a net reduction in capillary density and loss of contractile function. Here, we review the mechanisms and pathophysiology of the most significant cardiotoxic effects of ErbB2 inhibitors and antiangiogenic drugs. Moreover, we highlight the role of cardioncology in recognizing these toxicities, developing strategies to prevent or minimize cardiovascular toxicity, and reducing long-term cardiotoxic effects.

  11. 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

  12. Proposal of Dual Inhibitor Targeting ATPase Domains of Topoisomerase II and Heat Shock Protein 90

    PubMed Central

    Jun, Kyu-Yeon; Kwon, Youngjoo

    2016-01-01

    There is a conserved ATPase domain in topoisomerase II (topo II) and heat shock protein 90 (Hsp90) which belong to the GHKL (gyrase, Hsp90, histidine kinase, and MutL) family. The inhibitors that target each of topo II and Hsp90 are intensively studied as anti-cancer drugs since they play very important roles in cell proliferation and survival. Therefore the development of dual targeting anti-cancer drugs for topo II and Hsp90 is suggested to be a promising area. The topo II and Hsp90 inhibitors, known to bind to their ATP binding site, were searched. All the inhibitors investigated were docked to both topo II and Hsp90. Four candidate compounds as possible dual inhibitors were selected by analyzing the molecular docking study. The pharmacophore model of dual inhibitors for topo II and Hsp90 were generated and the design of novel dual inhibitor was proposed. PMID:27582553

  13. Isoxazole-Based-Scaffold Inhibitors Targeting Cyclooxygenases (COXs).

    PubMed

    Perrone, Maria Grazia; Vitale, Paola; Panella, Andrea; Ferorelli, Savina; Contino, Marialessandra; Lavecchia, Antonio; Scilimati, Antonio

    2016-06-01

    A new set of cyclooxygenase (COX) inhibitors endowed with an additional functionality was explored. These new compounds also contained either rhodamine 6G or 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, two moieties typical of efflux pump substrates and inhibitors, respectively. Among all the synthesized compounds, two new COX inhibitors with opposite selectivity were discovered: compound 8 [N-(9-{2-[(4-{2-[3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-yl]acetamido}butyl)carbamoyl]phenyl-6-(ethylamino)-2,7-dimethyl-3H-xanthen-3-ylidene}ethanaminium chloride] was found to be a selective COX-1 inhibitor, whereas 17 (2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]-1-[6,7-dimethoxy-3,4-dihydroisoquinolin-2-(1H)-yl]ethanone) was found to be a sub-micromolar selective COX-2 inhibitor. However, both were shown to interact with P-glycoprotein. Docking experiments helped to clarify the molecular aspects of the observed COX selectivity. PMID:27136372

  14. Steroid 5 α-reductase inhibitors targeting BPH and prostate cancer.

    PubMed

    Schmidt, Lucy J; Tindall, Donald J

    2011-05-01

    Steroid 5 alpha-reductase inhibitors (5ARIs) have been approved for use clinically in treatment of benign prostate hyperplasia (BPH) and accompanying lower urinary tract symptoms (LUTS) and have also been evaluated in clinical trials for prevention and treatment of prostate cancer. There are currently two steroidal inhibitors in use, finasteride and dutasteride, both with distinct pharmacokinetic properties. This review will examine the evidence presented by various studies supporting the use of these steroidal inhibitors in the prevention and treatment of prostate disease. Article from the Special issue on Targeted Inhibitors. PMID:20883781

  15. Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

    PubMed Central

    Lu, Lu; Yu, Fei; Cai, Lifeng; Debnath, Asim K.; Jiang, Shibo

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development. PMID:26324044

  16. The ALK gene, an attractive target for inhibitor development.

    PubMed

    Tartari, Carmen J; Scapozza, Leonardo; Gambacorti-Passerini, Carlo

    2011-01-01

    Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase that belongs to the Insulin receptor subfamily involved as full length receptor in neural development. Even if the expression of ALK protein is down-regulated in the adults, the ALK full length is expressed in different types of tumors. Moreover, chromosomal rearrangements, involving the alk gene, can occur leading the formation of different ALK fusion proteins characterized by the kinase domain of ALK fused to several partners that determine cellular localization. Structural investigation and characterization of the ALK kinase domain in absence of its crystal structure constituted the basis of development of ALK small molecule inhibitors. Here, we described normal function of the ALK receptor and its role in tumors; formation of the constitutively activated ALK fusion proteins and we reported an update of developed small molecule inhibitors of the ALK kinase activity. PMID:21513493

  17. Novel pyridazinone inhibitors for vascular adhesion protein-1 (VAP-1): old target-new inhibition mode.

    PubMed

    Bligt-Lindén, Eva; Pihlavisto, Marjo; Szatmári, István; Otwinowski, Zbyszek; Smith, David J; Lázár, László; Fülöp, Ferenc; Salminen, Tiina A

    2013-12-27

    Vascular adhesion protein-1 (VAP-1) is a primary amine oxidase and a drug target for inflammatory and vascular diseases. Despite extensive attempts to develop potent, specific, and reversible inhibitors of its enzyme activity, the task has proven challenging. Here we report the synthesis, inhibitory activity, and molecular binding mode of novel pyridazinone inhibitors, which show specificity for VAP-1 over monoamine and diamine oxidases. The crystal structures of three inhibitor-VAP-1 complexes show that these compounds bind reversibly into a unique binding site in the active site channel. Although they are good inhibitors of human VAP-1, they do not inhibit rodent VAP-1 well. To investigate this further, we used homology modeling and structural comparison to identify amino acid differences, which explain the species-specific binding properties. Our results prove the potency and specificity of these new inhibitors, and the detailed characterization of their binding mode is of importance for further development of VAP-1 inhibitors.

  18. Targeting mTOR: prospects for mTOR complex 2 inhibitors in cancer therapy

    PubMed Central

    Sparks, CA; Guertin, DA

    2010-01-01

    Small molecule inhibitors that selectively target cancer cells and not normal cells would be valuable anti-cancer therapeutics. The mammalian target of rapamycin complex 2 (mTORC2) is emerging as a promising candidate target for such an inhibitor. Recent studies in cancer biology indicate that mTORC2 activity is essential for the transformation and vitality of a number of cancer cell types, but in many normal cells, mTORC2 activity is less essential. These studies are intensifying interest in developing inhibitors that specifically target mTORC2. However, there are many open questions regarding the function and regulation of mTORC2 and its function in both normal and cancer cells. Here, we summarize exciting new research into the biology of mTORC2 signaling and highlight the current state and future prospects for mTOR-targeted therapy. PMID:20418915

  19. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors.

    PubMed

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R J

    2015-11-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy.

  20. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

    PubMed Central

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R.J.

    2015-01-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy. PMID:26713267

  1. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants.

    PubMed

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ9-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors' therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic-pituitary-adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted. PMID:26630956

  2. Effects of tobacco genetically modified to express protease inhibitor bovine spleen trypsin inhibitor on non-target soil organisms.

    PubMed

    O'Callaghan, Maureen; Brownbridge, Michael; Stilwell, Wendy B; Gerard, Emily M; Burgess, Elisabeth P J; Barraclough, Emma I; Christeller, John T

    2007-01-01

    Effects of tobacco genetically modified to express the protease inhibitor bovine spleen trypsin inhibitor (BSTI) were examined in laboratory assays against three earthworm and one collembolan species. BSTI is a serine protease inhibitor that can bind to the digestive trypsins of insects feeding on modified plants, resulting in reduced growth and survival. Protease inhibitors are active against a broad range of insects, so may have a large impact on non-target organisms. Survival and fecundity of the collembolan Folsomia candida were unaffected by consumption of artificial diet containing BSTI-expressing tobacco leaf or powdered freeze-dried BSTI-expressing tobacco leaf that was added to soil. Similarly, mortality and growth of earthworms Aporrectodea caliginosa and Lumbricus rubellus did not differ significantly between soil augmented with BSTI-expressing tobacco leaves or unmodified control leaves. The redworm Eisenia fetida gained less weight when provided with BSTI-expressing leaves in one assay, but when the experiment was repeated, there was no significant difference between treatments. BSTI-expressing tobacco and unmodified control leaves decomposed at the same rate, indicating that the inhibitor had no effect on the overall function of the decomposer community of micro-flora and fauna in soil. PMID:18001685

  3. Effects of tobacco genetically modified to express protease inhibitor bovine spleen trypsin inhibitor on non-target soil organisms.

    PubMed

    O'Callaghan, Maureen; Brownbridge, Michael; Stilwell, Wendy B; Gerard, Emily M; Burgess, Elisabeth P J; Barraclough, Emma I; Christeller, John T

    2007-01-01

    Effects of tobacco genetically modified to express the protease inhibitor bovine spleen trypsin inhibitor (BSTI) were examined in laboratory assays against three earthworm and one collembolan species. BSTI is a serine protease inhibitor that can bind to the digestive trypsins of insects feeding on modified plants, resulting in reduced growth and survival. Protease inhibitors are active against a broad range of insects, so may have a large impact on non-target organisms. Survival and fecundity of the collembolan Folsomia candida were unaffected by consumption of artificial diet containing BSTI-expressing tobacco leaf or powdered freeze-dried BSTI-expressing tobacco leaf that was added to soil. Similarly, mortality and growth of earthworms Aporrectodea caliginosa and Lumbricus rubellus did not differ significantly between soil augmented with BSTI-expressing tobacco leaves or unmodified control leaves. The redworm Eisenia fetida gained less weight when provided with BSTI-expressing leaves in one assay, but when the experiment was repeated, there was no significant difference between treatments. BSTI-expressing tobacco and unmodified control leaves decomposed at the same rate, indicating that the inhibitor had no effect on the overall function of the decomposer community of micro-flora and fauna in soil.

  4. Targeting Staphylococcus aureus Quorum Sensing with Nonpeptidic Small Molecule Inhibitors

    PubMed Central

    2014-01-01

    A series of 3-oxo-C12-HSL, tetramic acid, and tetronic acid analogues were synthesized to gain insights into the structural requirements for quorum sensing inhibition in Staphylococcus aureus. Compounds active against agr were noncompetitive inhibitors of the autoinducing peptide (AIP) activated AgrC receptor, by altering the activation efficacy of the cognate AIP-1. They appeared to act as negative allosteric modulators and are exemplified by 3-tetradecanoyltetronic acid 17, which reduced nasal cell colonization and arthritis in a murine infection model. PMID:24592914

  5. Imatinib in pulmonary arterial hypertension: c-Kit inhibition.

    PubMed

    Farha, Samar; Dweik, Raed; Rahaghi, Franck; Benza, Raymond; Hassoun, Paul; Frantz, Robert; Torres, Fernando; Quinn, Deborah A; Comhair, Suzy; Erzurum, Serpil; Asosingh, Kewal

    2014-09-01

    Pulmonary arterial hypertension (PAH) is a progressive disease characterized by severe remodeling of the pulmonary artery resulting in increased pulmonary artery pressure and right ventricular hypertrophy and, ultimately, failure. Bone marrow-derived progenitor cells play a critical role in vascular homeostasis and have been shown to be involved in the pathogenesis of PAH. A proliferation of c-Kit(+) hematopoietic progenitors and mast cells has been noted in the remodeled vessels in PAH. Imatinib, a tyrosine kinase inhibitor that targets c-Kit, has been shown to be beneficial for patients with PAH. Here we hypothesize that the clinical benefit of imatinib in PAH could be related to c-Kit inhibition of progenitor cell mobilization and maturation into mast cells. As a corollary to the phase 3 study using imatinib in PAH, blood samples were collected from 12 patients prior to starting study drug (baseline) and while on treatment at weeks 4 and 24. Eight were randomized to imatinib and 4 to placebo. Circulating c-Kit(+) and CD34(+)CD133(+) hematopoietic progenitors as well as biomarkers of mast cell numbers and activation were measured. Circulating CD34(+)CD133(+) and c-Kit(+) progenitor cells as well as c-Kit(+)/CD34(+)CD133(+) decreased with imatinib therapy (all P < 0.05). In addition, total tryptase, a marker of mast cell load, dropped with imatinib therapy (P = 0.02) and was related to pulmonary vascular resistance (R = 0.7, P = 0.02). The findings support c-Kit inhibition as a potential mechanism of action of imatinib in PAH and suggest that tryptase is a potential biomarker of response to therapy. PMID:25621158

  6. Targeted Radiosensitization by the Chk1 Inhibitor SAR-020106

    SciTech Connect

    Borst, Gerben R.; McLaughlin, Martin; Kyula, Joan N.; Neijenhuis, Sari; Khan, Aadil; Good, James; Zaidi, Shane; Powell, Ned G.; Meier, Pascal; Collins, Ian; Garrett, Michelle D.; Verheij, Marcel; Harrington, Kevin J.

    2013-03-15

    Purpose: To explore the activity of a potent Chk1 inhibitor (SAR-020106) in combination with radiation. Methods and Materials: Colony and mechanistic in vitro assays and a xenograft in vivo model. Results: SAR-020106 suppressed-radiation-induced G{sub 2}/M arrest and reduced clonogenic survival only in p53-deficient tumor cells. SAR-020106 promoted mitotic entry following irradiation in all cell lines, but p53-deficient cells were likely to undergo apoptosis or become aneuploid, while p53 wild-type cells underwent a postmitotic G{sub 1} arrest followed by subsequent normal cell cycle re-entry. Following combined treatment with SAR-020106 and radiation, homologous-recombination-mediated DNA damage repair was inhibited in all cell lines. A significant increase in the number of pan-γH2AX-staining apoptotic cells was observed only in p53-deficient cell lines. Efficacy was confirmed in vivo in a clinically relevant human head-and-neck cell carcinoma xenograft model. Conclusion: The Chk1 inhibitor SAR-020106 is a potent radiosensitizer in tumor cell lines defective in p53 signaling.

  7. Design of a peptidic inhibitor that targets the dimer interface of a prototypic galectin

    PubMed Central

    Vladoiu, Maria Claudia; Labrie, Marilyne; Létourneau, Myriam; Egesborg, Philippe; Gagné, Donald; Billard, Étienne; Grosset, Andrée-Anne; Doucet, Nicolas; Chatenet, David; St-Pierre, Yves

    2015-01-01

    Galectins are small soluble lectins that bind β-galactosides via their carbohydrate recognition domain (CRD). Their ability to dimerize is critical for the crosslinking of glycoprotein receptors and subsequent cellular signaling. This is particularly important in their immunomodulatory role via the induction of T-cell apoptosis. Because galectins play a central role in many pathologies, including cancer, they represent valuable therapeutic targets. At present, most inhibitors have been directed towards the CRD, a challenging task in terms of specificity given the high structural homology of the CRD among galectins. Such inhibitors are not effective at targeting CRD-independent functions of galectins. Here, we report a new class of galectin inhibitors that specifically binds human galectin-7 (hGal-7), disrupts the formation of homodimers, and inhibits the pro-apoptotic activity of hGal-7 on Jurkat T cells. In addition to representing a new means to achieve specificity when targeting galectins, such inhibitors provide a promising alternative to more conventional galectin inhibitors that target the CRD with soluble glycans or other small molecular weight allosteric inhibitors. PMID:26543238

  8. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants

    PubMed Central

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ9-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors’ therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic–pituitary–adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted. PMID:26630956

  9. Development of novel entry inhibitors targeting emerging viruses

    PubMed Central

    Zhou, Yanchen; Simmons, Graham

    2013-01-01

    Emerging viral diseases pose a unique risk to public health, and thus there is a need to develop therapies. A current focus of funding agencies, and hence research, is the development of broad-spectrum antivirals, and in particular, those targeting common cellular pathways. The scope of this article is to review screening strategies and recent advances in this area, with a particular emphasis on antivirals targeting the step of viral entry for emerging lipid-enveloped viruses such as Ebola virus and SARS-coronavirus. PMID:23199399

  10. Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.

    PubMed

    Song, Hongbin; He, Chun; Knaak, Christian; Guthridge, Joel M; Holers, V Michael; Tomlinson, Stephen

    2003-06-01

    In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the membrane complement inhibitors decay accelerating factor (DAF) or CD59. The targeted complement inhibitors bound to C3-opsonized cells, and all were significantly more effective (up to 20-fold) than corresponding untargeted inhibitors at protecting target cells from complement. CR2 fusion proteins also inhibited CR3-dependent adhesion of U937 cells to C3 opsonized erythrocytes, indicating a second potential anti-inflammatory mechanism of CR2 fusion proteins, since CR3 is involved in endothelial adhesion and diapedesis of leukocytes at inflammatory sites. Finally, the in vivo validity of the targeting strategy was confirmed by the demonstration that CR2-DAF, but not soluble DAF, targets to the kidney in mouse models of lupus nephritis that are associated with renal complement deposition.

  11. Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.

    PubMed

    Song, Hongbin; He, Chun; Knaak, Christian; Guthridge, Joel M; Holers, V Michael; Tomlinson, Stephen

    2003-06-01

    In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the membrane complement inhibitors decay accelerating factor (DAF) or CD59. The targeted complement inhibitors bound to C3-opsonized cells, and all were significantly more effective (up to 20-fold) than corresponding untargeted inhibitors at protecting target cells from complement. CR2 fusion proteins also inhibited CR3-dependent adhesion of U937 cells to C3 opsonized erythrocytes, indicating a second potential anti-inflammatory mechanism of CR2 fusion proteins, since CR3 is involved in endothelial adhesion and diapedesis of leukocytes at inflammatory sites. Finally, the in vivo validity of the targeting strategy was confirmed by the demonstration that CR2-DAF, but not soluble DAF, targets to the kidney in mouse models of lupus nephritis that are associated with renal complement deposition. PMID:12813023

  12. Pharmacological inhibitors of exocytosis and endocytosis: novel bullets for old targets.

    PubMed

    Ivanov, Andrei I

    2014-01-01

    Pharmacological inhibitors of vesicle trafficking possess great promise as valuable analytical tools for the study of a variety of biological processes and as potential therapeutic agents to fight microbial infections and cancer. However, many commonly used trafficking inhibitors are characterized by poor selectivity that diminishes their use in solving basic problems of cell biology or drug development. Recent high-throughput chemical screens intensified the search for novel modulators of vesicle trafficking, and successfully identified a number of small molecules that inhibit exocytosis and endocytosis in different types of mammalian cells. This chapter provides a systematic overview of recently discovered inhibitors of vesicle trafficking. It describes cellular effects and mechanisms of action of novel inhibitors of exocytosis and endocytosis. Furthermore, it pays special attention to the selectivity and possible off-target effects of these inhibitors. PMID:24947371

  13. Targeting inhibitors of apoptosis proteins (IAPs) for new breast cancer therapeutics.

    PubMed

    Wang, Shaomeng; Bai, Longchuan; Lu, Jianfeng; Liu, Liu; Yang, Chao-Yie; Sun, Haiying

    2012-12-01

    Apoptosis resistance is a hallmark of human cancer. Research in the last two decades has identified key regulators of apoptosis, including inhibitor of apoptosis proteins (IAPs). These critical apoptosis regulators have been targeted for the development of new cancer therapeutics. In this article, we will discuss three members of IAP proteins, namely XIAP, cIAP1 and cIAP2, as cancer therapeutic targets and the progress made in developing new cancer therapeutic agents to target these IAP proteins.

  14. The SCF/c-KIT system in the male: Survival strategies in fertility and cancer.

    PubMed

    Cardoso, Henrique J; Figueira, Marília I; Correia, Sara; Vaz, Cátia V; Socorro, Sílvia

    2014-12-01

    Maintaining the delicate balance between cell survival and death is of the utmost importance for the proper development of germ cells and subsequent fertility. On the other hand, the fine regulation of tissue homeostasis by mechanisms that control cell fate is a factor that can prevent carcinogenesis. c-KIT is a type III receptor tyrosine kinase activated by its ligand, stem cell factor (SCF). c-KIT signaling plays a crucial role in cell fate decisions, specifically controlling cell proliferation, differentiation, survival, and apoptosis. Indeed, deregulating the SCF/c-KIT system by attenuation or overactivation of its signaling strength is linked to male infertility and cancer, and rebalancing its activity via c-KIT inhibitors has proven beneficial in treating human tumors that contain gain-of-function mutations or overexpress c-KIT. This review addresses the roles of SCF and c-KIT in the male reproductive tract, and discusses the potential application of c-KIT target therapies in disorders of the reproductive system. PMID:25359157

  15. 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

  16. Curing a viral infection by targeting the host: The example of cyclophilin inhibitors

    PubMed Central

    Lin, Kai; Gallay, Philippe

    2015-01-01

    Every step of the viral life cycle is dependent on the host, which potentially can be explored for antiviral targets. Historically, however, drug discovery has focused mainly on viral targets, because of their perceived specificity. Efforts to pursue host targets have been largely hampered by concern over potential on-target toxicity, the lack of predictive cell culture and animal models, and the complexity of host–virus interactions. On the other hand, there are distinct advantages of targeting the host, such as creating a high barrier to resistance, providing broad coverage of different genotypes/serotypes and possibly even multiple viruses, and expanding the list of potential targets, when druggable viral targets are limited. Taking hepatitis C virus (HCV) as the example, there are more than 20 inhibitors of the viral protease, polymerase and NS5A protein currently in advanced clinical testing. However, resistance has become a main challenge with these direct-acting antivirals, because HCV, an RNA virus, is notoriously prone to mutation, and a single mutation in the viral target may prevent the binding of an inhibitor, and rendering it ineffective. Host cyclophilin inhibitors have shown promising effects both in vitro and in patients to prevent the emergence of resistance and to cure HCV infection, either alone or in combination with other agents. They are also capable of blocking the replication of a number of other viral pathogens. While the road to developing host-targeting antivirals has been less traveled, and significant challenges remain, delivering the most effective antiviral regimen, which may comprise inhibitors of both host and viral targets, should be well worth the effort. PMID:23578729

  17. A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

    SciTech Connect

    Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young; Chun, Sung Hak; Han, Jeong Yun; Kim, Sung Dae; Lee, Janet; Lee, Chang-Woo; Yang, Kwangmo; Lee, Chang Geun

    2013-01-25

    Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24{sup −}/CD44{sup +}) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer.

  18. Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

    PubMed

    Abdul-Hay, Samer O; Bannister, Thomas D; Wang, Hui; Cameron, Michael D; Caulfield, Thomas R; Masson, Amandine; Bertrand, Juliette; Howard, Erin A; McGuire, Michael P; Crisafulli, Umberto; Rosenberry, Terrone R; Topper, Caitlyn L; Thompson, Caroline R; Schürer, Stephan C; Madoux, Franck; Hodder, Peter; Leissring, Malcolm A

    2015-12-18

    Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 μM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

  19. Bruton tyrosine kinase inhibitors: a promising novel targeted treatment for B cell lymphomas

    PubMed Central

    Aalipour, Amin; Advani, Ranjana H.

    2015-01-01

    Summary Constitutive or aberrant signalling of the B cell receptor signalling cascade has been implicated in the propagation and maintenance of a variety of B cell malignancies. Small molecule inhibitors of Bruton tyrosine kinase (BTK), a protein early in this cascade and specifically expressed in B cells, have emerged as a new class of targeted agents. There are several BTK inhibitors, including ONO-WG-307, LFM-A13, dasatinib, CC-292, and PCI-32765 (ibrutinib), in preclinical and/or clinical development of which ibrutinib is currently in phase III trials. Recent clinical data suggest significant activity of ibrutinib as a first in class oral inhibitor of BTK. This review provides an overview of ongoing clinical studies of BTK inhibitors. PMID:24111579

  20. Role of ELA region in auto-activation of mutant KIT receptor: a molecular dynamics simulation insight.

    PubMed

    Purohit, Rituraj

    2014-01-01

    KIT receptor is the prime target in gastrointestinal stromal tumor (GISTs) therapy. Second generation inhibitor, Sunitinib, binds to an inactivated conformation of KIT receptor and stabilizes it in order to prevent tumor formation. Here, we investigated the dynamic behavior of wild type and mutant D816H KIT receptor, and emphasized the extended A-loop (EAL) region (805-850) by conducting molecular dynamics simulation (∼100 ns). We analyzed different properties such as root mean square cutoff or deviation, root mean square fluctuation, radius of gyration, solvent-accessible surface area, hydrogen bonding network analysis, and essential dynamics. Apart from this, clustering and cross-correlation matrix approach was used to explore the conformational space of the wild type and mutant EAL region of KIT receptor. Molecular dynamics analysis indicated that mutation (D816H) was able to alter intramolecular hydrogen bonding pattern and affected the structural flexibility of EAL region. Moreover, flexible secondary elements, specially, coil and turns were dominated in EAL region of mutant KIT receptor during simulation. This phenomenon increased the movement of EAL region which in turn helped in shifting the equilibrium towards the active kinase conformation. Our atomic investigation of mutant KIT receptor which emphasized on EAL region provided a better insight into the understanding of Sunitinib resistance mechanism of KIT receptor and would help to discover new therapeutics for KIT-based resistant tumor cells in GIST therapy.

  1. Targeting Mycobacterium tuberculosis topoisomerase I by small-molecule inhibitors.

    PubMed

    Godbole, Adwait Anand; Ahmed, Wareed; Bhat, Rajeshwari Subray; Bradley, Erin K; Ekins, Sean; Nagaraja, Valakunja

    2015-03-01

    We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

  2. Overcoming acquired BRAF inhibitor resistance in melanoma via targeted inhibition of Hsp90 with ganetespib.

    PubMed

    Acquaviva, Jaime; Smith, Donald L; Jimenez, John-Paul; Zhang, Chaohua; Sequeira, Manuel; He, Suqin; Sang, Jim; Bates, Richard C; Proia, David A

    2014-02-01

    Activating BRAF kinase mutations serve as oncogenic drivers in over half of all melanomas, a feature that has been exploited in the development of new molecularly targeted approaches to treat this disease. Selective BRAF(V600E) inhibitors, such as vemurafenib, typically induce initial, profound tumor regressions within this group of patients; however, durable responses have been hampered by the emergence of drug resistance. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90, in melanoma lines harboring the BRAF(V600E) mutation. Ganetespib exposure resulted in the loss of mutant BRAF expression and depletion of mitogen-activated protein kinase and AKT signaling, resulting in greater in vitro potency and antitumor efficacy compared with targeted BRAF and MAP-ERK kinase (MEK) inhibitors. Dual targeting of Hsp90 and BRAF(V600E) provided combinatorial benefit in vemurafenib-sensitive melanoma cells in vitro and in vivo. Importantly, ganetespib overcame mechanisms of intrinsic and acquired resistance to vemurafenib, the latter of which was characterized by reactivation of extracellular signal-regulated kinase (ERK) signaling. Continued suppression of BRAF(V600E) by vemurafenib potentiated sensitivity to MEK inhibitors after acquired resistance had been established. Ganetespib treatment reduced, but not abolished, elevations in steady-state ERK activity. Profiling studies revealed that the addition of a MEK inhibitor could completely abrogate ERK reactivation in the resistant phenotype, with ganetespib displaying superior combinatorial activity over vemurafenib. Moreover, ganetespib plus the MEK inhibitor TAK-733 induced tumor regressions in vemurafenib-resistant xenografts. Overall these data highlight the potential of ganetespib as a single-agent or combination treatment in BRAF(V600E)-driven melanoma, particularly as a strategy to overcome acquired resistance to selective BRAF inhibitors. PMID:24398428

  3. Methods for Investigation of Targeted Kinase Inhibitor Therapy using Chemical Proteomics and Phosphorylation Profiling

    PubMed Central

    Fang, Bin; Haura, Eric B.; Smalley, Keiran S.; Eschrich, Steven A.; Koomen, John M.

    2010-01-01

    Phosphorylation acts as a molecular switch for many regulatory events in signaling pathways that drive cell division, proliferation, and apoptosis. Because of the critical nature of these protein post-translational modifications in cancer, drug development programs often focus on inhibitors for kinases and phosphatases, which control protein phosphorylation. Numerous kinase inhibitors have entered clinical use, but prediction of their efficacy and a molecular basis for patient response remain uncertain. Chemical proteomics, the combination of drug affinity chromatography with mass spectrometry, identifies potential target proteins that bind to the drugs. Phosphorylation profiling can complement chemical proteomics by cataloging modifications in the target kinases and their downstream substrates using phosphopeptide enrichment and quantitative mass spectrometry. These experiments shed light on the mechanism of disease development and illuminate candidate biomarkers to guide personalized therapeutic strategies. In this review, commonly applied technologies and workflows are discussed to illustrate the role of proteomics in examining tumor biology and therapeutic intervention using kinase inhibitors. PMID:20361944

  4. Overcoming Chemical, Biological, and Computational Challenges in the Development of Inhibitors Targeting Protein-Protein Interactions

    PubMed Central

    Laraia, Luca; McKenzie, Grahame; Spring, David R.; Venkitaraman, Ashok R.; Huggins, David J.

    2015-01-01

    Protein-protein interactions (PPIs) underlie the majority of biological processes, signaling, and disease. Approaches to modulate PPIs with small molecules have therefore attracted increasing interest over the past decade. However, there are a number of challenges inherent in developing small-molecule PPI inhibitors that have prevented these approaches from reaching their full potential. From target validation to small-molecule screening and lead optimization, identifying therapeutically relevant PPIs that can be successfully modulated by small molecules is not a simple task. Following the recent review by Arkin et al., which summarized the lessons learnt from prior successes, we focus in this article on the specific challenges of developing PPI inhibitors and detail the recent advances in chemistry, biology, and computation that facilitate overcoming them. We conclude by providing a perspective on the field and outlining four innovations that we see as key enabling steps for successful development of small-molecule inhibitors targeting PPIs. PMID:26091166

  5. When Teaching Gets Tough--Professional Community Inhibitors of Teacher-Targeted Bullying and Turnover Intentions

    ERIC Educational Resources Information Center

    Pyhältö, Kirsi; Pietarinen, Janne; Soini, Tiina

    2015-01-01

    Bullying in school has become an international concern in recent decades. Yet, we know surprisingly little about inhibitors of teacher-targeted bullying. The study focused on exploring the interrelation between the teacher-working environment fit, bullying, experienced exhaustion and turnover intentions. Altogether 2310 comprehensive school…

  6. Structural and Biochemical Basis for Development of Influenza Virus Inhibitors Targeting the PA Endonuclease

    PubMed Central

    DuBois, Rebecca M.; Slavish, P. Jake; Baughman, Brandi M.; Yun, Mi-Kyung; Bao, Ju; Webby, Richard J.; Webb, Thomas R.; White, Stephen W.

    2012-01-01

    Emerging influenza viruses are a serious threat to human health because of their pandemic potential. A promising target for the development of novel anti-influenza therapeutics is the PA protein, whose endonuclease activity is essential for viral replication. Translation of viral mRNAs by the host ribosome requires mRNA capping for recognition and binding, and the necessary mRNA caps are cleaved or “snatched” from host pre-mRNAs by the PA endonuclease. The structure-based development of inhibitors that target PA endonuclease is now possible with the recent crystal structure of the PA catalytic domain. In this study, we sought to understand the molecular mechanism of inhibition by several compounds that are known or predicted to block endonuclease-dependent polymerase activity. Using an in vitro endonuclease activity assay, we show that these compounds block the enzymatic activity of the isolated PA endonuclease domain. Using X-ray crystallography, we show how these inhibitors coordinate the two-metal endonuclease active site and engage the active site residues. Two structures also reveal an induced-fit mode of inhibitor binding. The structures allow a molecular understanding of the structure-activity relationship of several known influenza inhibitors and the mechanism of drug resistance by a PA mutation. Taken together, our data reveal new strategies for structure-based design and optimization of PA endonuclease inhibitors. PMID:22876176

  7. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies

    PubMed Central

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-01-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  8. Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

    SciTech Connect

    Dias, Marcio V.B.; Snee, William C.; Bromfield, Karen M.; Payne, Richard J.; Palaninathan, Satheesh K.; Ciulli, Alessio; Howard, Nigel I.; Abell, Chris; Sacchettini, James C.; Blundell, Tom L.

    2011-09-06

    The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form {pi}-stacking interactions with the catalytic Tyr{sup 24} have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.

  9. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms

    PubMed Central

    Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

    2015-01-01

    Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor–ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ. PMID:26170631

  10. Suppression of microRNAs by dual-targeting and clustered Tough Decoy inhibitors

    PubMed Central

    Hollensen, Anne Kruse; Bak, Rasmus O.; Haslund, Didde; Mikkelsen, Jacob Giehm

    2013-01-01

    MicroRNAs (miRNAs) are ubiquitous regulators of gene expression that contribute to almost any cellular process. Methods for managing of miRNA activity are attracting increasing attention in relation to diverse experimental and therapeutic applications. DNA-encoded miRNA inhibitors expressed from plasmid or virus-based vectors provide persistent miRNA suppression and options of tissue-directed micromanaging. In this report, we explore the potential of exploiting short, hairpin-shaped RNAs for simultaneous suppression of two or more miRNAs. Based on the “Tough Decoy” (TuD) design, we create dual-targeting hairpins carrying two miRNA recognition sites and demonstrate potent co-suppression of different pairs of unrelated miRNAs by a single DNA-encoded inhibitor RNA. In addition, enhanced miRNA suppression is achieved by expression of RNA polymerase II-transcribed inhibitors carrying clustered TuD hairpins with up to a total of eight miRNA recognition sites. Notably, by expressing clustered TuD inhibitors harboring a single recognition site for each of a total of six miRNAs, we document robust parallel suppression of multiple miRNAs by inhibitor RNA molecules encoded by a single expression cassette. These findings unveil a new potential of TuD-based miRNA inhibitors and pave the way for standardizing synchronized suppression of families or clusters of miRNAs. PMID:23324610

  11. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms.

    PubMed

    Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

    2015-01-01

    Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor-ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ.

  12. 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

  13. Identification of a small molecule HIV-1 inhibitor that targets the capsid hexamer.

    PubMed

    Xu, Jimmy P; Branson, Jeffrey D; Lawrence, Rae; Cocklin, Simon

    2016-02-01

    The HIV-1 CA protein is an attractive therapeutic target for the development of new antivirals. An inter-protomer pocket within the hexamer configuration of the CA, which is a binding site for key host dependency factors, is an especially appealing region for small molecule targeting. Using a field-based pharmacophore derived from an inhibitor known to interact with this region, coupled to biochemical and biological assessment, we have identified a new compound that inhibits HIV-1 infection and that targets the assembled CA hexamer. PMID:26747394

  14. Design, Synthesis and Evaluation of Fe-S Targeted Adenosine 5′-Phosphosulfate Reductase Inhibitors

    PubMed Central

    Paritala, Hanumantharao; Suzuki, Yuta; Carroll, Kate S.

    2015-01-01

    Adenosine 5′-phosphosulfate reductase (APR) is an iron-sulfur enzyme that is vital for survival of Mycobacterium tuberculosis during dormancy and is an attractive target for the treatment of latent tuberculosis (TB) infection. The 4Fe-4S cluster is coordinated to APR by sulfur atoms of four cysteine residues, is proximal to substrate, adenosine 5′-phopsphosulfate (APS), and is essential for catalytic activity. Herein, we present an approach for the development of a new class of APR inhibitors. As an initial step, we have employed an improved solid-phase chemistry method to prepare a series of N6-substituted adenosine analogues and their 5′-phosphates as well as adenosine 5′-phosphate diesters bearing different Fe and S binding groups, such as thiols or carboxylic and hydroxamic acid moieties. Evaluation of the resulting compounds indicates a clearly defined spacing requirement between the Fe-S targeting group and adenosine scaffold and that smaller Fe-S targeting groups are better tolerated. Molecular docking analysis suggests that the S atom of the most potent inhibitor may establish a favorable interaction with an S atom in the cluster. In summary, this study showcases an improved solid-phase method that expedites the preparation of adenosine and related 5′-phosphate derivatives and presents a unique Fe-S targeting strategy for the development of APR inhibitors. PMID:25710356

  15. Hsp90 Inhibitors as New Leads To Target Parasitic Diarrheal Diseases

    PubMed Central

    Shahinas, Dea; Bryant, Clifford; Hirata, Ken; Miyamoto, Yukiko; Hwang, Grace; Gut, Jiri; Renslo, Adam R.; Pillai, Dylan R.; Eckmann, Lars; Reed, Sharon L.; McKerrow, James H.

    2014-01-01

    Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis. PMID:24820073

  16. Immunosuppressive potency of mechanistic target of rapamycin inhibitors in solid-organ transplantation

    PubMed Central

    Baroja-Mazo, Alberto; Revilla-Nuin, Beatriz; Ramírez, Pablo; Pons, José A

    2016-01-01

    Mammalian target of rapamycin, also known as mechanistic target of rapamycin (mTOR) is a protein kinase that belongs to the PI3K/AKT/mTOR signaling pathway, which is involved in several fundamental cellular functions such as cell growth, proliferation, and survival. This protein and its associated pathway have been implicated in cancer development and the regulation of immune responses, including the rejection response generated following allograft transplantation. Inhibitors of mTOR (mTORi) such as rapamycin and its derivative everolimus are potent immunosuppressive drugs that both maintain similar rates of efficacy and could optimize the renal function and diminish the side effects compared with calcineurin inhibitors. These drugs are used in solid-organ transplantationtoinduceimmunosuppression while also promoting the expansion of CD4+CD25+FOXP3+ regulatory T-cells that could favor a scenery of immunological tolerance. In this review, we describe the mechanisms by which inhibitors of mTOR induce suppression by regulation of these pathways at different levels of the immune response. In addition, we particularly emphasize about the main methods that are used to assess the potency of immunosuppressive drugs, highlighting the studies carried out about immunosuppressive potency of inhibitors of mTOR. PMID:27011916

  17. Targeting Tat Inhibitors in the Assembly of Human Immunodeficiency Virus Type 1 Transcription Complexes▿ †

    PubMed Central

    D'Orso, Iván; Grunwell, Jocelyn R.; Nakamura, Robert L.; Das, Chandreyee; Frankel, Alan D.

    2008-01-01

    Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein, which relieves a block to elongation by recruiting an elongation factor, P-TEFb, to the viral promoter. Here, we report the discovery of potent Tat inhibitors that utilize a localization signal to target a dominant negative protein to its site of action. Fusing the Tat activation domain to some splicing factors, particularly to the Arg-Ser (RS) domain of U2AF65, creates Tat inhibitors that localize to subnuclear speckles, sites where pre-mRNA processing factors are stored for assembly into transcription complexes. A U2AF65 fusion named T-RS interacts with the nonphosphorylated C-terminal domain of RNA polymerase II (RNAP II) via its RS domain and is loaded into RNAP II holoenzyme complexes. T-RS is recruited efficiently to the HIV-1 promoter in a TAR-independent manner before RNAP II hyperphosphorylation but not to cellular promoters. The “preloading” of T-RS into HIV-1 preinitiation complexes prevents the entry of active Tat molecules, leaving the complexes in an elongation-incompetent state and effectively suppressing HIV-1 replication. The ability to deliver inhibitors to transcription complexes through the use of targeting/localization signals may provide new avenues for designing viral and transcription inhibitors. PMID:18667497

  18. Rational design of small molecule inhibitors targeting the Ras GEF, SOS1

    PubMed Central

    Evelyn, Chris R.; Duan, Xin; Biesiada, Jacek; Seibel, William L.; Meller, Jaroslaw; Zheng, Yi

    2014-01-01

    Summary Ras GTPases regulate intracellular signaling involved in cell proliferation. Elevated Ras signaling activity has been associated with human cancers. Ras activation is catalyzed by guanine-nucleotide exchange factors (GEFs), of which SOS1 is a major member that transduces receptor tyrosine kinase signaling to Ras. We have developed a rational approach coupling virtual screening with experimental screening in identifying small-molecule inhibitors targeting the catalytic site of SOS1 and SOS1-regulated Ras activity. A lead inhibitor, NSC-658497, is found to bind to SOS1, competitively suppresses SOS1-Ras interaction, and dose-dependently inhibits SOS1 GEF activity. Mutagenesis and structure-activity relationship studies map the NSC-658497 site of action to the SOS1 catalytic site, and define the chemical moieties in the inhibitor essential for the activity. NSC-658497 showed dose-dependent efficacy in inhibiting Ras, downstream signaling activities, and associated cell proliferation. These studies establish a proof of principle for rational design of small-molecule inhibitors targeting Ras GEF enzymatic activity. PMID:25455859

  19. Rational design of small molecule inhibitors targeting the Ras GEF, SOS1.

    PubMed

    Evelyn, Chris R; Duan, Xin; Biesiada, Jacek; Seibel, William L; Meller, Jaroslaw; Zheng, Yi

    2014-12-18

    Ras GTPases regulate intracellular signaling involved in cell proliferation. Elevated Ras signaling activity has been associated with human cancers. Ras activation is catalyzed by guanine nucleotide exchange factors (GEFs), of which SOS1 is a major member that transduces receptor tyrosine kinase signaling to Ras. We have developed a rational approach coupling virtual screening with experimental screening in identifying small-molecule inhibitors targeting the catalytic site of SOS1 and SOS1-regulated Ras activity. A lead inhibitor, NSC-658497, was found to bind to SOS1, competitively suppress SOS1-Ras interaction, and dose-dependently inhibit SOS1 GEF activity. Mutagenesis and structure-activity relationship studies map the NSC-658497 site of action to the SOS1 catalytic site, and define the chemical moieties in the inhibitor essential for the activity. NSC-658497 showed dose-dependent efficacy in inhibiting Ras, downstream signaling activities, and associated cell proliferation. These studies establish a proof of principle for rational design of small-molecule inhibitors targeting Ras GEF enzymatic activity.

  20. Tyrosine kinase inhibitors target cancer stem cells in renal cell cancer.

    PubMed

    Czarnecka, Anna M; Solarek, Wojciech; Kornakiewicz, Anna; Szczylik, Cezary

    2016-03-01

    This study was designed to analyze the impact of multi-targeted tyrosine kinase inhibitors on the cancer stem cell subpopulation in renal cell cancer. The second objective was to evaluate the effect of tumor growth inhibition related to a tumor niche factor - oxygen deprivation - as hypoxia develops along with the anti-angiogenic activity of tyrosine kinase inhibitors in renal tumors. Cells were treated with tyrosine kinase inhibitors, sunitinib, sorafenib and axitinib, in 2D and 3D culture conditions. Cell proliferation along with drug toxicity were evaluated. It was shown that the proliferation rate of cancer stem cells was decreased by the tyrosine kinase inhibitors. The efficacy of the growth inhibition was limited by hypoxic conditions and 3D intratumoral cell-cell interactions. We conclude that understanding the complex molecular interaction feedback loops between differentiated cancer cells, cancer stem cells and the tumor microenvironment in 3D culture should aid the identification of novel treatment targets and to evalute the efficacy of renal cancer therapies. Cell-cell interaction may represent a critical microenvironmental factor regulating cancer stem cell self-renewal potential, enhancing the stem cell phenotype and limiting drug toxicity. At the same time the role of hypoxia in renal cancer stem cell biology is also significant.

  1. Inhibitors of emerging epigenetic targets for cancer therapy: a patent review (2010-2014).

    PubMed

    Tanaka, Minoru; Roberts, Justin M; Qi, Jun; Bradner, James E

    2015-01-01

    Gene regulatory pathways comprise an emerging and active area of chemical probe discovery and investigational drug development. Emerging insights from cancer genome sequencing and chromatin biology have identified leveraged opportunities for development of chromatin-directed small molecules as cancer therapies. At present, only six agents in two epigenetic target classes have been approved by the US FDA, limited to treatment of hematological malignancies. Recently, new classes of epigenetic inhibitors have appeared in literatures. First-in-class compounds have successfully transitioned to clinical investigation, importantly also in solid tumors and pediatric malignancies. This review considers patent applications for small-molecule inhibitors of selected epigenetic targets from 2010 to 2014. Included are exemplary classes of chromatin-associated epigenomic writers (DOT1L and EZH2), erasers (LSD1) and readers (BRD4).

  2. The RNA Polymerase ‘‘Switch Region’’ Is a Target for Inhibitors

    SciTech Connect

    Mukhopadhyay, J.; Das, K; Ismail, S; Koppstein, D; Jang, M; Hudson, B; Sarafianos, S; Tuske, S; Patel, J; et. al.

    2008-01-01

    The ?-pyrone antibiotic myxopyronin (Myx) inhibits bacterial RNA polymerase (RNAP). Here, through a combination of genetic, biochemical, and structural approaches, we show that Myx interacts with the RNAP 'switch region'-the hinge that mediates opening and closing of the RNAP active center cleft-to prevent interaction of RNAP with promoter DNA. We define the contacts between Myx and RNAP and the effects of Myx on RNAP conformation and propose that Myx functions by interfering with opening of the RNAP active-center cleft during transcription initiation. We further show that the structurally related ?-pyrone antibiotic corallopyronin (Cor) and the structurally unrelated macrocyclic-lactone antibiotic ripostatin (Rip) function analogously to Myx. The RNAP switch region is distant from targets of previously characterized RNAP inhibitors, and, correspondingly, Myx, Cor, and Rip do not exhibit crossresistance with previously characterized RNAP inhibitors. The RNAP switch region is an attractive target for identification of new broad-spectrum antibacterial therapeutic agents.

  3. Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor.

    PubMed

    Christensen, Camilla L; Kwiatkowski, Nicholas; Abraham, Brian J; Carretero, Julian; Al-Shahrour, Fatima; Zhang, Tinghu; Chipumuro, Edmond; Herter-Sprie, Grit S; Akbay, Esra A; Altabef, Abigail; Zhang, Jianming; Shimamura, Takeshi; Capelletti, Marzia; Reibel, Jakob B; Cavanaugh, Jillian D; Gao, Peng; Liu, Yan; Michaelsen, Signe R; Poulsen, Hans S; Aref, Amir R; Barbie, David A; Bradner, James E; George, Rani E; Gray, Nathanael S; Young, Richard A; Wong, Kwok-Kin

    2014-12-01

    Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive to transcription-targeting drugs, in particular to THZ1, a recently identified covalent inhibitor of cyclin-dependent kinase 7. We find that expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a prototype drug for tailored SCLC therapy.

  4. Characterization of a serine hydrolase targeted by acyl-protein thioesterase inhibitors in Toxoplasma gondii.

    PubMed

    Kemp, Louise E; Rusch, Marion; Adibekian, Alexander; Bullen, Hayley E; Graindorge, Arnault; Freymond, Céline; Rottmann, Matthias; Braun-Breton, Catherine; Baumeister, Stefan; Porfetye, Arthur T; Vetter, Ingrid R; Hedberg, Christian; Soldati-Favre, Dominique

    2013-09-20

    In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein. PMID:23913689

  5. Discovery of covalent inhibitors of glyceraldehyde-3-phosphate dehydrogenase, a target for the treatment of malaria.

    PubMed

    Bruno, Stefano; Pinto, Andrea; Paredi, Gianluca; Tamborini, Lucia; De Micheli, Carlo; La Pietra, Valeria; Marinelli, Luciana; Novellino, Ettore; Conti, Paola; Mozzarelli, Andrea

    2014-09-11

    We developed a new class of covalent inhibitors of Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase, a validated target for the treatment of malaria, by screening a small library of 3-bromo-isoxazoline derivatives that inactivate the enzyme through a covalent, selective bond to the catalytic cysteine, as demonstrated by mass spectrometry. Substituents on the isoxazolinic ring modulated the potency up to 20-fold, predominantly due to an electrostatic effect, as assessed by computational analysis. PMID:25137375

  6. Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

    PubMed Central

    Kemp, Louise E.; Rusch, Marion; Adibekian, Alexander; Bullen, Hayley E.; Graindorge, Arnault; Freymond, Céline; Rottmann, Matthias; Braun-Breton, Catherine; Baumeister, Stefan; Porfetye, Arthur T.; Vetter, Ingrid R.; Hedberg, Christian; Soldati-Favre, Dominique

    2013-01-01

    In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein. PMID:23913689

  7. Kits in Motion

    ERIC Educational Resources Information Center

    Gee, Maureen

    1975-01-01

    Discusses three kits developed by museums in British Columbia for use in rural classrooms. The science kit on marine biology consists of modules which included specimens, books, audiovisual materials and student activities. (BR)

  8. Biodistribution of Fracture-Targeted GSK3β Inhibitor-Loaded Micelles for Improved Fracture Healing.

    PubMed

    Low, Stewart A; Galliford, Chris V; Yang, Jiyuan; Low, Philip S; Kopeček, Jindřich

    2015-10-12

    Bone fractures constitute a major cause of morbidity and mortality especially in the elderly. Complications associated with osteoporosis drugs and the age of the patient slow bone turnover and render such fractures difficult to heal. Increasing the speed of fracture repair by administration of a fracture-targeted bone anabolic agent could find considerable application. Aspartic acid oligopeptides are negatively charged molecules at physiological pH that adsorb to hydroxyapatite, the mineral portion of bone. This general adsorption is the strongest where bone turnover is highest or where hydroxyapatite is freshly exposed. Importantly, both of these conditions are prominent at fracture sites. GSK3β inhibitors are potent anabolic agents that can promote tissue repair when concentrated in a damaged tissue. Unfortunately, they can also cause significant toxicity when administered systemically and are furthermore difficult to deliver due to their strong hydrophobicity. In this paper, we solve both problems by conjugating the hydrophobic GSK3β inhibitor to a hydrophilic aspartic acid octapeptide using a hydrolyzable bond, thereby generating a bone fracture-targeted water-soluble form of the drug. The resulting amphiphile is shown to assemble into micelles, extending its circulation time while maintaining its fracture-targeting abilities. For measurement of pharmacokinetics, an 125I was introduced at the location of the bromine in the GSK3β inhibitor to minimize any structural differences. Biodistribution studies demonstrate a greater than 4-fold increase in fracture accumulation over healthy bone. PMID:26331790

  9. Biodistribution of fracture-targeted GSK3β inhibitor-loaded micelles for improved fracture healing

    PubMed Central

    Low, Stewart A.; Galliford, Chris V.; Yang, Jiyuan; Low, Philip S.; Kopeček, Jindřich

    2016-01-01

    Bone fractures constitute a major cause of morbidity and mortality especially in the elderly. Complications associated with osteoporosis drugs and the age of the patient slow bone turnover and render such fractures difficult to heal. Increasing the speed of fracture repair by administration of a fracture-targeted bone anabolic agent could find considerable application. Aspartic acid oligopeptides are negatively charged molecules at physiological pH that adsorb to hydroxyapatite, the mineral portion of bone. This general adsorption is the strongest where bone turnover is highest or where hydroxyapatite is freshly exposed. Importantly, both of these conditions are prominent at fracture sites. GSK3β inhibitors are potent anabolic agents that can promote tissue repair when concentrated in a damaged tissue. Unfortunately, they can also cause significant toxicity when administered systemically and are furthermore difficult to deliver due to their strong hydrophobicity. In this paper, we solve both problems by conjugating the hydrophobic GSK3β inhibitor to a hydrophilic aspartic acid octapeptide using a hydrolyzable bond, thereby generating a bone fracture-targeted water-soluble form of the drug. The resulting amphiphile is shown to assemble into micelles, extending its circulation time while maintaining its fracture-targeting abilities. For measurement of pharmacokinetics, an 125I was introduced at the location of the bromine in the GSK3β inhibitor to minimize any structural differences. Biodistribution studies demonstrate a greater than 4-fold increase in fracture accumulation over healthy bone. PMID:26331790

  10. Identification of alsterpaullone as a novel small molecule inhibitor to target group 3 medulloblastoma

    PubMed Central

    Faria, Claudia C.; Agnihotri, Sameer; Mack, Stephen C.; Golbourn, Brian J.; Diaz, Roberto J.; Olsen, Samantha; Bryant, Melissa; Bebenek, Matthew; Wang, Xin; Bertrand, Kelsey C.; Kushida, Michelle; Head, Renee; Clark, Ian; Dirks, Peter; Smith, Christian A.; Taylor, Michael D.; Rutka, James T.

    2015-01-01

    Advances in the molecular biology of medulloblastoma revealed four genetically and clinically distinct subgroups. Group 3 medulloblastomas are characterized by frequent amplifications of the oncogene MYC, a high incidence of metastasis, and poor prognosis despite aggressive therapy. We investigated several potential small molecule inhibitors to target Group 3 medulloblastomas based on gene expression data using an in silico drug screen. The Connectivity Map (C-MAP) analysis identified piperlongumine as the top candidate drug for non-WNT medulloblastomas and the cyclin-dependent kinase (CDK) inhibitor alsterpaullone as the compound predicted to have specific antitumor activity against Group 3 medulloblastomas. To validate our findings we used these inhibitors against established Group 3 medulloblastoma cell lines. The C-MAP predicted drugs reduced cell proliferation in vitro and increased survival in Group 3 medulloblastoma xenografts. Alsterpaullone had the highest efficacy in Group 3 medulloblastoma cells. Genomic profiling of Group 3 medulloblastoma cells treated with alsterpaullone confirmed inhibition of cell cycle-related genes, and down-regulation of MYC. Our results demonstrate the preclinical efficacy of using a targeted therapy approach for Group 3 medulloblastomas. Specifically, we provide rationale for advancing alsterpaullone as a targeted therapy in Group 3 medulloblastoma. PMID:26061748

  11. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    PubMed Central

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-01-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency. PMID:27432161

  12. Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells.

    PubMed

    Taipale, Mikko; Krykbaeva, Irina; Whitesell, Luke; Santagata, Sandro; Zhang, Jianming; Liu, Qingsong; Gray, Nathanael S; Lindquist, Susan

    2013-07-01

    The interaction between the HSP90 chaperone and its client kinases is sensitive to the conformational status of the kinase, and stabilization of the kinase fold by small molecules strongly decreases chaperone interaction. Here we exploit this observation and assay small-molecule binding to kinases in living cells, using chaperones as 'thermodynamic sensors'. The method allows determination of target specificities of both ATP-competitive and allosteric inhibitors in the kinases' native cellular context in high throughput. We profile target specificities of 30 diverse kinase inhibitors against >300 kinases. Demonstrating the value of the assay, we identify ETV6-NTRK3 as a target of the FDA-approved drug crizotinib (Xalkori). Crizotinib inhibits proliferation of ETV6-NTRK3-dependent tumor cells with nanomolar potency and induces the regression of established tumor xenografts in mice. Finally, we show that our approach is applicable to other chaperone and target classes by assaying HSP70/steroid hormone receptor and CDC37/kinase interactions, suggesting that chaperone interactions will have broad application in detecting drug-target interactions in vivo.

  13. Levitation Kits Demonstrate Superconductivity.

    ERIC Educational Resources Information Center

    Worthy, Ward

    1987-01-01

    Describes the "Project 1-2-3" levitation kit used to demonstrate superconductivity. Summarizes the materials included in the kit. Discusses the effect demonstrated and gives details on how to obtain kits. Gives an overview of the documentation that is included. (CW)

  14. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  15. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  16. Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

    SciTech Connect

    Hast, Michael A.; Nichols, Connie B.; Armstrong, Stephanie M.; Kelly, Shannon M.; Hellinga, Homme W.; Alspaugh, J. Andrew; Beese, Lorena S.

    2012-09-17

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

  17. 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.

  18. Computer-assisted identification of novel small molecule inhibitors targeting GLUT1

    NASA Astrophysics Data System (ADS)

    Wan, Zhining; Li, Xin; Sun, Rong; Li, Yuanyuan; Wang, Xiaoyun; Li, Xinru; Rong, Li; Shi, Zheng; Bao, Jinku

    2015-12-01

    Glucose transporters (GLUTs) are the main carriers of glucose that facilitate the diffusion of glucose in mammalian cells, especially GLUT1. Notably, GLUT1 is a rate-limiting transporter for glucose uptake, and its overexpression is a common characteristic in most cancers. Thus, the inhibition of GLUT1 by novel small compounds to lower glucose levels for cancer cells has become an emerging strategy. Herein, we employed high-throughput screening approaches to identify potential inhibitors against the sugar-binding site of GLUT1. Firstly, molecular docking screening was launched against the specs products, and three molecules (ZINC19909927, ZINC19908826, and ZINC19815451) were selected as candidate GLUT1 inhibitors for further analysis. Then, taking the initial ligand β-NG as a reference, molecular dynamic (MD) simulations and molecular mechanics/generalized born surface area (MM/GBSA) method were applied to evaluate the binding stability and affinity of the three candidates towards GLUT1. Finally, we found that ZINC19909927 might have the highest affinity to occupy the binding site of GLUT1. Meanwhile, energy decomposition analysis identified several residues located in substrate-binding site that might provide clues for future inhibitor discovery towards GLUT1. Taken together, these results in our study may provide valuable information for identifying new inhibitors targeting GLUT1-mediated glucose transport and metabolism for cancer therapeutics.

  19. Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers

    PubMed Central

    Mullarky, Edouard; Lucki, Natasha C.; Beheshti Zavareh, Reza; Anglin, Justin L.; Gomes, Ana P.; Nicolay, Brandon N.; Wong, Jenny C. Y.; Christen, Stefan; Takahashi, Hidenori; Singh, Pradeep K.; Blenis, John; Fendt, Sarah-Maria; Asara, John M.; DeNicola, Gina M.; Lyssiotis, Costas A.; Lairson, Luke L.; Cantley, Lewis C.

    2016-01-01

    Cancer cells reprogram their metabolism to promote growth and proliferation. The genetic evidence pointing to the importance of the amino acid serine in tumorigenesis is striking. The gene encoding the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first committed step of serine biosynthesis, is overexpressed in tumors and cancer cell lines via focal amplification and nuclear factor erythroid-2-related factor 2 (NRF2)-mediated up-regulation. PHGDH-overexpressing cells are exquisitely sensitive to genetic ablation of the pathway. Here, we report the discovery of a selective small molecule inhibitor of PHGDH, CBR-5884, identified by screening a library of 800,000 drug-like compounds. CBR-5884 inhibited de novo serine synthesis in cancer cells and was selectively toxic to cancer cell lines with high serine biosynthetic activity. Biochemical characterization of the inhibitor revealed that it was a noncompetitive inhibitor that showed a time-dependent onset of inhibition and disrupted the oligomerization state of PHGDH. The identification of a small molecule inhibitor of PHGDH not only enables thorough preclinical evaluation of PHGDH as a target in cancers, but also provides a tool with which to study serine metabolism. PMID:26831078

  20. The BIRC6 gene as a novel target for therapy of prostate cancer: dual targeting of inhibitors of apoptosis

    PubMed Central

    Iris Luk, Sze Ue; Xue, Hui; Cheng, Hongwei; Lin, Dong; Gout, Peter W.; Fazli, Ladan; Collins, Colin C.; Gleave, Martin E.; Wang, Yuzhuo

    2014-01-01

    Treatment resistance, the major challenge in the management of advanced prostate cancer, is in part based on resistance to apoptosis. The Inhibitor of Apoptosis (IAP) protein family is thought to play key roles in survival and drug resistance of cancer via inhibition of apoptosis. Of the IAP family members, cIAP1, cIAP2, XIAP and survivin are known to be up-regulated in prostate cancer. BIRC6, a much less studied IAP member, was recently shown to be elevated in castration-resistant prostate cancer (CRPC). In the present study, we showed a correlation between elevated BIRC6 expression in clinical prostate cancer specimens and poor patient prognostic factors, as well as co-upregulation of certain IAP members. In view of this, we designed antisense oligonucleotides that simultaneously target BIRC6 and another co-upregulated IAP member (dASOs). Two dASOs, targeting BIRC6+cIAP1 and BIRC6+survivin, showed substantial inhibition of CRPC cell proliferation, exceeding that obtained with single BIRC6 targeting. The growth inhibition was associated with increased apoptosis, cell cycle arrest and suppression of NFkB activation. Moreover, treatment with either dASO led to significantly lower viable tumor volume in vivo, without major host toxicity. This study shows that BIRC6-based dual IAP-targeting ASOs represent potential novel therapeutic agents against advanced prostate cancer. PMID:25071009

  1. Development of antibody-based c-Met inhibitors for targeted cancer therapy.

    PubMed

    Lee, Dongheon; Sung, Eun-Sil; Ahn, Jin-Hyung; An, Sungwon; Huh, Jiwon; You, Weon-Kyoo

    2015-01-01

    Signaling pathways mediated by receptor tyrosine kinases (RTKs) and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics.

  2. Targeting the Sonic Hedgehog Signaling Pathway: Review of Smoothened and GLI Inhibitors

    PubMed Central

    Rimkus, Tadas K.; Carpenter, Richard L.; Qasem, Shadi; Chan, Michael; Lo, Hui-Wen

    2016-01-01

    The sonic hedgehog (Shh) signaling pathway is a major regulator of cell differentiation, cell proliferation, and tissue polarity. Aberrant activation of the Shh pathway has been shown in a variety of human cancers, including, basal cell carcinoma, malignant gliomas, medulloblastoma, leukemias, and cancers of the breast, lung, pancreas, and prostate. Tumorigenesis, tumor progression and therapeutic response have all been shown to be impacted by the Shh signaling pathway. Downstream effectors of the Shh pathway include smoothened (SMO) and glioma-associated oncogene homolog (GLI) family of zinc finger transcription factors. Both are regarded as important targets for cancer therapeutics. While most efforts have been devoted towards pharmacologically targeting SMO, developing GLI-targeted approach has its merit because of the fact that GLI proteins can be activated by both Shh ligand-dependent and -independent mechanisms. To date, two SMO inhibitors (LDE225/Sonidegib and GDC-0449/Vismodegib) have received FDA approval for treating basal cell carcinoma while many clinical trials are being conducted to evaluate the efficacy of this exciting class of targeted therapy in a variety of cancers. In this review, we provide an overview of the biology of the Shh pathway and then detail the current landscape of the Shh-SMO-GLI pathway inhibitors including those in preclinical studies and clinical trials. PMID:26891329

  3. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  4. The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer

    PubMed Central

    Sadowski, Martin C.; Pouwer, Rebecca H.; Gunter, Jennifer H.; Lubik, Amy A.; Quinn, Ronald J.; Nelson, Colleen C.

    2014-01-01

    Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer. PMID:25313139

  5. Targeting insulin amyloid assembly by small aromatic molecules: toward rational design of aggregation inhibitors.

    PubMed

    Levy-Sakin, Michal; Shreberk, Michal; Daniel, Yael; Gazit, Ehud

    2009-01-01

    Amyloid fibril formation is a common event in more than twenty human diseases and in some normal physiological processes. The mechanism of this ordered aggregation process and the molecular forces driving it are therefore of great importance. One of the strategies used in this field is targeting the fibrillization process by different factors, like, short peptides, organic molecules, etc. Here, we targeted insulin fibril formation by a range of small aromatic molecules, with different numbers of aromatic rings and various substituent groups. Using Thioflavin T fluorescence assay and transmission electron microscopy, we found that all dicyclic and tricyclic compounds in our screen were efficient inhibitors of insulin fibril formation. A common notion regarding amyloid inhibitors is that two functional groups are essentials for interfering with the amyloid formation process; a recognition motif and a bulky group for inducing a steric interference. However, here, we showed that some monocyclic compounds as small as toluene were also found to inhibit fibrillization. In addition, we found that substituent of benzene ring have a great influence on the inhibitory potency. Specifically, cyano, methyl and nitro groups increased the inhibitory potency. The results introduced here may contribute to future rational design of amyloid inhibitors.

  6. Effective Targeting of Estrogen Receptor Negative Breast Cancers with the Protein Kinase D inhibitor CRT0066101

    PubMed Central

    Borges, Sahra; Perez, Edith A.; Thompson, E. Aubrey; Radisky, Derek C.; Geiger, Xochiquetzal J.; Storz, Peter

    2015-01-01

    Invasive ductal carcinomas (IDCs) of the breast are associated with altered expression of hormone receptors (HR), amplification or overexpression of HER2, or a triple-negative phenotype. The most aggressive cases of IDC are characterized by a high proliferation rate, a great propensity to metastasize and their ability to resist to standard chemotherapy, hormone therapy or HER2 targeted therapy. Using progression tissue microarrays we here demonstrate that the serine/threonine kinase Protein Kinase D3 (PKD3) is highly up-regulated in estrogen receptor (ER)-negative tumors. We identify direct binding of the estrogen receptor to the PRKD3 gene promoter as a mechanism of inhibition of PKD3 expression. Loss of ER results in upregulation of PKD3 leading to all hallmarks of aggressive IDC, including increased cell proliferation, migration and invasion. This identifies ER-negative breast cancers as ideal for treatment with the PKD inhibitor CRT0066101. We show that similar to a knockdown of PKD3, treatment with this inhibitor targets all tumorigenic processes in vitro and decreases growth of primary tumors and metastasis in vivo. Our data strongly support the development of PKD inhibitors for clinical use for ER-negative breast cancers, including the triple-negative phenotype. PMID:25852060

  7. Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.

    PubMed

    Tso, Shih-Chia; Qi, Xiangbing; Gui, Wen-Jun; Wu, Cheng-Yang; Chuang, Jacinta L; Wernstedt-Asterholm, Ingrid; Morlock, Lorraine K; Owens, Kyle R; Scherer, Philipp E; Williams, Noelle S; Tambar, Uttam K; Wynn, R Max; Chuang, David T

    2014-02-14

    Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 μM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes. PMID:24356970

  8. The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer.

    PubMed

    Sadowski, Martin C; Pouwer, Rebecca H; Gunter, Jennifer H; Lubik, Amy A; Quinn, Ronald J; Nelson, Colleen C

    2014-10-15

    Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer.

  9. An Intriguing Correlation Based on the Superimposition of Residue Pairs with Inhibitors that Target Protein-Protein Interfaces

    PubMed Central

    Nakadai, Masakazu; Tomida, Shuta; Sekimizu, Kazuhisa

    2016-01-01

    Druggable sites on protein-protein interfaces are difficult to predict. To survey inhibitor-binding sites onto which residues are superimposed at protein-protein interfaces, we analyzed publicly available information for 39 inhibitors that target the protein-protein interfaces of 8 drug targets. By focusing on the differences between residues that were superimposed with inhibitors and non-superimposed residues, we observed clear differences in the distances and changes in the solvent-accessible surface areas (∆SASA). Based on the observation that two or more residues were superimposed onto inhibitors in 37 (95%) of 39 protein-inhibitor complexes, we focused on the two-residue relationships. Application of a cross-validation procedure confirmed a linear negative correlation between the absolute value of the dihedral angle and the sum of the ∆SASAs of the residues. Finally, we applied the regression equation of this correlation to four inhibitors that bind to new sites not bound by the 39 inhibitors as well as additional inhibitors of different targets. Our results shed light on the two-residue correlation between the absolute value of the dihedral angle and the sum of the ∆SASA, which may be a useful relationship for identifying the key two-residues as potential targets of protein-protein interfaces. PMID:26730437

  10. Programmed activation of cancer cell apoptosis: A tumor-targeted phototherapeutic topoisomerase I inhibitor.

    PubMed

    Shin, Weon Sup; Han, Jiyou; Kumar, Rajesh; Lee, Gyung Gyu; Sessler, Jonathan L; Kim, Jong-Hoon; Kim, Jong Seung

    2016-01-01

    We report here a tumor-targeting masked phototherapeutic agent 1 (PT-1). This system contains SN-38-a prodrug of the topoisomerase I inhibitor irinotecan. Topoisomerase I is a vital enzyme that controls DNA topology during replication, transcription, and recombination. An elevated level of topoisomerase I is found in many carcinomas, making it an attractive target for the development of effective anticancer drugs. In addition, PT-1 contains both a photo-triggered moiety (nitrovanillin) and a cancer targeting unit (biotin). Upon light activation in cancer cells, PT-1 interferes with DNA re-ligation, diminishes the expression of topoisomerase I, and enhances the expression of inter alia mitochondrial apoptotic genes, death receptors, and caspase enzymes, inducing DNA damage and eventually leading to apoptosis. In vitro and in vivo studies showed significant inhibition of cancer growth and the hybrid system PT-1 thus shows promise as a programmed photo-therapeutic ("phototheranostic"). PMID:27374023

  11. Programmed activation of cancer cell apoptosis: A tumor-targeted phototherapeutic topoisomerase I inhibitor

    NASA Astrophysics Data System (ADS)

    Shin, Weon Sup; Han, Jiyou; Kumar, Rajesh; Lee, Gyung Gyu; Sessler, Jonathan L.; Kim, Jong-Hoon; Kim, Jong Seung

    2016-07-01

    We report here a tumor-targeting masked phototherapeutic agent 1 (PT-1). This system contains SN-38—a prodrug of the topoisomerase I inhibitor irinotecan. Topoisomerase I is a vital enzyme that controls DNA topology during replication, transcription, and recombination. An elevated level of topoisomerase I is found in many carcinomas, making it an attractive target for the development of effective anticancer drugs. In addition, PT-1 contains both a photo-triggered moiety (nitrovanillin) and a cancer targeting unit (biotin). Upon light activation in cancer cells, PT-1 interferes with DNA re-ligation, diminishes the expression of topoisomerase I, and enhances the expression of inter alia mitochondrial apoptotic genes, death receptors, and caspase enzymes, inducing DNA damage and eventually leading to apoptosis. In vitro and in vivo studies showed significant inhibition of cancer growth and the hybrid system PT-1 thus shows promise as a programmed photo-therapeutic (“phototheranostic”).

  12. Programmed activation of cancer cell apoptosis: A tumor-targeted phototherapeutic topoisomerase I inhibitor

    PubMed Central

    Shin, Weon Sup; Han, Jiyou; Kumar, Rajesh; Lee, Gyung Gyu; Sessler, Jonathan L.; Kim, Jong-Hoon; Kim, Jong Seung

    2016-01-01

    We report here a tumor-targeting masked phototherapeutic agent 1 (PT-1). This system contains SN-38—a prodrug of the topoisomerase I inhibitor irinotecan. Topoisomerase I is a vital enzyme that controls DNA topology during replication, transcription, and recombination. An elevated level of topoisomerase I is found in many carcinomas, making it an attractive target for the development of effective anticancer drugs. In addition, PT-1 contains both a photo-triggered moiety (nitrovanillin) and a cancer targeting unit (biotin). Upon light activation in cancer cells, PT-1 interferes with DNA re-ligation, diminishes the expression of topoisomerase I, and enhances the expression of inter alia mitochondrial apoptotic genes, death receptors, and caspase enzymes, inducing DNA damage and eventually leading to apoptosis. In vitro and in vivo studies showed significant inhibition of cancer growth and the hybrid system PT-1 thus shows promise as a programmed photo-therapeutic (“phototheranostic”). PMID:27374023

  13. H3K36 methyltransferases as cancer drug targets: rationale and perspectives for inhibitor development.

    PubMed

    Rogawski, David S; Grembecka, Jolanta; Cierpicki, Tomasz

    2016-09-01

    Methylation at histone 3, lysine 36 (H3K36) is a conserved epigenetic mark regulating gene transcription, alternative splicing and DNA repair. Genes encoding H3K36 methyltransferases (KMTases) are commonly overexpressed, mutated or involved in chromosomal translocations in cancer. Molecular biology studies have demonstrated that H3K36 KMTases regulate oncogenic transcriptional programs. Structural studies of the catalytic SET domain of H3K36 KMTases have revealed intriguing opportunities for design of small molecule inhibitors. Nevertheless, potent inhibitors for most H3K36 KMTases have not yet been developed, underlining the challenges associated with this target class. As we now have strong evidence linking H3K36 KMTases to cancer, drug development efforts are predicted to yield novel compounds in the near future. PMID:27548565

  14. 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.

  15. Targeting abnormal DNA double strand break repair in tyrosine kinase inhibitor-resistant chronic myeloid leukemias

    PubMed Central

    Tobin, Lisa A.; Robert, Carine; Rapoport, Aaron P.; Gojo, Ivana; Baer, Maria R.; Tomkinson, Alan E.; Rassool, Feyruz V.

    2013-01-01

    Resistance to imatinib (IM) and other BCR-ABL1 tyrosine kinase inhibitors (TKI)s is an increasing problem in leukemias caused by expression of BCR-ABL1. Since chronic myeloid leukemia (CML) cell lines expressing BCR-ABL1 utilize an alternative non-homologous end-joining pathway (ALT NHEJ) to repair DNA double strand breaks (DSB)s, we asked whether this repair pathway is a novel therapeutic target in TKI-resistant disease. Notably, the steady state levels of two ALT NHEJ proteins, poly-(ADP-ribose) polymerase 1 (PARP1) and DNA ligase IIIα were increased in the BCR-ABL1-positive CML cell line K562 and, to a greater extent, in its imatinib resistant (IMR) derivative. Incubation of these cell lines with a combination of DNA ligase and PARP inhibitors inhibited ALT NHEJ and selectively decreased survival with the effect being greater in the IMR derivative. Similar results were obtained with TKI-resistant derivatives of two hematopoietic cell lines that had been engineered to stably express BCR-ABL1. Together our results show that the sensitivity of cell lines expressing BCR-ABL1 to the combination of DNA ligase and PARP inhibitors correlates with the steady state levels of PARP1 and DNA ligase IIIα, and ALT NHEJ activity. Importantly, analysis of clinical samples from CML patients confirmed that the expression levels of PARP1 and DNA ligase IIIα correlated with sensitivity to the DNA repair inhibitor combination. Thus, the expression levels of PARP1 and DNA ligase IIIα serve as biomarkers to identify a subgroup of CML patients who may be candidates for therapies that target the ALT NHEJ pathway when treatment with TKIs has failed. PMID:22641215

  16. PI-3K Inhibitors Preferentially Target CD15+ Cancer Stem Cell Population in SHH Driven Medulloblastoma

    PubMed Central

    Singh, Alok R.; Joshi, Shweta; Zulcic, Muamera; Alcaraz, Michael; Garlich, Joseph R.; Morales, Guillermo A.; Cho, Yoon J.; Bao, Lei; Levy, Michael L.; Newbury, Robert; Malicki, Denise; Messer, Karen; Crawford, John; Durden, Donald L.

    2016-01-01

    Sonic hedgehog (SHH) medulloblastoma (MB) subtype is driven by a proliferative CD15+ tumor propagating cell (TPC), also considered in the literature as a putative cancer stem cell (CSC). Despite considerable research, much of the biology of this TPC remains unknown. We report evidence that phosphatase and tensin homolog (PTEN) and phosphoinositide 3-kinase (PI-3K) play a crucial role in the propagation, survival and potential response to therapy in this CD15+ CSC/TPC-driven malignant disease. Using the ND2-SmoA1 transgenic mouse model for MB, mouse genetics and patient-derived xenografts (PDXs), we demonstrate that the CD15+TPCs are 1) obligately required for SmoA1Tg-driven tumorigenicity 2) regulated by PTEN and PI-3K signaling 3) selectively sensitive to the cytotoxic effects of pan PI-3K inhibitors in vitro and in vivo but resistant to chemotherapy 4) in the SmoA1Tg mouse model are genomically similar to the SHH human MB subgroup. The results provide the first evidence that PTEN plays a role in MB TPC signaling and biology and that PI-3K inhibitors target and suppress the survival and proliferation of cells within the mouse and human CD15+ cancer stem cell compartment. In contrast, CD15+ TPCs are resistant to cisplatinum, temozolomide and the SHH inhibitor, NVP-LDE-225, agents currently used in treatment of medulloblastoma. These studies validate the therapeutic efficacy of pan PI-3K inhibitors in the treatment of CD15+ TPC dependent medulloblastoma and suggest a sequential combination of PI-3K inhibitors and chemotherapy will have augmented efficacy in the treatment of this disease. PMID:26938241

  17. PI-3K Inhibitors Preferentially Target CD15+ Cancer Stem Cell Population in SHH Driven Medulloblastoma.

    PubMed

    Singh, Alok R; Joshi, Shweta; Zulcic, Muamera; Alcaraz, Michael; Garlich, Joseph R; Morales, Guillermo A; Cho, Yoon J; Bao, Lei; Levy, Michael L; Newbury, Robert; Malicki, Denise; Messer, Karen; Crawford, John; Durden, Donald L

    2016-01-01

    Sonic hedgehog (SHH) medulloblastoma (MB) subtype is driven by a proliferative CD15+ tumor propagating cell (TPC), also considered in the literature as a putative cancer stem cell (CSC). Despite considerable research, much of the biology of this TPC remains unknown. We report evidence that phosphatase and tensin homolog (PTEN) and phosphoinositide 3-kinase (PI-3K) play a crucial role in the propagation, survival and potential response to therapy in this CD15+ CSC/TPC-driven malignant disease. Using the ND2-SmoA1 transgenic mouse model for MB, mouse genetics and patient-derived xenografts (PDXs), we demonstrate that the CD15+TPCs are 1) obligately required for SmoA1Tg-driven tumorigenicity 2) regulated by PTEN and PI-3K signaling 3) selectively sensitive to the cytotoxic effects of pan PI-3K inhibitors in vitro and in vivo but resistant to chemotherapy 4) in the SmoA1Tg mouse model are genomically similar to the SHH human MB subgroup. The results provide the first evidence that PTEN plays a role in MB TPC signaling and biology and that PI-3K inhibitors target and suppress the survival and proliferation of cells within the mouse and human CD15+ cancer stem cell compartment. In contrast, CD15+ TPCs are resistant to cisplatinum, temozolomide and the SHH inhibitor, NVP-LDE-225, agents currently used in treatment of medulloblastoma. These studies validate the therapeutic efficacy of pan PI-3K inhibitors in the treatment of CD15+ TPC dependent medulloblastoma and suggest a sequential combination of PI-3K inhibitors and chemotherapy will have augmented efficacy in the treatment of this disease. PMID:26938241

  18. ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines

    SciTech Connect

    Zask, Arie; Verheijen, Jeroen C.; Curran, Kevin; Kaplan, Joshua; Richard, David J.; Nowak, Pawel; Malwitz, David J.; Brooijmans, Natasja; Bard, Joel; Svenson, Kristine; Lucas, Judy; Toral-Barza, Lourdes; Zhang, Wei-Guo; Hollander, Irwin; Gibbons, James J.; Abraham, Robert T.; Ayral-Kaloustian, Semiramis; Mansour, Tarek S.; Yu, Ker

    2009-09-18

    The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.

  19. Mammary-Derived Growth Inhibitor Targeting Peptide-Modified PEG-PLA Nanoparticles for Enhanced Targeted Glioblastoma Therapy.

    PubMed

    Feng, Xingye; Gao, Xiaoling; Kang, Ting; Jiang, Di; Yao, Jianhui; Jing, Yixian; Song, Qingxiang; Jiang, Xinguo; Liang, Jianying; Chen, Jun

    2015-08-19

    Targeting delivery of chemotherapeutics to neovasculature represents a promising means for tumor therapy since angiogenesis has been a featured hallmark of glioblastma. However, anti-angiogenic therapy would induce the occurrence of metastatic tumor and even neoplasm recurrence. Simultaneous targeting of tumor cells and neovasculature perfectly overcome such defects and has been proven to be an efficacious strategy for suppressing tumor growth. In the present study, a tumor homing peptide CooP selective binding to mammary-derived growth inhibitor that overexpressed in glioma cells and blood vessel endothelial cells was decorated on the surface of paclitaxel-loading PEG-PLA nanoparticles (NP-PTX) to obtain the dual targeting nanovector CooP-NP-PTX. In vitro antiproliferation study showed that HUVEC cells and U87MG cells were much more sensitive to CooP-NP-PTX than NP-PTX. In vivo imaging demonstrated that CooP-NP accumulated more selectively and penetrated deeper into the tumor site. In addition, the glioma-bearing mice treated with CooP-NP-PTX achieved the longest survival time compared to NP-PTX and Taxol. The findings observed above indicated that CooP peptide-functionalized anti-neoplastic agent-loaded nanoparticles might possess promising potential for glioblastoma therapy. PMID:26222392

  20. Targeting angiogenesis: a review of angiogenesis inhibitors in the treatment of lung cancer.

    PubMed

    Sridhar, Srikala S; Shepherd, Frances A

    2003-12-01

    It has now been almost 30 years since Dr J. Folkman first proposed that inhibition of angiogenesis could play a key role in treating cancer; however, it is only recently that anti-angiogenesis agents have entered the clinical setting. The search for novel therapies is particularly important in lung cancer, where the majority of patients succumb to their disease despite aggressive treatments. Several classes of agents now exist that target the different steps involved in angiogenesis. These include drugs inhibiting matrix breakdown, the matrix metalloproteinase inhibitors (MMPIs), such as marimastat, prinomastat, BMS275291, BAY12-9566, and neovastat drugs that block endothelial cell signaling via vascular endothelial growth factor (VEGF) and its receptor (VEGFR) including rhuMAb VEGF, SU5416, SU6668, ZD6474, CP-547,632 and ZD4190. Drugs that are similar to endogenous inhibitors of angiogenesis including endostatin, angiostatin and interferons. There has also been renewed interest in thalidomide. Drugs such as squalamine, celecoxib, ZD6126, TNP-470 and those targeting the integrins are also being evaluated in lung cancer. Despite early enthusiasm for many of these agents, Phase III trials have not yet demonstrated significant increases in overall survival and toxicity remains an issue. It is hoped that as our understanding of the complex process of angiogenesis increases, so will our ability to design more effective targeted therapies. PMID:14611919

  1. Impacting tumor cell-fate by targeting the inhibitor of apoptosis protein survivin.

    PubMed

    Kelly, Ronan J; Lopez-Chavez, Ariel; Citrin, Deborah; Janik, John E; Morris, John C

    2011-04-06

    Survivin (BIRC5), a member of the inhibitor of apoptosis protein (IAP) family that inhibits caspases and blocks cell death is highly expressed in cancer and is associated with a poorer clinical outcome. Functioning simultaneously during cell division and apoptosis inhibition, survivin plays a pivotal role in determining cell survival. Survivin has consistently been identified by molecular profiling analysis to be associated with higher tumor grade, more advanced disease, abbreviated survival, accelerated rates of recurrence, and chemotherapy and radiation resistance. Survivin's differential expression in cancer compared to normal tissue and its role as a nodal protein in a number of cellular pathways make it a highly flexible therapeutic target, suitable for small-molecule inhibitiors, molecular antagonists, and vaccination-based therapies. By targeting survivin it is hoped that multiple tumor signaling circuitries may be simultaneously disabled. This effect may be applicable to many tumor histologies irrespective of specific genetic makeup. To date, survivin inhibitors have shown modest activity as single agents, but it is anticipated that when given in combination with cytotoxic chemotherapy or monoclonal antibodies they may exhibit enhanced efficacy. This review discusses the complex circuitry of survivin in human cancers and highlights clinical trials involving novel agents that target this important protein.

  2. En Route to New Therapeutic Options for Iron Overload Diseases: Matriptase-2 as a Target for Kunitz-Type Inhibitors.

    PubMed

    Beckmann, Anna-Madeleine; Maurer, Eva; Lülsdorff, Verena; Wilms, Annika; Furtmann, Norbert; Bajorath, Jürgen; Gütschow, Michael; Stirnberg, Marit

    2016-04-01

    The cell-surface serine protease matriptase-2 is a critical stimulator of iron absorption by negatively regulating hepcidin, the key hormone of iron homeostasis. Thus, it has attracted much attention as a target in primary and secondary iron overload diseases. Here, we have characterised Kunitz-type inhibitors hepatocyte growth factor activator inhibitor 1 (HAI-1) and HAI-2 as powerful, slow-binding matriptase-2 inhibitors. The binding modes of the matriptase-2-HAI complexes were suggested by molecular modelling. Different assays, including cell-free and cell-based measurements of matriptase-2 activity, determination of inhibition constants and evaluation of matriptase-2 inhibition by analysis of downstream effects in human liver cells, demonstrated that matriptase-2 is an excellent target for Kunitz inhibitors. In particular, HAI-2 is considered a promising scaffold for the design of potent and selective matriptase-2 inhibitors.

  3. Identification of a new JNK inhibitor targeting the JNK-JIP interaction site

    PubMed Central

    Stebbins, John L.; De, Surya K.; Machleidt, Thomas; Becattini, Barbara; Vazquez, Jesus; Kuntzen, Christian; Chen, Li-Hsing; Cellitti, Jason F.; Riel-Mehan, Megan; Emdadi, Aras; Solinas, Giovanni; Karin, Michael; Pellecchia, Maurizio

    2008-01-01

    JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics. PMID:18922779

  4. Histone deacetylase inhibitors disrupt the mitotic spindle assembly checkpoint by targeting histone and nonhistone proteins.

    PubMed

    Gabrielli, Brian; Brown, Mellissa

    2012-01-01

    Histone deacetylase inhibitors exhibit pleiotropic effects on cell functions, both in vivo and in vitro. One of the more dramatic effects of these drugs is their ability to disrupt normal mitotic division, which is a significant contributor to the anticancer properties of these drugs. The most important feature of the disrupted mitosis is that drug treatment overcomes the mitotic spindle assembly checkpoint and drives mitotic slippage, but in a manner that triggers apoptosis. The mechanism by which histone deacetylase inhibitors affect mitosis is now becoming clearer through the identification of a number of chromatin and nonchromatin protein targets that are critical to the regulation of normal mitotic progression and cell division. These proteins are directly regulated by acetylation and deacetylation, or in some cases indirectly through the acetylation of essential partner proteins. There appears to be little contribution from deacetylase inhibitor-induced transcriptional changes to the mitotic effects of these drugs. The overall mitotic phenotype of drug treatment appears to be the sum of these disrupted mechanisms. PMID:23088867

  5. Middle East respiratory syndrome coronavirus (MERS-CoV) entry inhibitors targeting spike protein.

    PubMed

    Xia, Shuai; Liu, Qi; Wang, Qian; Sun, Zhiwu; Su, Shan; Du, Lanying; Ying, Tianlei; Lu, Lu; Jiang, Shibo

    2014-12-19

    The recent outbreak of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) infection has led to more than 800 laboratory-confirmed MERS cases with a high case fatality rate (∼35%), posing a serious threat to global public health and calling for the development of effective and safe therapeutic and prophylactic strategies to treat and prevent MERS-CoV infection. Here we discuss the most recent studies on the structure of the MERS-CoV spike protein and its role in virus binding and entry, and the development of MERS-CoV entry/fusion inhibitors targeting the S1 subunit, particularly the receptor-binding domain (RBD), and the S2 subunit, especially the HR1 region, of the MERS-CoV spike protein. We then look ahead to future applications of these viral entry/fusion inhibitors, either alone or in combination with specific and nonspecific MERS-CoV replication inhibitors, for the treatment and prevention of MERS-CoV infection. PMID:25451066

  6. KSHV targeted therapy: an update on inhibitors of viral lytic replication.

    PubMed

    Coen, Natacha; Duraffour, Sophie; Snoeck, Robert; Andrei, Graciela

    2014-11-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing.

  7. Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors

    NASA Astrophysics Data System (ADS)

    Bhowmick, Tuhin; Ghosh, Soumitra; Dixit, Karuna; Ganesan, Varsha; Ramagopal, Udupi A.; Dey, Debayan; Sarma, Siddhartha P.; Ramakumar, Suryanarayanarao; Nagaraja, Valakunja

    2014-06-01

    The nucleoid-associated protein HU plays an important role in maintenance of chromosomal architecture and in global regulation of DNA transactions in bacteria. Although HU is essential for growth in Mycobacterium tuberculosis (Mtb), there have been no reported attempts to perturb HU function with small molecules. Here we report the crystal structure of the N-terminal domain of HU from Mtb. We identify a core region within the HU-DNA interface that can be targeted using stilbene derivatives. These small molecules specifically inhibit HU-DNA binding, disrupt nucleoid architecture and reduce Mtb growth. The stilbene inhibitors induce gene expression changes in Mtb that resemble those induced by HU deficiency. Our results indicate that HU is a potential target for the development of therapies against tuberculosis.

  8. The replication focus targeting sequence (RFTS) domain is a DNA-competitive inhibitor of Dnmt1.

    PubMed

    Syeda, Farisa; Fagan, Rebecca L; Wean, Matthew; Avvakumov, George V; Walker, John R; Xue, Sheng; Dhe-Paganon, Sirano; Brenner, Charles

    2011-04-29

    Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.

  9. The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1

    SciTech Connect

    Syeda, Farisa; Fagan, Rebecca L.; Wean, Matthew; Avvakumov, George V.; Walker, John R.; Xue, Sheng; Dhe-Paganon, Sirano; Brenner, Charles

    2015-11-30

    Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA- and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.

  10. Effect of cell division inhibitors on polyclonal activation can vary according to the target cell used.

    PubMed

    Löwy, I; Chedid, L

    1979-01-01

    The effects of inhibitors of cell division on polyclonal stimulation induced either by bacterial lipopolysaccharide (LPS) or by a synthetic adjuvant, MDP, were compared, using different target cells. Doses of colchicine that prevented 3H-thymidine incorporation also prevented the induction of antibodies against TNP and against an altered self antigen: bromelain-treated mouse red blood cells (br-MRBC). Under identical conditions, incubation with cytosine arabinoside (CA) strongly prevented the induction of anti-TNP PFC and to a lesser degree anti-SRBC PFC. However, the number of anti br-MRBC PFC was unchanged even when a dose of CA which inhibits totally the incorporation of 3H-thymidine was used. Our findings indicate that the general term "polyclonal stimulation" may concern at least two different types of cell populations and therefore we strongly stress the importance of choosing similar targets in comparative experiments.

  11. Biochemical and Structural Analysis of Inhibitors Targeting the ADC-7 Cephalosporinase of Acinetobacter baumannii

    PubMed Central

    2015-01-01

    β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in β-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpC numbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that ΔH driven compounds may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-β-lactam inhibitors by ADC enzymes and offers a starting point for the structure-based optimization of this class of novel β-lactamase inhibitors against a key resistance target. PMID:25380506

  12. Creation of learning kits

    NASA Technical Reports Server (NTRS)

    Stow, D. A.; Estes, J. E.; Mertz, F. C.

    1981-01-01

    A learning kit is an essential part of any remote sensing workshop, course, or in-house training program to provide the "hands-on" experience of working with remotely sensed imagery. This is the objective of laboratory and field exercises as well as the reason behind the production of imagery/map kits. The way in which these learning kits (containing conventional remotely sensed and collateral data products) are put together is described and some concerns that influence the creation of learning kits are discussed. These include budgetary constraints, number of imagery types, and number of collateral data types.

  13. Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans.

    PubMed

    Arras, Samantha D M; Fraser, James A

    2016-01-01

    The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80Δ genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well. PMID:27643854

  14. Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans

    PubMed Central

    Arras, Samantha D. M.; Fraser, James A.

    2016-01-01

    The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80Δ genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well. PMID:27643854

  15. Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans.

    PubMed

    Arras, Samantha D M; Fraser, James A

    2016-01-01

    The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80Δ genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well.

  16. Cellular Reporter Screens for Inhibitors of Burkholderia pseudomallei Targets in Pseudomonas aeruginosa

    PubMed Central

    Moir, D. T.; Di, M.; Moore, R. A.; Schweizer, H. P.; Woods, D. E.

    2009-01-01

    Summary To facilitate the discovery of new therapeutics for Burkholderia pseudomallei infections, we have developed cellular reporter screens for inhibitors of B. pseudomallei targets in the surrogate host Pseudomonas aeruginosa. P. aeruginosa strains carrying deletions of essential genes were engineered to be dependent on the IPTG-regulated expression of their B. pseudomallei orthologs on a broad-host-range plasmid. P. aeruginosa genes which are upregulated in response to depletion of each target gene product were fused to the Photorhabdus luminescens luxCDABE operon via pGSV3-lux-SpR to generate reporter strains with increased bioluminescence upon target inhibition. A total of 11 of 19 B. pseudomallei genes complemented deletions of their orthologs in P. aeruginosa. The dependence of growth on IPTG levels varied from complete dependence (ftsQ, gyrA, glmU, secA), to slower growth in the absence of IPTG (coaD, efp, mesJ), to apparently normal growth in the absence of IPTG (ligA, lpxA, folA, ipk). Reporter screening strains have been constructed for three gene targets (gyrA, glmU, secA), and one (gyrA) has been applied to 68,000 compounds resulting in a primary hit rate of 0.5% and a confirmed hit rate of 0.06% including several fluoroquinolones. These results provide proof of principle for surrogate cellular reporter screens as a useful approach to identify inhibitors of essential gene products. PMID:19121678

  17. Cellular reporter screens for inhibitors of Burkholderia pseudomallei targets in Pseudomonas aeruginosa.

    PubMed

    Moir, Donald T; Di, Ming; Moore, Richard A; Schweizer, Herbert P; Woods, Donald E

    2008-12-01

    To facilitate the discovery of new therapeutics for Burkholderia pseudomallei infections, we have developed cellular reporter screens for inhibitors of B. pseudomallei targets in the surrogate host Pseudomonas aeruginosa. Pseudomonas aeruginosa strains carrying deletions of essential genes were engineered to be dependent on the isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated expression of their B. pseudomallei orthologues on a broad-host-range plasmid. Pseudomonas aeruginosa genes which are upregulated in response to depletion of each target gene product, were fused to the Photorhabdus luminescens luxCDABE operon via pGSV3-lux-Sp(R) to generate reporter strains with increased bioluminescence upon target inhibition. A total of 11 of 19 B. pseudomallei genes complemented deletions of their orthologues in P. aeruginosa. The dependence of growth on IPTG levels varied from complete dependence (ftsQ, gyrA, glmU, secA) to slower growth in the absence of IPTG (coaD, efp, mesJ), to apparently normal growth in the absence of IPTG (ligA, lpxA, folA, ipk). Reporter screening strains have been constructed for three gene targets (gyrA, glmU, secA), and one (gyrA) has been applied to 68,000 compounds resulting in a primary hit rate of 0.5% and a confirmed hit rate of 0.06%, including several fluoroquinolones. These results provide proof of principle for surrogate cellular reporter screens as a useful approach to identify inhibitors of essential gene products. PMID:19121678

  18. Selective Covalent Targeting of Anti-Apoptotic BFL-1 by Cysteine-Reactive Stapled Peptide Inhibitors.

    PubMed

    Huhn, Annissa J; Guerra, Rachel M; Harvey, Edward P; Bird, Gregory H; Walensky, Loren D

    2016-09-22

    Anti-apoptotic BCL-2 family proteins block cell death by trapping the critical α-helical BH3 domains of pro-apoptotic members in a surface groove. Cancer cells hijack this survival mechanism by overexpressing a spectrum of anti-apoptotic members, mounting formidable apoptotic blockades that resist chemotherapeutic treatment. Drugging the BH3-binding pockets of anti-apoptotic proteins has become a highest-priority goal, fueled by the clinical success of ABT-199, a selective BCL-2 inhibitor, in reactivating apoptosis in BCL-2-dependent cancers. BFL-1 is a BCL-2 homolog implicated in melanoma, lymphoma, and other cancers, and remains undrugged. A natural juxtaposition of two unique cysteines at the binding interface of the NOXA BH3 helix and BFL-1 pocket informed the development of stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1 by covalent targeting. Given the frequent proximity of native cysteines to regulatory binding surfaces, covalent stapled peptide inhibitors provide a new therapeutic strategy for targeting pathologic protein interactions. PMID:27617850

  19. Inhibitor designing, virtual screening, and docking studies for methyltransferase: A potential target against dengue virus

    PubMed Central

    Singh, Jagbir; Kumar, Mahesh; Mansuri, Rani; Sahoo, Ganesh Chandra; Deep, Aakash

    2016-01-01

    Aim: Aim of this work was to design and identify some S-adenosyl-L-homocysteine (SAH) analogs as inhibitors of S-adenosyl-L-methionine-dependent methyltransferase (MTase) protein using computational approaches. Introduction: According to the current scenario the dengue has been a global burden. The people are being killed by dengue virus in an abundant number. Despite of lot of research being going on dengue worldwide, there is no single drug which can kill its virus. This creates an urge for new drug target identification and designing. MTase has been reported as an effective target against dengue virus as it catalyzes an essential step in methylation and capping of viral RNA for viral replication. Materials and Methods: The crystal structure of MTase in complex with SAH was used for designing new analogs of SAH. SAH analogs designed were analyzed on the basis of docking, ADMET, and toxicity analysis done using Discovery Studio 3.5. Results: Seventeen analogs found noncarcinogenic, nonmutagenic, as well as good ADMET properties and good drug-like profile. Conclusion: These SAH analogs, inhibitors of MTase may act as drugs against dengue virus. Further synthesis and biological testing against dengue virus is under observation. PMID:27413346

  20. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

    PubMed

    Zhang, Tinghu; Kwiatkowski, Nicholas; Olson, Calla M; Dixon-Clarke, Sarah E; Abraham, Brian J; Greifenberg, Ann K; Ficarro, Scott B; Elkins, Jonathan M; Liang, Yanke; Hannett, Nancy M; Manz, Theresa; Hao, Mingfeng; Bartkowiak, Bartlomiej; Greenleaf, Arno L; Marto, Jarrod A; Geyer, Matthias; Bullock, Alex N; Young, Richard A; Gray, Nathanael S

    2016-10-01

    Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities. PMID:27571479

  1. Identification of Polyketide Inhibitors Targeting 3-Dehydroquinate Dehydratase in the Shikimate Pathway of Enterococcus faecalis

    PubMed Central

    Hernandez-Valladares, Maria; Go, Maybelle Kho; Tung, Alvin; Aguda, Adeleke H.; Robinson, Robert C.; Yew, Wen Shan

    2014-01-01

    Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the “drug of last resort”. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis. PMID:25072253

  2. Targeting of KRAS mutant tumors by HSP90 inhibitors involves degradation of STK33

    PubMed Central

    Azoitei, Ninel; Hoffmann, Christopher M.; Ellegast, Jana M.; Ball, Claudia R.; Obermayer, Kerstin; Gößele, Ulrike; Koch, Britta; Faber, Katrin; Genze, Felicitas; Schrader, Mark; Kestler, Hans A.; Döhner, Hartmut; Chiosis, Gabriela; Glimm, Hanno

    2012-01-01

    Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS, the most commonly mutated human oncogene, have not been successful. Cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, identifying STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33 are not yet available. Here, using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex binds to and stabilizes STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon tumor-initiating cells harboring mutant KRAS. These findings provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, and identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential. PMID:22451720

  3. Targeting of KRAS mutant tumors by HSP90 inhibitors involves degradation of STK33.

    PubMed

    Azoitei, Ninel; Hoffmann, Christopher M; Ellegast, Jana M; Ball, Claudia R; Obermayer, Kerstin; Gößele, Ulrike; Koch, Britta; Faber, Katrin; Genze, Felicitas; Schrader, Mark; Kestler, Hans A; Döhner, Hartmut; Chiosis, Gabriela; Glimm, Hanno; Fröhling, Stefan; Scholl, Claudia

    2012-04-01

    Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS, the most commonly mutated human oncogene, have not been successful. Cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, identifying STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33 are not yet available. Here, using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex binds to and stabilizes STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon tumor-initiating cells harboring mutant KRAS. These findings provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, and identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential.

  4. Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors

    PubMed Central

    Owen, Jeremy G.; Charlop-Powers, Zachary; Smith, Alexandra G.; Ternei, Melinda A.; Calle, Paula Y.; Reddy, Boojala Vijay B.; Montiel, Daniel; Brady, Sean F.

    2015-01-01

    In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not computationally demanding. To demonstrate the power of this approach to natural product discovery we conducted a computational search for epoxyketone proteasome inhibitors within 185 globally distributed soil metagenomes. This led to the identification of 99 unique epoxyketone sequence tags, falling into 6 phylogenetically distinct clades. Complete gene clusters associated with nine unique tags were recovered from four saturating soil metagenomic libraries. Using heterologous expression methodologies, seven potent epoxyketone proteasome inhibitors (clarepoxcins A–E and landepoxcins A and B) were produced from these pathways, including compounds with different warhead structures and a naturally occurring halohydrin prodrug. This study provides a template for the targeted expansion of bacterially derived natural products using the global metagenome. PMID:25831524

  5. Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors.

    PubMed

    Owen, Jeremy G; Charlop-Powers, Zachary; Smith, Alexandra G; Ternei, Melinda A; Calle, Paula Y; Reddy, Boojala Vijay B; Montiel, Daniel; Brady, Sean F

    2015-04-01

    In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not computationally demanding. To demonstrate the power of this approach to natural product discovery we conducted a computational search for epoxyketone proteasome inhibitors within 185 globally distributed soil metagenomes. This led to the identification of 99 unique epoxyketone sequence tags, falling into 6 phylogenetically distinct clades. Complete gene clusters associated with nine unique tags were recovered from four saturating soil metagenomic libraries. Using heterologous expression methodologies, seven potent epoxyketone proteasome inhibitors (clarepoxcins A-E and landepoxcins A and B) were produced from these pathways, including compounds with different warhead structures and a naturally occurring halohydrin prodrug. This study provides a template for the targeted expansion of bacterially derived natural products using the global metagenome.

  6. Inhibitors of apoptosis proteins (IAPs) as potential molecular targets for therapy of hematological malignancies.

    PubMed

    Smolewski, P; Robak, T

    2011-11-01

    Apoptosis, a programmed cell death, plays a key role in the regulation of tissue homeostasis. However, impairment of its regulation may promote formation and progression of malignancy. An important part of the apoptotic machinery are the inhibitor of apoptosis protein (IAP) family, regulating caspase activity, cell division or cell survival pathways through binding to their baculovirus AIP repeat (BIR) domains and/or by their ubiquitin-ligase RING zinc finger (RZF) activity. The following IAPs have been described so far: NAIP (neuronal apoptosis inhibitory protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome binding IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7) and Ts-IAP (testis-specific IAP; BIRC8). Several studies suggested a potential contribution of IAPs to oncogenesis and resistance to anti-tumor treatment. Increased IAP expression was found in variety of human cancers, including hematological malignancies, such as leukemias and B-cell lymphomas. A correlation between the progression of those diseases and high levels of survivin or XIAP has been reported. Overexpression of XIAP in acute myeloid leukemia or survivin in acute lymphoblastic leukemia and diffuse large B-cell lymphoma have been indicated as an unfavorable prognostic factors. Elevated cellular levels of cIAP1, cIAP2, XIAP and survivin correlated with a progressive course of chronic lymphocytic leukemia. Thus, targeting IAPs with small-molecule inhibitors by their antisense approaches or natural IAP antagonist mimetics, may be an attractive strategy of anti-cancer treatment. Such agents can either directly induce apoptosis of tumor cells or sensitize them to other cytotoxic agents, hence overcoming drug-resistance. This review demonstrates the current knowledge on IAP molecular biology, as well as the mechanisms of action and the development of IAP-targeting agents for treatment of hematological

  7. Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

    PubMed Central

    Reddy, B. K. Kishore; Landge, Sudhir; Ravishankar, Sudha; Patil, Vikas; Shinde, Vikas; Tantry, Subramanyam; Kale, Manoj; Raichurkar, Anandkumar; Menasinakai, Sreenivasaiah; Mudugal, Naina Vinay; Ambady, Anisha; Ghosh, Anirban; Tunduguru, Ragadeepthi; Kaur, Parvinder; Singh, Ragini; Kumar, Naveen; Bharath, Sowmya; Sundaram, Aishwarya; Bhat, Jyothi; Sambandamurthy, Vasan K.; Björkelid, Christofer; Jones, T. Alwyn; Das, Kaveri; Bandodkar, Balachandra; Malolanarasimhan, Krishnan; Mukherjee, Kakoli

    2014-01-01

    Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis. PMID:24687493

  8. Synthetic heterovalent inhibitors targeting recognition E3 components of the N-end rule pathway.

    PubMed

    Lee, Min Jae; Pal, Krishnendu; Tasaki, Takafumi; Roy, Sayantani; Jiang, Yonghua; An, Jee Young; Banerjee, Rajkumar; Kwon, Yong Tae

    2008-01-01

    Multivalent binding allows high selectivity and affinity in a ligand-protein interaction. The N-end rule pathway is a ubiquitin (Ub)-dependent proteolytic system in which specific E3s, called N-recognins, mediate ubiquitylation through the recognition of types 1 and 2, destabilizing N-terminal residues of substrates. We recently identified a set of E3 Ub ligases (named UBR1-UBR7) containing the 70-residue UBR box, and we demonstrated that UBR1, UBR2, UBR4, and UBR5 can bind to destabilizing N-terminal residues. To explore a model of heterovalent interaction to the N-recognin family, we synthesized the small-molecule compound RF-C11, which bears two heterovalent ligands designed to target N-recognins, together with control molecules with two homovalent ligands. We demonstrate that heterovalent ligands of RF-C11 selectively and cooperatively bind cognate-binding sites of multiple N-recognins and thereby inhibit both types 1 and 2 N-end rule activities. Furthermore, the efficacy of heterovalent RF-C11 was substantially higher than homovalent inhibitors, which can target either a type 1 or type 2 site, providing the molecular basis of designing multivalent inhibitors for the control of specific intracellular pathways. In addition, RF-C11 exhibited higher efficacy and stability, compared with dipeptides bearing destabilizing N-terminal residues, which are known competitive inhibitors of the pathway. We also used the heterovalent compound to study the function of N-recognins in cardiac signaling. Using mouse and rat cardiomyocytes, we demonstrate that the N-end rule pathway has a cell-autonomous function in cardiac proliferation and hypertrophy, explaining our earlier results implicating the pathway in cardiac development and proteolysis of multiple cardiovascular regulators. PMID:18162545

  9. Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery.

    PubMed

    Tykvart, Jan; Schimer, Jiří; Bařinková, Jitka; Pachl, Petr; Poštová-Slavětínská, Lenka; Majer, Pavel; Konvalinka, Jan; Šácha, Pavel

    2014-08-01

    Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure-activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in Ki value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII. PMID:24954515

  10. Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery.

    PubMed

    Tykvart, Jan; Schimer, Jiří; Bařinková, Jitka; Pachl, Petr; Poštová-Slavětínská, Lenka; Majer, Pavel; Konvalinka, Jan; Šácha, Pavel

    2014-08-01

    Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure-activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in Ki value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.

  11. Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

    PubMed

    Słowińska, Mariola; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej

    2015-08-01

    A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

  12. Identification of Targets of the HIF-1 Inhibitor IDF-11774 Using Alkyne-Conjugated Photoaffinity Probes.

    PubMed

    Ban, Hyun Seung; Naik, Ravi; Kim, Hwan Mook; Kim, Bo-Kyung; Lee, Hongsub; Kim, Inhyub; Ahn, Heechul; Jang, Yerin; Jang, Kyusik; Eo, Yumi; Song, Kyung Bin; Lee, Kyeong; Won, Misun

    2016-08-17

    We developed a hypoxia-inducible factor-1 (HIF-1) inhibitor, IDF-11774, as a clinical candidate for cancer therapy. To understand the mechanism of action of IDF-11774, we attempted to isolate target proteins of IDF-11774 using bioconjugated probes. Multifunctional chemical probes containing sites for click conjugation and photoaffinity labeling were designed and synthesized. After fluorescence and photoaffinity labeling of proteins, two-dimensional electrophoresis (2DE) was performed to isolate specific molecular targets of IDF-11774. Heat shock protein (HSP) 70 was identified as a target protein of IDF-11774. We revealed that IDF-11774 inhibited HSP70 chaperone activity by binding to its allosteric pocket, rather than the ATP-binding site in its nucleotide-binding domain (NBD). Moreover, IDF-11774 reduced the oxygen consumption rate (OCR) and ATP production, thereby increasing intracellular oxygen tension. This result suggests that the inhibition of HSP70 chaperone activity by IDF-11774 suppresses HIF-1α refolding and stimulates HIF-1α degradation. Taken together, these findings indicate that IDF-11774-derived chemical probes successfully identified IDF-11774's target molecule, HSP70, and elucidated the mode of action of IDF-11774 in inhibiting HSP70 chaperone activity and stimulating HIF-1α degradation in cancer cells. PMID:27386732

  13. In Silico Screening for Inhibitors of P-Glycoprotein That Target the Nucleotide Binding Domains

    PubMed Central

    Brewer, Frances K.; Follit, Courtney A.; Vogel, Pia D.

    2014-01-01

    Multidrug resistances and the failure of chemotherapies are often caused by the expression or overexpression of ATP-binding cassette transporter proteins such as the multidrug resistance protein, P-glycoprotein (P-gp). P-gp is expressed in the plasma membrane of many cell types and protects cells from accumulation of toxins. P-gp uses ATP hydrolysis to catalyze the transport of a broad range of mostly hydrophobic compounds across the plasma membrane and out of the cell. During cancer chemotherapy, the administration of therapeutics often selects for cells which overexpress P-gp, thereby creating populations of cancer cells resistant to a variety of chemically unrelated chemotherapeutics. The present study describes extremely high-throughput, massively parallel in silico ligand docking studies aimed at identifying reversible inhibitors of ATP hydrolysis that target the nucleotide-binding domains of P-gp. We used a structural model of human P-gp that we obtained from molecular dynamics experiments as the protein target for ligand docking. We employed a novel approach of subtractive docking experiments that identified ligands that bound predominantly to the nucleotide-binding domains but not the drug-binding domains of P-gp. Four compounds were found that inhibit ATP hydrolysis by P-gp. Using electron spin resonance spectroscopy, we showed that at least three of these compounds affected nucleotide binding to the transporter. These studies represent a successful proof of principle demonstrating the potential of targeted approaches for identifying specific inhibitors of P-gp. PMID:25270578

  14. Compstatin: a C3-targeted complement inhibitor reaching its prime for bedside intervention

    PubMed Central

    Mastellos, Dimitrios C.; Yancopoulou, Despina; Kokkinos, Petros; Huber-Lang, Markus; Hajishengallis, George; Biglarnia, Ali Reza; Lupu, Florea; Nilsson, Bo; Risitano, Antonio M.; Ricklin, Daniel; Lambris, John D.

    2015-01-01

    There is a growing awareness that complement plays an integral role in human physiology and disease, transcending its traditional perception as an accessory system for pathogen clearance and opsonic cell killing. As the list of pathologies linked to dysregulated complement activation grows longer, it has become clear that targeted modulation of this innate immune system opens new windows of therapeutic opportunity for anti-inflammatory drug design. Indeed, the introduction of the first complement-targeting drugs has reignited a vibrant interest in the clinical translation of complement-based inhibitors. Compstatin was discovered as a cyclic peptide that inhibits complement activation by binding C3 and interfering with convertase formation and C3 cleavage. As the convergence point of all activation pathways and a molecular hub for crosstalk with multiple pathogenic pathways, C3 represents an attractive target for therapeutic modulation of the complement cascade. A multidisciplinary drug optimization effort encompassing rational “wet” and in silico synthetic approaches and an array of biophysical, structural, and analytical tools has culminated in an impressive structure-function refinement of compstatin, yielding a series of analogs that show promise for a wide spectrum of clinical applications. These new derivatives have improved inhibitory potency and pharmacokinetic profiles and show efficacy in clinically relevant primate models of disease. This review provides an up-to-date survey of the drug design effort placed on the compstatin family of C3 inhibitors, highlighting the most promising drug candidates. It also discusses translational challenges in complement drug discovery and peptide drug development and reviews concerns related to systemic C3 interception. PMID:25678219

  15. PIF-pocket as a target for C. albicans Pkh selective inhibitors.

    PubMed

    Pastor-Flores, Daniel; Schulze, Jörg O; Bahí, Anna; Giacometti, Romina; Ferrer-Dalmau, Jofre; Passeron, Susana; Engel, Matthias; Süss, Evelyn; Casamayor, Antonio; Biondi, Ricardo M

    2013-10-18

    The phosphoinositide-dependent protein kinase 1, PDK1, is a master kinase that phosphorylates the activation loop of up to 23 AGC kinases. S. cerevisiae has three PDK1 orthologues, Pkh1-3, which also phosphorylate AGC kinases (e.g., Ypk, Tpk, Pkc1, and Sch9). Pkh1 and 2 are redundant proteins involved in multiple essential cellular functions, including endocytosis and cell wall integrity. Based on similarities with the budding yeast, the Pkh of fungal infectious species was postulated as a novel target for antifungals. Here, we found that depletion of Pkh eventually induces oxidative stress and DNA double-strand breaks, leading to programmed cell death. This finding supports Pkh as an antifungal target since pharmacological inhibition of Pkh would lead to the death of yeast cells, the ultimate goal of antifungals. It was therefore of interest to further investigate the possibility to develop Pkh inhibitors with selectivity for Candida Pkh that would not inhibit the human ortholog. Here, we describe C. albicans Pkh2 biochemically, structurally and by using chemical probes in comparison to human PDK1. We found that a regulatory site on the C. albicans Pkh2 catalytic domain, the PIF-pocket, diverges from human PDK1. Indeed, we identified and characterized PS77, a new small allosteric inhibitor directed to the PIF-pocket, which has increased selectivity for C. albicans Pkh2. Together, our results describe novel features of the biology of Pkh and chemical biology approaches that support the validation of Pkh as a drug target for selective antifungals. PMID:23911092

  16. Novel Inhibitors of AKT: Assessment of a Different Approach Targeting the Pleckstrin Homology Domain

    PubMed Central

    Meuillet, E.J.

    2014-01-01

    Protein kinase B/AKT plays a central role in cancer. The serine/threonine kinase is overexpressed or constitutively active in many cancers and has been validated as a therapeutic target for cancer treatment. However, targeting the kinase activity has revealed itself to be a challenge due to non-selectivity of the compounds towards other kinases. This review summarizes other approaches scientists have developed to inhibit the activity and function of AKT. They consist of targeting the pleckstrin homology (PH) domain of AKT. Indeed, upon the generation of 3-phosphorylated phosphatidylinositol phosphates (PI3Ps) by PI3-kinase (PI3K), AKT translocates from the cytosol to the plasma membrane and binds to the PI3Ps via its PH domain. Thus, several analogs of PI3Ps (PI Analogs or PIAs), alkylphospholipids (APLs), such as edelfosine or inositol phosphates (IPs) have been described to inhibit the binding of the PH domain to PI3Ps. Recent allostetic inhibitors and small molecules that do not bind the kinase domain but affect the kinase activity of AKT, presumably by interacting with the PH domain, have been also identified. Finally, several drug screening studies spawned novel chemical scaffolds that bind the PH domain of AKT. Together, these approaches have been more or less sucessful in vitro and to some extent translated in preclinical studies. Several of these new AKT PH domain inhibitors exhibit promising anti-tumor activity in mouse models and some of them show synergy with ionizing radiation and chemotherapy. Early clinical trials have started and results will attest to the validity and efficacy of such approaches in the near future. PMID:21649580

  17. Benchmarking Tool Kit.

    ERIC Educational Resources Information Center

    Canadian Health Libraries Association.

    Nine Canadian health libraries participated in a pilot test of the Benchmarking Tool Kit between January and April, 1998. Although the Tool Kit was designed specifically for health libraries, the content and approach are useful to other types of libraries as well. Used to its full potential, benchmarking can provide a common measuring stick to…

  18. Communications Survival Kit.

    ERIC Educational Resources Information Center

    Williamson, Shirley Porter; Braden, Bill

    The purpose of the Communications Survival Kit is to serve as a guide for counselors to assist in planning, developing and implementing a public relations program for the guidance department at the local school level. The kit contains practical information in three categories. First is a rationale for a public relations (PR) program, what PR is…

  19. Community Consultation Kit.

    ERIC Educational Resources Information Center

    Boulder Area Growth Study Commission, CO.

    This kit, designed for leaders and participants, provides a model for organizing and taking part in Community Consultation Groups. The kit was designed to be used in connection with community concerns about growth in Boulder, Colorado. These groups build upon a previous survey to assist the Commission in determining specific growth concerns in the…

  20. Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

    PubMed

    Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

    2016-01-01

    The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib. PMID:27467080

  1. Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT

    PubMed Central

    Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

    2016-01-01

    The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors’ sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib. PMID:27467080

  2. Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

    PubMed

    Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

    2016-01-01

    The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

  3. Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition.

    PubMed

    Shi, Xiarong; Sousa, Leiliane P; Mandel-Bausch, Elizabeth M; Tome, Francisco; Reshetnyak, Andrey V; Hadari, Yaron; Schlessinger, Joseph; Lax, Irit

    2016-08-16

    Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. PMID:27482095

  4. The Qiagen Investigator® Quantiplex HYres as an alternative kit for DNA quantification.

    PubMed

    Frégeau, Chantal J; Laurin, Nancy

    2015-05-01

    The Investigator® Quantiplex HYres kit was evaluated as a potential replacement for dual DNA quantification of casework samples. This kit was determined to be highly sensitive with a limit of quantification and limit of detection of 0.0049ng/μL and 0.0003ng/μL, respectively, for both human and male DNA, using full or half reaction volumes. It was also accurate in assessing the amount of male DNA present in 96 mock and actual casework male:female mixtures (various ratios) processed in this exercise. The close correlation between the male/human DNA ratios expressed in percentages derived from the Investigator® Quantiplex HYres quantification results and the male DNA proportion calculated in mixed AmpFlSTR® Profiler® Plus or AmpFlSTR® Identifiler® Plus profiles, using the Amelogenin Y peak and STR loci, allowed guidelines to be developed to facilitate decisions regarding when to submit samples to Y-STR rather than autosomal STR profiling. The internal control (IC) target was shown to be more sensitive to inhibitors compared to the human and male DNA targets included in the Investigator® Quantiplex HYres kit serving as a good quality assessor of DNA extracts. The new kit met our criteria of enhanced sensitivity, accuracy, consistency, reliability and robustness for casework DNA quantification. PMID:25603128

  5. The Indonesia Kit. A Study Kit.

    ERIC Educational Resources Information Center

    Briere, Elaine; Gage, Susan

    This document is designed for Canadians interested in the South Pacific island chain nation of Indonesia. The kit includes information, photographs, and illustrations concerning Indonesia, West Papua (Irian Jaya), and East Timor. There are discussions of Indonesia's environment, its transmigration program, development refugees, and ties with…

  6. Polynucleotide kinase as a potential target for enhancing cytotoxicity by ionizing radiation and topoisomerase I inhibitors

    PubMed Central

    Bernstein, N. K.; Karimi-Busheri, F.; Rasouli-Nia, A.; Mani, R.; Dianov, G.; Glover, J. N. M.; Weinfeld, M.

    2010-01-01

    The cytotoxicity of many antineoplastic agents is due to their capacity to damage DNA and there is evidence indicating that DNA repair contributes to the cellular resistance to such agents. DNA strand breaks constitute a significant proportion of the lesions generated by a broad range of genotoxic agents, either directly, or during the course of DNA repair. Strand breaks that are caused by many agents including ionizing radiation, topoisomerase I inhibitors, and DNA repair glycosylases such as NEIL1 and NEIL2, often contain 5’-hydroxyl and/or 3’-phosphate termini. These ends must be converted to 5’-phosphate and 3’-hydroxyl termini in order to allow DNA polymerases and ligases to catalyze repair synthesis and strand rejoining. A key enzyme involved in this end-processing is polynucleotide kinase (PNK), which possesses two enzyme activities, a DNA 5’-kinase activity and a 3’-phosphatase activity. PNK participates in the single-strand break repair pathway and the non-homologous end joining pathway for double-strand break repair. RNAi-mediated down-regulation of PNK renders cells more sensitive to ionizing radiation and camptothecin, a topoisomerase I inhibitor. Structural analysis of PNK revealed the protein is composed of three domains, the kinase domain at the C-terminus, the phosphatase domain in the centre and a forkhead associated (FHA) domain at the N-terminus. The FHA domain plays a critical role in the binding of PNK to other DNA repair proteins. Thus each PNK domain may be a suitable target for small molecule inhibition to effectively reduce resistance to ionizing radiation and topoisomerase I inhibitors. PMID:18473721

  7. The PCSK9 Inhibitors: A Novel Therapeutic Target Enters Clinical Practice

    PubMed Central

    Lepor, Norman E.; Kereiakes, Dean J.

    2015-01-01

    There is a critical need for alternative, potent agents that can reduce low-density lipoprotein cholesterol (LDL-C) levels in patients with heterozygous familial hyperlipidemia and statin intolerance and those not reaching lipid-lowering treatment goals who are at high risk for cardiovascular (CV) events. The first proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor was approved in July 2015 by the US Food and Drug Administration as an adjunct to diet and maximally tolerated statin therapy for treatment of adults with heterozygous familial hyperlipidemia or clinical atherosclerotic CV disease, who require additional lowering of LDL-C levels. In clinical trials, PCSK9 inhibitors have been shown to reduce LDL-C levels by as much as 60% to 70% when administered as monotherapy or as an add-on treatment to statins and other lipid-lowering therapies. In studies of PCSK9 genetic mutations, loss of function in the PCSK9 allele was associated with a relative decrease of 88% in the risk for atherosclerotic CV events during 15 years of patient follow-up. The use of PCSK9 inhibitors may eventually support the LDL-C hypothesis that the lower the LDL-C level, the lower the CV risk. Although some recent clinical practice guidelines have deemphasized the importance of numeric LDL-C targets, many clinicians are reluctant to discard them, and this position is supported by recent clinical evidence. We eagerly await the results of the ODYSSEY, FOURIER, and SPIRE clinical outcome trials, which we anticipate will provide further validation that “lower is better” with respect to reducing LDL-C levels and improving clinical outcomes. PMID:26834934

  8. The PCSK9 Inhibitors: A Novel Therapeutic Target Enters Clinical Practice.

    PubMed

    Lepor, Norman E; Kereiakes, Dean J

    2015-12-01

    There is a critical need for alternative, potent agents that can reduce low-density lipoprotein cholesterol (LDL-C) levels in patients with heterozygous familial hyperlipidemia and statin intolerance and those not reaching lipid-lowering treatment goals who are at high risk for cardiovascular (CV) events. The first proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor was approved in July 2015 by the US Food and Drug Administration as an adjunct to diet and maximally tolerated statin therapy for treatment of adults with heterozygous familial hyperlipidemia or clinical atherosclerotic CV disease, who require additional lowering of LDL-C levels. In clinical trials, PCSK9 inhibitors have been shown to reduce LDL-C levels by as much as 60% to 70% when administered as monotherapy or as an add-on treatment to statins and other lipid-lowering therapies. In studies of PCSK9 genetic mutations, loss of function in the PCSK9 allele was associated with a relative decrease of 88% in the risk for atherosclerotic CV events during 15 years of patient follow-up. The use of PCSK9 inhibitors may eventually support the LDL-C hypothesis that the lower the LDL-C level, the lower the CV risk. Although some recent clinical practice guidelines have deemphasized the importance of numeric LDL-C targets, many clinicians are reluctant to discard them, and this position is supported by recent clinical evidence. We eagerly await the results of the ODYSSEY, FOURIER, and SPIRE clinical outcome trials, which we anticipate will provide further validation that "lower is better" with respect to reducing LDL-C levels and improving clinical outcomes. PMID:26834934

  9. Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

    PubMed

    Gal, Maayan; Bloch, Itai; Shechter, Nelia; Romanenko, Olga; Shir, Ofer M

    2016-01-01

    Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

  10. Transforming growth factor-beta requires its target plasminogen activator inhibitor-1 for cytostatic activity.

    PubMed

    Kortlever, Roderik M; Nijwening, Jeroen H; Bernards, René

    2008-09-01

    The cytokine transforming growth factor beta (TGFbeta) has strong antiproliferative activity in most normal cells but contributes to tumor progression in the later stages of oncogenesis. It is not fully understood which TGFbeta target genes are causally involved in mediating its cytostatic activity. We report here that suppression of the TGFbeta target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference leads to escape from the cytostatic activity of TGFbeta both in human keratinocytes (HaCaTs) and primary mouse embryo fibroblasts. Consistent with this, PAI-1 knock-out mouse embryo fibroblasts are also resistant to TGFbeta growth arrest. Conversely, we show that ectopic expression of PAI-1 in proliferating HaCaT cells induces a growth arrest. PAI-1 knockdown does not interfere with canonical TGFbeta signaling as judged by SMAD phosphorylation and induction of bona fide TGFbeta target genes. Instead, knockdown of PAI-1 results in sustained activation of protein kinase B. Significantly, we find that constitutive protein kinase B activity leads to evasion of the growth-inhibitory action of TGFbeta. Our data are consistent with a model in which induction of PAI-1 by TGFbeta is critical for the induction of proliferation arrest.

  11. A Novel Small-Molecule Inhibitor Targeting the IL-6 Receptor β Subunit, Glycoprotein 130.

    PubMed

    Hong, Soon-Sun; Choi, Jung Ho; Lee, Sung Yoon; Park, Yeon-Hwa; Park, Kyung-Yeon; Lee, Joo Young; Kim, Juyoung; Gajulapati, Veeraswamy; Goo, Ja-Il; Singh, Sarbjit; Lee, Kyeong; Kim, Young-Kook; Im, So Hee; Ahn, Sung-Hoon; Rose-John, Stefan; Heo, Tae-Hwe; Choi, Yongseok

    2015-07-01

    IL-6 is a major causative factor of inflammatory disease. Although IL-6 and its signaling pathways are promising targets, orally available small-molecule drugs specific for IL-6 have not been developed. To discover IL-6 antagonists, we screened our in-house chemical library and identified LMT-28, a novel synthetic compound, as a candidate IL-6 blocker. The activity, mechanism of action, and direct molecular target of LMT-28 were investigated. A reporter gene assay showed that LMT-28 suppressed activation of STAT3 induced by IL-6, but not activation induced by leukemia inhibitory factor. In addition, LMT-28 downregulated IL-6-stimulated phosphorylation of STAT3, gp130, and JAK2 protein and substantially inhibited IL-6-dependent TF-1 cell proliferation. LMT-28 antagonized IL-6-induced TNF-α production in vivo. In pathologic models, oral administration of LMT-28 alleviated collagen-induced arthritis and acute pancreatitis in mice. Based on the observation of upstream IL-6 signal inhibition by LMT-28, we hypothesized IL-6, IL-6Rα, or gp130 to be putative molecular targets. We subsequently demonstrated direct interaction of LMT-28 with gp130 and specific reduction of IL-6/IL-6Rα complex binding to gp130 in the presence of LMT-28, which was measured by surface plasmon resonance analysis. Taken together, our data suggest that LMT-28 is a novel synthetic IL-6 inhibitor that functions through direct binding to gp130. PMID:26026064

  12. Target-Based Identification of Whole-Cell Active Inhibitors of Biotin Biosynthesis in Mycobacterium tuberculosis

    PubMed Central

    Park, Sae Woong; Casalena, Dominick; Wilson, Daniel; Dai, Ran; Nag, Partha; Liu, Feng; Boyce, Jim P.; Bittker, Joshua; Schreiber, Stuart; Finzel, Barry C.; Schnappinger, Dirk; Aldrich, Courtney C.

    2014-01-01

    SUMMARY Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counter-screen in either biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counter-screen proved crucial to filter out compounds whose whole-cell activity was off-target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were co-crystallized with BioA to provide a framework for future structure-based drug design efforts. PMID:25556942

  13. Identification and characterization of carprofen as a multi-target FAAH/COX inhibitor

    PubMed Central

    Favia, Angelo D.; Habrant, Damien; Scarpelli, Rita; Migliore, Marco; Albani, Clara; Bertozzi, Sine Mandrup; Dionisi, Mauro; Tarozzo, Glauco; Piomelli, Daniele; Cavalli, Andrea; De Vivo, Marco

    2013-01-01

    Pain and inflammation are major therapeutic areas for drug discovery. Current drugs for these pathologies have limited efficacy, however, and often cause a number of unwanted side effects. In the present study, we identify the non-steroid anti-inflammatory drug, carprofen, as a multi-target-directed ligand that simultaneously inhibits cyclooxygenase-1 (COX-1), COX-2 and fatty acid amide hydrolase (FAAH). Additionally, we synthesized and tested several racemic derivatives of carprofen, sharing this multi-target activity. This may result in improved analgesic efficacy and reduced side effects (Naidu, et al (2009) J Pharmacol Exp Ther 329, 48-56; Fowler, C.J. et al. (2012) J Enzym Inhib Med Chem Jan 6; Sasso, et al (2012) Pharmacol Res 65, 553). The new compounds are among the most potent multi-target FAAH/COXs inhibitors reported so far in the literature, and thus may represent promising starting points for the discovery of new analgesic and anti-inflammatory drugs. PMID:23043222

  14. Identification of novel targets for PGC-1{alpha} and histone deacetylase inhibitors in neuroblastoma cells

    SciTech Connect

    Cowell, Rita M. Talati, Pratik; Blake, Kathryn R.; Meador-Woodruff, James H.; Russell, James W.

    2009-02-06

    Recent evidence suggests that the transcriptional coactivator peroxisome proliferator activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) is involved in the pathology of Huntington's Disease (HD). While animals lacking PGC-1{alpha} express lower levels of genes involved in antioxidant defense and oxidative phosphorylation in the brain, little is known about other targets for PGC-1{alpha} in neuronal cells and whether there are ways to pharmacologically target PGC-1{alpha} in neurons. Here, PGC-1{alpha} overexpression in SH-SY5Y neuroblastoma cells upregulated expression of genes involved in mitochondrial function, glucose transport, fatty acid metabolism, and synaptic function. Overexpression also decreased vulnerability to hydrogen peroxide-induced cell death and caspase 3 activation. Treatment of cells with the histone deacetylase inhibitors (HDACi's) trichostatin A and valproic acid upregulated PGC-1{alpha} and glucose transporter 4 (GLUT4). These results suggest that PGC-1{alpha} regulates multiple pathways in neurons and that HDACi's may be good candidates to target PGC-1{alpha} and GLUT4 in HD and other neurological disorders.

  15. Histone Deacetylase Inhibitors Delivery using Nanoparticles with Intrinsic Passive Tumor Targeting Properties for Tumor Therapy

    PubMed Central

    el Bahhaj, Fatima; Denis, Iza; Pichavant, Loic; Delatouche, Régis; Collette, Floraine; Linot, Camille; Pouliquen, Daniel; Grégoire, Marc; Héroguez, Valérie; Blanquart, Christophe; Bertrand, Philippe

    2016-01-01

    Fast clearance, metabolism and systemic toxicity are major limits for the clinical use of anti-cancer drugs. Histone deacetylase inhibitors (HDACi) present these defects despite displaying promising anti-tumor properties on tumor cells in vitro and in in vivo model of cancers. Specific delivery of anti-cancer drugs into the tumor should improve their clinical benefit by limiting systemic toxicity and by increasing the anti-tumor effect. In this work, we describe a simple and flexible polymeric nanoparticle platform highly targeting the tumor in vivo and triggering impressive tumor weight reduction when functionalized with HDACi. Our nanoparticles were produced by Ring-Opening Metathesis Polymerization of azido-polyethylene oxide-norbornene macromonomers and functionalized using click chemistry. Using an orthotopic model of peritoneal invasive cancer, a highly selective accumulation of the particles in the tumor was obtained. A combination of epigenetic drugs involving a pH-responsive histone deacetylase inhibitor (HDACi) polymer conjugated to these particles gave 80% reduction of tumor weight without toxicity whereas the free HDACi has no effect. Our work demonstrates that the use of a nanovector with theranostic properties leads to an optimized delivery of potent HDACi in tumor and then, to an improvement of their anti-tumor properties in vivo. PMID:27162550

  16. Inhibitor of Apoptosis Proteins (IAPs) as therapeutic targets in Multiple Myeloma (MM)

    PubMed Central

    Ramakrishnan, Vijay; Painuly, Utkarsh; Kimlinger, Teresa; Haug, Jessica; Rajkumar, S. Vincent; Kumar, Shaji

    2014-01-01

    The inhibitor of apoptosis (IAP) proteins plays a critical role in the control of apoptotic machinery, and has been explored as a therapeutic target. Here, we have examined the functional importance of IAPs in multiple myeloma (MM) by using a Smac-mimetic LCL161. We observed that LCL161 was able to potently induce apoptosis in some MM cell lines but not in others. Examining the levels of XIAP, cIAP1 and cIAP2 post LCL161 treatment indicated clear down regulation of both XIAP activity and cIAP1 levels in both the sensitive and less sensitive (resistant) cell lines. cIAP2, however, was not down regulated in the cell line resistant to the drug. siRNA mediated silencing of cIAP2 significantly enhanced the effect of LCL161 indicating the importance of down regulating all IAPs simultaneously for induction of apopotsis in MM cells. LCL161 induced marked up regulation of the Jak2/Stat3 pathway in the resistant MM cell lines. Combining LCL161 with a Jak2 specific inhibitor resulted in synergistic cell death in MM cell lines and patient cells. In addition, combining LCL161 with death inducing ligands clearly showed that LCL161 sensitized MM cells to both FAS-L and TRAIL. PMID:24402161

  17. Inhibitor focusing: direct selection of drug targets from proteomes using activity-based probes.

    PubMed

    Nomanbhoy, Tyzoon K; Rosenblum, Jonathan; Aban, Arwin; Burbaum, Jonathan J

    2003-02-01

    In the latter stages of drug discovery and development, assays that establish drug selectivity and toxicity are important when side effects, which are often due to lack of specificity, determine drug candidate viability. There has been no comprehensive or systematic methodology to measure these factors outside of whole-animal assays, and such phenomenological assays generally fail to establish the additional targets of a given small molecule, or the molecular origin of toxicity. Consequently, small-molecule development programs destined for failure often reach advanced stages of testing, and the money and time invested in such programs could be saved if information on selectivity were available early in the process. Here, we present a methodology that utilizes chemical ABPs in combination with small-molecule inhibitors to selectively label small-molecule binding sites in whole proteomic samples. In principle, the ABP and small molecule will compete for similar binding sites, such that the small molecule will protect against modification by the ABP. Thus, after removal of the small molecule, the binding site for the ABP will be revealed, and a second probe can then be used to label the small-molecule binding sites selectively. To demonstrate this experimentally, we mapped the binding sites of the DPP4 inhibitor, IT, in a number of different tissue types. PMID:15090140

  18. Linking off-target kinase pharmacology to the differential cellular effects observed among PARP inhibitors.

    PubMed

    Antolín, Albert A; Mestres, Jordi

    2014-05-30

    PARP inhibitors hold promise as a novel class of targeted anticancer drugs. However, their true mechanism of action is still not well understood following recent reports that show marked differences in cellular effects. Here, we demonstrate that three PARP drug candidates, namely, rucaparib, veliparib, and olaparib, have a clearly different in vitro affinity profile across a panel of diverse kinases selected using a computational approach that relates proteins by ligand similarity. In this respect, rucaparib inhibits nine kinases with micromolar affinity, including PIM1, PIM2, PRKD2, DYRK1A, CDK1, CDK9, HIPK2, CK2, and ALK. In contrast, olaparib does not inhibit any of the sixteen kinases tested. In between, veliparib inhibits only two, namely, PIM1 and CDK9. The differential kinase pharmacology observed among PARP inhibitors provides a plausible explanation to their different cellular effects and offers unexplored opportunities for this drug class, but alerts also on the risk associated to transferring directly both preclinical and clinical outcomes from one PARP drug candidate to another. PMID:24632590

  19. Hedgehog inhibitors selectively target cell migration and adhesion of mantle cell lymphoma in bone marrow microenvironment

    PubMed Central

    Zhang, Han; Chen, Zheng; Neelapu, Sattva S.; Romaguera, Jorge; McCarty, Nami

    2016-01-01

    The clinical benefits of a Hedgehog (Hh) inhibitor, LDE225 (NPV-LDE-225, Erismodegib), have been unclear in hematological cancers. Here, we report that LDE225 selectively inhibited migration and adhesion of mantle cell lymphoma (MCL) to bone marrows via very late antigen-4 (VLA-4) mediated inactivation of focal adhesion kinase (FAK) signaling. LDE225 treatment not only affected MCL cells, but also modulated stromal cells within the bone marrow microenvironment by decreasing their production of SDF-1, IL-6 and VCAM-1, the ligand for VLA-4. Surprisingly, LDE225 treatment alone did not suppress cell proliferation due to increased CXCR4 expression mediated by reactive oxygen species (ROS). The increased ROS/CXCR4 further stimulated autophagy formation. The combination of LDE225 with the autophagy inhibitors further enhanced MCL cell death. Our data, for the first time, revealed LDE225 selectively targets MCL cells migration and adhesion to bone marrows. The ineffectiveness of LDE225 in MCL is due to autophagy formation, which in turn increases cell viability. Inhibiting autophagy will be an effective adjuvant therapy for LDE225 in MCL, especially for advanced MCL patients with bone marrow involvement. PMID:26885608

  20. Strategies to target long-lived plasma cells for treating hemophilia A inhibitors.

    PubMed

    Liu, Chao Lien; Lyle, Meghan J; Shin, Simon C; Miao, Carol H

    2016-03-01

    Long-lived plasma cells (LLPCs) can persistently produce anti-factor VIII (FVIII) antibodies which disrupt therapeutic effect of FVIII in hemophilia A patients with inhibitors. The migration of plasma cells to BM where they become LLPCs is largely controlled by an interaction between the chemokine ligand CXCL12 and its receptor CXCR4. AMD3100 combined with G-CSF inhibit their interactions, thus facilitating the mobilization of CD34(+) cells and blocking the homing of LLPCs. These reagents were combined with anti-CD20 to reduce B-cells and the specific IL-2/IL-2mAb (JES6-1) complexes to induce Treg expansion for targeting anti-FVIII immune responses. Groups of mice primed with FVIII plasmid and protein respectively were treated with the combined regimen for six weeks, and a significant reduction of anti-FVIII inhibitor titers was observed, associated with the dramatic decrease of circulating and bone marrow CXCR4(+) plasma cells. The combination regimens are highly promising in modulating pre-existing anti-FVIII antibodies in FVIII primed subjects.

  1. Hedgehog inhibitors selectively target cell migration and adhesion of mantle cell lymphoma in bone marrow microenvironment.

    PubMed

    Zhang, Han; Chen, Zheng; Neelapu, Sattva S; Romaguera, Jorge; McCarty, Nami

    2016-03-22

    The clinical benefits of a Hedgehog (Hh) inhibitor, LDE225 (NPV-LDE-225, Erismodegib), have been unclear in hematological cancers. Here, we report that LDE225 selectively inhibited migration and adhesion of mantle cell lymphoma (MCL) to bone marrows via very late antigen-4 (VLA-4) mediated inactivation of focal adhesion kinase (FAK) signaling. LDE225 treatment not only affected MCL cells, but also modulated stromal cells within the bone marrow microenvironment by decreasing their production of SDF-1, IL-6 and VCAM-1, the ligand for VLA-4. Surprisingly, LDE225 treatment alone did not suppress cell proliferation due to increased CXCR4 expression mediated by reactive oxygen species (ROS). The increased ROS/CXCR4 further stimulated autophagy formation. The combination of LDE225 with the autophagy inhibitors further enhanced MCL cell death. Our data, for the first time, revealed LDE225 selectively targets MCL cells migration and adhesion to bone marrows. The ineffectiveness of LDE225 in MCL is due to autophagy formation, which in turn increases cell viability. Inhibiting autophagy will be an effective adjuvant therapy for LDE225 in MCL, especially for advanced MCL patients with bone marrow involvement. PMID:26885608

  2. Targeting radioresistant breast cancer cells by single agent CHK1 inhibitor via enhancing replication stress

    PubMed Central

    Du, Zhanwen; Gao, Jinnan; Yang, Shuming; Gorityala, Shashank; Xiong, Xiahui; Deng, Ou; Ma, Zhefu; Yan, Chunhong; Susana, Gonzalo; Xu, Yan; Zhang, Junran

    2016-01-01

    Radiotherapy (RT) remains a standard therapeutic modality for breast cancer patients. However, intrinsic or acquired resistance limits the efficacy of RT. Here, we demonstrate that CHK1 inhibitor AZD7762 alone significantly inhibited the growth of radioresistant breast cancer cells (RBCC). Given the critical role of ATR/CHK1 signaling in suppressing oncogene-induced replication stress (RS), we hypothesize that CHK1 inhibition leads to the specific killing for RBCC due to its abrogation in the suppression of RS induced by oncogenes. In agreement, the expression of oncogenes c-Myc/CDC25A/c-Src/H-ras/E2F1 and DNA damage response (DDR) proteins ATR/CHK1/BRCA1/CtIP were elevated in RBCC. AZD7762 exposure led to significantly higher levels of RS in RBCC, compared to the parental cells. The mechanisms by which CHK1 inhibition led to specific increase of RS in RBCC were related to the interruptions in the replication fork dynamics and the homologous recombination (HR). In summary, RBCC activate oncogenic pathways and thus depend upon mechanisms controlled by CHK1 signaling to maintain RS under control for survival. Our study provided the first example where upregulating RS by CHK1 inhibitor contributes to the specific killing of RBCC, and highlight the importance of the CHK1 as a potential target for treatment of radioresistant cancer cells. PMID:27167194

  3. PFI-1 – A highly Selective Protein Interaction Inhibitor Targeting BET Bromodomains

    PubMed Central

    Picaud, Sarah; Costa, David Da; Thanasopoulou, Angeliki; Filippakopoulos, Panagis; Fish, Paul V.; Philpott, Martin; Fedorov, Oleg; Brennan, Paul; Bunnage, Mark E.; Owen, Dafydd R.; Bradner, James E.; Taniere, Philippe; O’Sullivan, Brendan; Müller, Susanne; Schwaller, Juerg; Stankovic, Tatjana; Knapp, Stefan

    2013-01-01

    Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are transcriptional regulators required for efficient expression of several growth promoting and anti-apoptotic genes as well as for cell cycle progression. BET proteins are recruited to transcriptionally active chromatin via their two N-terminal bromodomains (BRDs), a protein interaction module that specifically recognizes acetylated lysine residues in histones H3 and H4. Inhibition of the BET-histone interaction results in transcriptional down-regulation of a number of oncogenes providing a novel pharmacological strategy for the treatment of cancer. Here we present a potent and highly selective dihydroquinazoline-2-one inhibitor, PFI-1 that efficiently blocks the interaction of BET BRDs with acetylated histone tails. Co-crystal structures showed that PFI-1 acts as an acetyl-lysine (Kac) mimetic inhibitor efficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukaemic cell lines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell cycle arrest, down-regulation of MYC expression as well as induction of apoptosis and induces differentiation of primary leukaemic blasts. Intriguingly, cells exposed to PFI-1 showed significant down-regulation of Aurora B kinase, thus attenuating phosphorylation of the Aurora substrate H3S10 providing an alternative strategy for the specific inhibition of this well established oncology target. PMID:23576556

  4. Targeting BRCA1- and BRCA2-deficient cells with RAD52 small molecule inhibitors.

    PubMed

    Huang, Fei; Goyal, Nadish; Sullivan, Katherine; Hanamshet, Kritika; Patel, Mikir; Mazina, Olga M; Wang, Charles X; An, W Frank; Spoonamore, James; Metkar, Shailesh; Emmitte, Kyle A; Cocklin, Simon; Skorski, Tomasz; Mazin, Alexander V

    2016-05-19

    RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair. PMID:26873923

  5. Novel inhibitor against Malassezia globosa LIP1 (SMG1), a potential anti-dandruff target.

    PubMed

    Guo, Shaohua; Huang, Wenkang; Zhang, Jian; Wang, Yonghua

    2015-09-01

    Compelling evidence have demonstrated the role of lipase activity in the pathogenicity of Malassezia globosa toward dandruff and seborrheic dermatitis (D/SD). As a representative secreted lipase from M. globosa CBS 7966, Malassezia globosa LIP1 (SMG1) is considered a potential anti-dandruff target. In this study, homology modeling, docking-based virtual screening and in vitro lipase-based assay were integrated to identify the first hit compound against SMG1, with an IC50 of 20 μM against synthetic lipase substrate, and of 0.19 μM when using natural lipase substrate. Evaluation of similar compounds, along with docking, offered information on the binding patterns of the hit compound. This work is expected to serve as a starting point for the rational design of more potent inhibitors against SMG1. PMID:26199121

  6. The inhibitor of apoptosis protein family (IAPs): an emerging therapeutic target in cancer.

    PubMed

    Nachmias, Boaz; Ashhab, Yaqoub; Ben-Yehuda, Dina

    2004-08-01

    Apoptosis is a crucial biological process that prevents uncontrolled cell proliferation and eliminates harmful cells. Resistance to apoptotic stimuli is a hallmark feature of various cancers. One of the mechanisms through which tumor cells are believed to acquire resistance to apoptosis is by overexpression of inhibitor of apoptosis proteins (IAPs). IAPs are a group of structurally related proteins that were initially identified in baculoviruses. Mammalian IAPs block apoptosis either by binding and inhibiting caspases or through caspase-independent mechanisms. This family of proteins has become increasingly prominent in the field of cancer biology. To date, overexpression of several IAPs has been detected in various cancers. This paper reviews the recent advances in the research of IAPs. The differential expression and the biological significance of each IAP in various cancer types will be discussed. Finally, we review the most recent advances in the research efforts aimed at using IAPs as potential targets for cancer therapy.

  7. Targeting inhibitor of apoptosis proteins (IAPs) for diagnosis and treatment of human diseases.

    PubMed

    Fulda, Simone; Debatin, Klaus-Michael

    2006-01-01

    Since cell death by apoptosis plays a key role in the regulation of tissue homeostasis, any defect in this intrinsic death program may result in tumor formation. "Inhibitor of apoptosis proteins" (IAPs) block apoptosis at the core of the apoptotic machinery by inhibiting effector caspases. Aberrant expression and/or function of IAPs have been implied to be involved in the pathogenesis and progression of various human diseases including cancer, autoimmune disorders or neurodegeneration. Recent insights into the regulation of IAPs have provided the basis for various exciting discoveries aimed at modulating expression or dysfunction of IAPs. Thus, targeting IAPs, e.g. by antisense approaches, RNA interference or small molecules, may proof to be a novel strategy for the diagnosis and treatment of human diseases.

  8. [Possibilities for inhibiting tumor-induced angiogenesis: results with multi-target tyrosine kinase inhibitors].

    PubMed

    Török, Szilvia; Döme, Balázs

    2012-03-01

    Functional blood vasculature is essential for tumor progression. The main signalization pathways that play a key role in the survival and growth of tumor vessels originate from the VEGF-, PDGF- and FGF tyrosine kinase receptors. In the past decade, significant results have been published on receptor tyrosine kinase inhibitors (RTKIs). In this paper, the mechanisms of action and the results so far available of experimental and clinical studies on multi-target antiangiogenic TKIs are discussed. On the one hand, notable achievements have been made recently and these drugs are already used in clinical practice in some patient populations. On the other hand, the optimal combination and dosage of these drugs, selection of the apropriate biomarker and better understanding of the conflicting role of PDGFR and FGFR signaling in angiogenesis remain future challenges. PMID:22403757

  9. Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells

    PubMed Central

    Di Martile, Marta; Desideri, Marianna; De Luca, Teresa; Gabellini, Chiara; Buglioni, Simonetta; Eramo, Adriana; Sette, Giovanni; Milella, Michele; Rotili, Dante; Mai, Antonello; Carradori, Simone; Secci, Daniela; De Maria, Ruggero; Del Bufalo, Donatella; Trisciuoglio, Daniela

    2016-01-01

    Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC. PMID:26870991

  10. Connexin targeting peptides as inhibitors of voltage- and intracellular Ca2+-triggered Cx43 hemichannel opening.

    PubMed

    Wang, Nan; De Bock, Marijke; Decrock, Elke; Bol, Mélissa; Gadicherla, Ashish; Bultynck, Geert; Leybaert, Luc

    2013-12-01

    Connexins form gap junctions that function as intercellular channels and hemichannels that form a conduit between the cytoplasm and extracellular fluid when open. Peptide inhibitors of connexin channels, especially those identical to defined connexin sequences, are interesting experimental, and possibly also therapeutic tools because they may have better selectivity than general inhibitors like carbenoxolone. Over the past ten years, several peptides have been demonstrated to block hemichannels, including Gap26, Gap27, peptide5, L2 and Gap19; some of these specifically block hemichannels but not gap junctions. Most of these peptides have only recently been investigated towards their actions at the single hemichannel level, bringing up interesting information on how they interact with the connexin protein and how they affect hemichannel gating. Hemichannels can be opened by electrical, mechanical and chemical stimuli. We here review the effect of the prototypic peptides Gap26/27 and L2/Gap19 with specific focus on their inhibition of Cx43 hemichannel opening triggered by positive membrane potentials and changes in intracellular Ca2+ concentration. Both Gap26/27 and L2/Gap19 peptide families block Cx43 hemichannel opening triggered by voltage as well as intracellular Ca2+ stimulation. Interestingly, these peptides as well as intracellular Ca2+ elevation modulate the voltage activation threshold for hemichannel opening, pointing to a common target. Moreover, L2 and Gap19 peptides are part of a sequence on the cytoplasmic loop that acts as a Ca2+/calmodulin interaction site. We here review the interesting network of interactions between Cx43 targeting peptides, voltage gating and intracellular Ca2+ as major modulators of hemichannel function. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

  11. A survival Kit for pancreatic beta cells: stem cell factor and c-Kit receptor tyrosine kinase.

    PubMed

    Feng, Zhi-Chao; Riopel, Matthew; Popell, Alex; Wang, Rennian

    2015-04-01

    The interactions between c-Kit and its ligand, stem cell factor (SCF), play an important role in haematopoiesis, pigmentation and gametogenesis. c-Kit is also found in the pancreas, and recent studies have revealed that c-Kit marks a subpopulation of highly proliferative pancreatic endocrine cells that may harbour islet precursors. c-Kit governs and maintains pancreatic endocrine cell maturation and function via multiple signalling pathways. In this review we address the importance of c-Kit signalling within the pancreas, including its profound role in islet morphogenesis, islet vascularisation, and beta cell survival and function. We also discuss the impact of c-Kit signalling in pancreatic disease and the use of c-Kit as a potential target for the development of cell-based and novel drug therapies in the treatment of diabetes.

  12. Small Molecule MYC Inhibitor Conjugated to Integrin-Targeted Nanoparticles Extends Survival in a Mouse Model of Disseminated Multiple Myeloma

    PubMed Central

    Cui, Grace; Senpan, Angana; Yang, Xiaoxia; Lu, Lan; Weilbaecher, Katherine N.; Prochownik, Edward V.; Lanza, Gregory M.; Tomasson, Michael H.

    2015-01-01

    Multiple myeloma pathogenesis is driven by the MYC oncoprotein, its dimerization with MAX, and the binding of this heterodimer to E-Boxes in the vicinity of target genes. The systemic utility of potent small molecule inhibitors of MYC-MAX dimerization was limited by poor bioavailability, rapid metabolism, and inadequate target site penetration. We hypothesized that new lipid-based MYC-MAX dimerization inhibitor prodrugs delivered via integrin-targeted nanoparticles (NP) would overcome prior shortcomings of MYC inhibitor approaches and prolong survival in a mouse model of cancer. An Sn 2 lipase-labile prodrug inhibitor of MYC-MAX dimerization (MI1-PD) was developed which decreased cell proliferation and induced apoptosis in cultured multiple myeloma cell lines alone (P < 0.05) and when incorporated into integrin-targeted lipid-encapsulated NPs (P < 0.05). Binding and efficacy of NPs closely correlated with integrin expression of the target multiple myeloma cells. Using a KaLwRij metastatic multiple myeloma mouse model, VLA-4–targeted NPs (20 nm and 200 nm) incorporating MI1-PD (D) NPs conferred significant survival benefits compared with respective NP controls, targeted (T) no-drug (ND), and untargeted (NT) control NPs (T/D 200: 46 days vs. NT/ND: 28 days, P < 0.05 and T/D 20: 52 days vs. NT/ND: 29 days, P = 0.001). The smaller particles performed better of the two sizes. Neither MI1 nor MI1-PD provided survival benefit when administered systemically as free compounds. These results demonstrate for the first time that a small molecule inhibitor of the MYC transcription factor can be an effective anticancer agent when delivered using a targeted nanotherapy approach. PMID:25824336

  13. Dual targeting of glioblastoma multiforme with a proteasome inhibitor (Velcade) and a phosphatidylinositol 3-kinase inhibitor (ZSTK474).

    PubMed

    Lin, Lehang; Gaut, Daria; Hu, Kaishun; Yan, Haiyan; Yin, Dong; Koeffler, H Phillip

    2014-02-01

    Proteasome inhibitors have been proven to be effective anticancer compounds in many tumor models, including glioblastoma multiforme (GBM). In this study, we found that the proteasome inhibitor Velcade (PS-341/bortezomib) caused GBM cell death while simultaneously activating the PI3K/Akt pathway. Therefore, we sought to investigate if the PI3K inhibitor ZSTK474 would enhance the effectiveness of Velcade in anticancer therapy. Two GBM cell lines were used to detect the effects of Velcade and ZSTK474 alone or in combination in vitro. The combination of Velcade and ZSTK474 synergistically inhibited the proliferation of GBM cell lines. Cell apoptosis was increased when exposed to Velcade and ZSTK474 in combination as shown by Annexin V analysis. Treatment with both drugs led to downregulation of the p-Akt, p-4EBP1 and p-mTOR proteins as determined by western blot analysis. The anticancer ability of Velcade for glioblastoma multiforme was, therefore, enhanced by combination with the PI3K pathway inhibitor ZSTK474 in glioblastoma multiforme.

  14. Metabolic Targeting of Malignant Tumors: Small-Molecule Inhibitors of Bioenergetic Flux

    PubMed Central

    Mathupala, Saroj P.

    2012-01-01

    Metabolism in tumors deviates significantly from that of normal tissues. Increasingly, the underlying aberrant metabolic pathways are being considered as novel targets for cancer therapy. Denoted “metabolic targeting”, small molecule drugs are under investigation for focused inhibition of key metabolic steps that are utilized by tumors, since such inhibitors should harbor minimal toxicity towards surrounding normal tissues.This review will examine the primary biochemical pathways that tumors harness to enhance their bioenergetic capacity, which in turn, help their rapid proliferation and metastasis within the host. It is hoped that “metabolite-mimetic” drugs can be utilized to interfere with metabolic flux pathways active within the tumor, and across tumor-microenvironment boundary. In fact, the major pathways of mammalian metabolism, i.e., the carbohydrate, amino-acid, and fatty-acid metabolic pathways have been examined as putative targets for drug development, with some drug candidates advancing to phase II/III stages. In this regard, glucose metabolism, i.e., the glycolytic pathway - that predominates the bio-energetic flux in tumors, and the associated mitochondrial metabolism have received the most attention as suitable “druggable” targets, focused either at the pathway enzymes or at the plasma-membrane-bound metabolite transporters. Outlined in this review are pre-clinical studies that have led to the discovery of promising drug candidates to target tumor-metabolic flux, and ensuing patents, with descriptions of the biochemical rationale for the combinatorial strategy of a particular metabolic pathway-drug candidate pair. PMID:21110820

  15. Targeting prostate cancer cells with a multivalent PSMA inhibitor-guided streptavidin conjugate.

    PubMed

    Liu, Tiancheng; Nedrow-Byers, Jessie R; Hopkins, Mark R; Wu, Lisa Y; Lee, Jeonghoon; Reilly, Peter T A; Berkman, Clifford E

    2012-06-15

    Prostate-specific membrane antigen (PSMA), a type II membrane glycoprotein, its high expression is associated with prostate cancer progression, and has been becoming an active target for imaging or therapeutic applications for prostate cancer. On the other hand, streptavidin-biotin system has been successfully employed in pretargeting therapy towards multiple cancers. Herein, we describe the synthesis of bifunctional ligands (biotin-CTT54, biotin-PEG(4)-CTT54, and biotin-PEG(12)-CTT54) possessing two functional motifs separated by a length-varied polyethylene glycol (PEG) spacer: one (CTT54) binds tumor-marker PSMA and the other (biotin) binds streptavidin or avidin. All three compounds exhibited high potencies (IC(50) values: 1.21, 2.53, and 10nM, respectively) and irreversibility; but only biotin-PEG(12)-CTT54 demonstrated specifically labeling PSMA-positive prostate cancer cells in a two-step pretargeting procedure. Additionally, the pre-formulated complex between biotin-PEG(12)-CTT54 and Cy5-streptavidin displayed the improved inhibitory potency (IC(50)=1.86 nM) and irreversibility against PSMA and rapid uptake of streptavidin conjugate into PSMA-positive prostate cancer cells through PSMA-associated internalization. Together, all these results supported a proof-concept that combination of streptavidin and PSMA's biotinylated inhibitor may lead to development of a novel strategy of tumor-targeting imaging or drug delivery towards prostate cancer.

  16. Targeted inhibition of complement using complement receptor 2-conjugated inhibitors attenuates EAE.

    PubMed

    Hu, Xianzhen; Tomlinson, Stephen; Barnum, Scott R

    2012-11-30

    Multiple sclerosis (MS) is the most common autoimmune demyelinating disease, affecting millions of individuals worldwide. In the last two decades, many therapeutic options for the treatment of MS have become available, however they are limited in terms of effectiveness and some remain plagued by safety issues. The currently available treatment options target relapsing remitting forms of MS and are not effective against the more progressive forms of the disease. These limitations highlight a significant unmet treatment need for MS. In experimental autoimmune encephalomyelitis (EAE) studies from our laboratory, we have previously shown, using a number of complement mutant and transgenic mice, that inhibition of the alternative complement pathway and the C3 convertase confers significant protection from disease. We report here that targeted inhibition of complement activation using complement receptor 2 (CR2)-conjugated inhibitors significantly attenuates EAE. Administration of CR2-Crry (blocks all complement pathways at C3 activation) and CR2-fH (specifically blocks the alternative pathway) just prior to and during the onset of EAE blocks progression of both acute and chronic disease. These data indicate that inhibition of complement may offer an effective therapeutic approach to treating both acute and chronic forms of demyelinating disease through blocking the alternative pathway or complement convertases. PMID:23079547

  17. Targeting Fusarium graminearum control via polyamine enzyme inhibitors and polyamine analogs.

    PubMed

    Crespo-Sempere, A; Estiarte, N; Marín, S; Sanchis, V; Ramos, A J

    2015-08-01

    Fusarium graminearum not only reduces yield and seed quality but also constitutes a risk to public or animal health owing to its ability to contaminate grains with mycotoxins. Resistance problems are emerging and control strategies based on new targets are needed. Polyamines have a key role in growth, development and differentiation. In this work, the possibility of using polyamine metabolism as a target to control F. graminearum has been assessed. It was found that putrescine induces mycotoxin production, correlating with an over expression of TRI5 and TRI6 genes. In addition, a homolog of the Saccharomyces cerevisiae TPO4 involved in putrescine excretion was up-regulated as putrescine concentration increased while DUR3 and SAM3 homologues, involved in putrescine uptake, were down-regulated. When 2.5 mM D, l-α-difluoromethylornithine (DFMO) was added to the medium, DON production decreased from 3.2 to 0.06 ng/mm(2) of colony and growth was lowered by up to 70 per cent. However, exogenous putrescine could overcome DFMO effects. Five polyamine transport inhibitors were also tested against F. graminearum. AMXT-1505 was able to completely inhibit in vitro growth and DON production. Additionally, AMXT-1505 blocked F. graminearum growth in inoculated wheat spikes reducing DON mycotoxin contamination from 76.87 μg/g to 0.62 μg/g.

  18. Targeting transcription regulation in cancer with a covalent CDK7 inhibitor

    PubMed Central

    Kwiatkowski, Nicholas; Zhang, Tinghu; Rahl, Peter B; Abraham, Brian J; Reddy, Jessica; Ficarro, Scott B; Dastur, Anahita; Amzallag, Arnaud; Ramaswamy, Sridhar; Tesar, Bethany; Jenkins, Christopher R; Hannett, Nancy M; McMillin, Douglas; Sanda, Takaomi; Sim, Taebo; Kim, Nam Doo; Look, Thomas; Mitsiades, Constantine; Weng, Andrew P; Brown, Jennifer R; Benes, Cyril H.; Marto, Jarrod A; Young, Richard A; Gray, Nathanael S.

    2014-01-01

    Tumor oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state1, but direct pharmacological inhibition of transcription factors has thus far proven difficult2. However, the transcriptional machinery contains various enzymatic co-factors that can be targeted for development of new therapeutic candidates3, including cyclin-dependent kinases (CDKs)4. Here we present the discovery and characterization of the first covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell line profiling indicates that a subset of cancer cell lines, including T-ALL, exhibit exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and this transcription factor’s key role in the core transcriptional regulatory circuitry of these tumor cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumor types exhibiting extreme dependencies on transcription for maintenance of the oncogenic state. PMID:25043025

  19. Transcription and translation are primary targets of Pim kinase inhibitor SGI-1776 in mantle cell lymphoma.

    PubMed

    Yang, Qingshan; Chen, Lisa S; Neelapu, Sattva S; Miranda, Roberto N; Medeiros, L Jeffrey; Gandhi, Varsha

    2012-10-25

    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 molecule 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

  20. Targeting inhibitor of apoptosis proteins in combination with ErbB antagonists in breast cancer

    PubMed Central

    Foster, Fiona M; Owens, Thomas W; Tanianis-Hughes, Jolanta; Clarke, Robert B; Brennan, Keith; Bundred, Nigel J; Streuli, Charles H

    2009-01-01

    Introduction Inhibitor of apoptosis (IAPs) proteins are a family of proteins that can block apoptosis in normal cells and have been suggested to cause resistance to apoptosis in cancer. Overexpression of oncogenic receptor tyrosine kinases is common in breast cancer; in particular 20% of all cases show elevated Her2. Despite clinical success with the use of targeted therapies, such as Trastuzumab, only up to 35% of Her2-positive patients initially respond. We reasoned that IAP-mediated apoptosis resistance might contribute to this insensitivity to receptor tyrosine kinase therapy, in particular ErbB antagonists. Here we examine the levels of IAPs in breast cancer and evaluate whether targeting IAPs can enhance apoptosis in response to growth factor receptor antagonists and TRAIL. Methods IAP levels were examined in a breast cancer cell line panel and in patient samples. IAPs were inhibited using siRNA or cell permeable mimetics of endogenous inhibitors. Cells were then exposed to TRAIL, Trastuzumab, Lapatinib, or Gefitinib for 48 hours. Examining nuclear morphology and staining for cleaved caspase 3 was used to score apoptosis. Proliferation was examined by Ki67 staining. Results Four members of the IAP family, Survivin, XIAP, cIAP1 and cIAP2, were all expressed to varying extents in breast cancer cell lines or tumours. MDAMB468, BT474 and BT20 cells all expressed XIAP to varying extents. Depleting the cells of XIAP overcame the intrinsic resistance of BT20 and MDAMB468 cells to TRAIL. Moreover, siRNA-based depletion of XIAP or use of a Smac mimetic to target multiple IAPs increased apoptosis in response to the ErbB antagonists, Trastuzumab, Lapatinib or Gefitinib in Her2-overexpressing BT474 cells, or Gefitinib in EGFR-overexpressing MDAMB468 cells. Conclusions The novel findings of this study are that multiple IAPs are concomitantly expressed in breast cancers, and that, in combination with clinically relevant Her2 treatments, IAP antagonists promote apoptosis

  1. Small Molecules, Inhibitors of DNA-PK, Targeting DNA Repair, and Beyond

    PubMed Central

    Davidson, David; Amrein, Lilian; Panasci, Lawrence; Aloyz, Raquel

    2012-01-01

    Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining a major mechanism for the repair of double-strand breaks (DSB) in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK). The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA-PK. Computer based drug

  2. A novel assay for screening inhibitors targeting HIV-1 integrase dimerization based on Ni-NTA magnetic agarose beads.

    PubMed

    Zhang, Dawei; He, Hongqiu; Liu, Mengmeng; Meng, Zhixia; Guo, Shunxing

    2016-05-03

    Human immunodeficiency virus (HIV)-1 integrase (IN), which mediates integration of viral cDNA into the cellular chromosome, is a validated antiviral drug target. Three IN inhibitors, raltegravir, elvitegravir and dolutegravir, have been clinically approved since 2008. However, drug resistance have emerged in infected patients receiving treatment using these drugs which share the same mechanism of action and have a low genetic barrier for resistance. Therefore, there is an urgent need to develop drugs with novel mechanism. IN requires a precise and dynamic equilibrium between several oligomeric species for its activities. The modulation of the process which is termed as IN oligomerization, presents an interesting allosteric target for drug development. In this research, we developed a magnetic beads based approach to assay the IN dimerization. Then, using the assay we screened a library of 1000 Food and Drug Administration (FDA)-approved drugs for IN dimerization inhibitors and identified dexlansoprazole as a potential IN dimerization inhibitor. In conclusion, the assay presented here has been proven to be sensitive and specific for the detection of IN dimerization as well as for the identification of antiviral drugs targeting IN dimerization. Moreover, a FDA-approved proton-pump inhibitors, dexlansoprazole, was identified as a potential inhibitor for IN dimerization.

  3. A novel assay for screening inhibitors targeting HIV-1 integrase dimerization based on Ni-NTA magnetic agarose beads

    PubMed Central

    Zhang, Dawei; He, Hongqiu; Liu, Mengmeng; Meng, Zhixia; Guo, Shunxing

    2016-01-01

    Human immunodeficiency virus (HIV)-1 integrase (IN), which mediates integration of viral cDNA into the cellular chromosome, is a validated antiviral drug target. Three IN inhibitors, raltegravir, elvitegravir and dolutegravir, have been clinically approved since 2008. However, drug resistance have emerged in infected patients receiving treatment using these drugs which share the same mechanism of action and have a low genetic barrier for resistance. Therefore, there is an urgent need to develop drugs with novel mechanism. IN requires a precise and dynamic equilibrium between several oligomeric species for its activities. The modulation of the process which is termed as IN oligomerization, presents an interesting allosteric target for drug development. In this research, we developed a magnetic beads based approach to assay the IN dimerization. Then, using the assay we screened a library of 1000 Food and Drug Administration (FDA)-approved drugs for IN dimerization inhibitors and identified dexlansoprazole as a potential IN dimerization inhibitor. In conclusion, the assay presented here has been proven to be sensitive and specific for the detection of IN dimerization as well as for the identification of antiviral drugs targeting IN dimerization. Moreover, a FDA-approved proton-pump inhibitors, dexlansoprazole, was identified as a potential inhibitor for IN dimerization. PMID:27137477

  4. Non-target-site resistance to ALS inhibitors in waterhemp (Amaranthus tuberculatus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A waterhemp population (MCR) previously characterized as resistant to 4-hyroxyphenylpyruvate dioxygenase (HPPD) and photosystem II (PSII) inhibitors was found to have two different resistance responses to acetolactate synthase (ALS) inhibitors. Plants from the MCR population exhibiting high resistan...

  5. Target prices for mass production of tyrosine kinase inhibitors for global cancer treatment

    PubMed Central

    Hill, Andrew; Gotham, Dzintars; Fortunak, Joseph; Meldrum, Jonathan; Erbacher, Isabelle; Martin, Manuel; Shoman, Haitham; Levi, Jacob; Powderly, William G; Bower, Mark

    2016-01-01

    Objective To calculate sustainable generic prices for 4 tyrosine kinase inhibitors (TKIs). Background TKIs have proven survival benefits in the treatment of several cancers, including chronic myeloid leukaemia, breast, liver, renal and lung cancer. However, current high prices are a barrier to treatment. Mass production of low-cost generic antiretrovirals has led to over 13 million people being on HIV/AIDS treatment worldwide. This analysis estimates target prices for generic TKIs, assuming similar methods of mass production. Methods Four TKIs with patent expiry dates in the next 5 years were selected for analysis: imatinib, erlotinib, lapatinib and sorafenib. Chemistry, dosing, published data on per-kilogram pricing for commercial transactions of active pharmaceutical ingredient (API), and quotes from manufacturers were used to estimate costs of production. Analysis included costs of excipients, formulation, packaging, shipping and a 50% profit margin. Target prices were compared with current prices. Global numbers of patients eligible for treatment with each TKI were estimated. Results API costs per kg were $347–$746 for imatinib, $2470 for erlotinib, $4671 for lapatinib, and $3000 for sorafenib. Basing on annual dose requirements, costs of formulation/packaging and a 50% profit margin, target generic prices per person-year were $128–$216 for imatinib, $240 for erlotinib, $1450 for sorafenib, and $4020 for lapatinib. Over 1 million people would be newly eligible to start treatment with these TKIs annually. Conclusions Mass generic production of several TKIs could achieve treatment prices in the range of $128–$4020 per person-year, versus current US prices of $75161–$139 138. Generic TKIs could allow significant savings and scaling-up of treatment globally, for over 1 million eligible patients. PMID:26817636

  6. MCM5 as a target of BET inhibitors in thyroid cancer cells.

    PubMed

    Mio, Catia; Lavarone, Elisa; Conzatti, Ketty; Baldan, Federica; Toffoletto, Barbara; Puppin, Cinzia; Filetti, Sebastiano; Durante, Cosimo; Russo, Diego; Orlacchio, Arturo; Di Cristofano, Antonio; Di Loreto, Carla; Damante, Giuseppe

    2016-04-01

    Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer subtype, refractory to the current medical treatment. Among various epigenetic anticancer drugs, bromodomain and extra-terminal inhibitors (BETis) are considered to be an appealing novel class of compounds. BETi target the bromodomain and extra-terminal of BET proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlies the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated the effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETis, JQ1 and I-BET762, decreased cell viability, reduced cell cycle S-phase, and determined cell death. In order to find BETi effectors, FRO and SW1736 were subjected to a global transcriptome analysis after JQ1 treatment. A significant portion of deregulated genes belongs to cell cycle regulators. Among them, MCM5 was decreased at both mRNA and protein levels in both tested cell lines. Chromatin immunoprecipitation (ChIP) experiments indicate that MCM5 is directly bound by the BET protein BRD4. MCM5 silencing reduced cell proliferation, thus underlining its involvement in the block of proliferation induced by BETis. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its overexpression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also overexpressed in a murine model of ATC, and JQ1 treatment reduced Mcm5 mRNA expression in two murine ATC cell lines. Thus, MCM5 could represent a new target in the therapeutic approach against ATC. PMID:26911376

  7. A Small-Molecule Inhibitor Targeting the Mitotic Spindle Checkpoint Impairs the Growth of Uterine Leiomyosarcoma

    PubMed Central

    Shan, Weiwei; Akinfenwa, Patricia Y.; Savannah, Kari B.; Kolomeyevskaya, Nonna; Laucirica, Rudolfo; Thomas, Dafydd G.; Odunsi, Kunle; Creighton, Chad J.; Lev, Dina C.; Anderson, Matthew L.

    2016-01-01

    Purpose Uterine leiomyosarcoma (ULMS) is a poorly understood cancer with few effective treatments. This study explores the molecular events involved in ULMS with the goal of developing novel therapeutic strategies. Experimental Design Genome-wide transcriptional profiling, Western blotting, and real-time PCR were used to compare specimens of myometrium, leiomyoma, and leiomyosarcoma. Aurora A kinase was targeted in cell lines derived from metastatic ULMS using siRNA or MK-5108, a highly specific small-molecule inhibitor. An orthotopic model was used to evaluate the ability of MK-5108 to inhibit ULMS growth in vivo. Results We found that 26 of 50 gene products most overexpressed in ULMS regulate mitotic centrosome and spindle functions. These include UBE2C, Aurora A and B kinase, TPX2, and Polo-like kinase 1 (PLK1). Targeting Aurora A inhibited proliferation and induced apoptosis in LEIO285, LEIO505, and SK-LMS1, regardless of whether siRNA or MK-5108 was used. In vitro, MK-5108 did not consistently synergize with gemcitabine or docetaxel. Gavage of an orthotopic ULMS model with MK-5108 at 30 or 60 mg/kg decreased the number and size of tumor implants compared with sham-fed controls. Oral MK-5108 also decreased the rate of proliferation, increased intratumoral apoptosis, and increased expression of phosphohistone H3 in ULMS xenografts. Conclusions Our results show that dysregulated centrosome function and spindle assembly are a robust feature of ULMS that can be targeted to slow its growth both in vitro and in vivo. These observations identify novel directions that can be potentially used to improve clinical outcomes for this disease. PMID:22535157

  8. FGF signaling inhibitor, SPRY4, is evolutionarily conserved target of WNT signaling pathway in progenitor cells.

    PubMed

    Katoh, Yuriko; Katoh, Masaru

    2006-03-01

    WNT, FGF and Hedgehog signaling pathways network together during embryogenesis, tissue regeneration, and carcinogenesis. FGF16, FGF18, and FGF20 genes are targets of WNT-mediated TCF/LEF-beta-catenin-BCL9/BCL9L-PYGO transcriptional complex. SPROUTY (SPRY) and SPRED family genes encode inhibitors for receptor tyrosine kinase signaling cascades, such as those of FGF receptor family members and EGF receptor family members. Here, transcriptional regulation of SPRY1, SPRY2, SPRY3, SPRY4, SPRED1, SPRED2, and SPRED3 genes by WNT/beta-catenin signaling cascade was investigated by using bioinformatics and human intelligence (humint). Because double TCF/LEF-binding sites were identified within the 5'-promoter region of human SPRY4 gene, comparative genomics analyses on SPRY4 orthologs were further performed. SPRY4-FGF1 locus at human chromosome 5q31.3 and FGF2-NUDT6-SPATA5-SPRY1 locus at human chromosome 4q27-q28.1 were paralogous regions within the human genome. Chimpanzee SPRY4 gene was identified within NW_107083.1 genome sequence. Human, chimpanzee, rat and mouse SPRY4 orthologs, consisting of three exons, were well conserved. SPRY4 gene was identified as the evolutionarily conserved target of WNT/beta-catenin signaling pathway based on the conservation of double TCF/LEF-binding sites within 5'-promoter region of mammalian SPRY4 orthologs. Human SPRY4 mRNA was expressed in embryonic stem (ES) cells, brain, pancreatic islet, colon cancer, head and neck tumor, melanoma, and pancreatic cancer. WNT signaling activation in progenitor cells leads to the growth regulation of progenitor cells themselves through SPRY4 induction, and also to the growth stimulation of proliferating cells through FGF secretion. Epigenetic silencing and loss-of-function mutations of SPRY4 gene in progenitor cells could lead to carcinogenesis. SPRY4 is the pharmacogenomics target in the fields of oncology and regenerative medicine. PMID:16465403

  9. Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor.

    PubMed

    Guo, W; Liu, R; Bhardwaj, G; Yang, J C; Changou, C; Ma, A-H; Mazloom, A; Chintapalli, S; Xiao, K; Xiao, W; Kumaresan, P; Sanchez, E; Yeh, C-T; Evans, C P; Patterson, R; Lam, K S; Kung, H-J

    2014-01-01

    Btk and Etk/BMX are Tec-family non-receptor tyrosine kinases. Btk has previously been reported to be expressed primarily in B cells and has an important role in immune responses and B-cell malignancies. Etk has been shown previously to provide a strong survival and metastasis signal in human prostate cancer cells, and to confer androgen independence and drug resistance. While the role of Etk in prostate carcinogenesis is well established, the functions of Btk in prostate cancer have never been investigated, likely due to the perception that Btk is a hematopoietic, but not epithelial, kinase. Herein, we found that Btk is overexpressed in prostate cancer tissues and prostate cancer cells. The level of Btk in prostate cancer tissues correlates with cancer grades. Knockdown of Btk expression selectively inhibits the growth of prostate cancer cells, but not that of the normal prostate epithelial cells, which express very little Btk. Dual inhibition of Btk and Etk has an additive inhibitory effect on prostate cancer cell growth. To explore Btk and Etk as targets for prostate cancer, we developed a small molecule dual inhibitor of Btk and Etk, CTN06. Treatment of PC3 and other prostate cancer cells, but not immortalized prostate epithelial cells with CTN06 resulted in effective cell killing, accompanied by the attenuation of Btk/Etk signals. The killing effect of CTN06 is more potent than that of commonly used inhibitors against Src, Raf/VEGFR and EGFR. CTN06 induces apoptosis as well as autophagy in human prostate cancer cells, and is a chemo-sensitizer for docetaxel (DTX), a standard of care for metastatic prostate cancer patients. CTN06 also impeded the migration of human prostate cancer cells based on a 'wound healing' assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model. PMID:25188519

  10. Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor

    PubMed Central

    Guo, W; Liu, R; Bhardwaj, G; Yang, J C; Changou, C; Ma, A-H; Mazloom, A; Chintapalli, S; Xiao, K; Xiao, W; Kumaresan, P; Sanchez, E; Yeh, C-T; Evans, C P; Patterson, R; Lam, K S; Kung, H-J

    2014-01-01

    Btk and Etk/BMX are Tec-family non-receptor tyrosine kinases. Btk has previously been reported to be expressed primarily in B cells and has an important role in immune responses and B-cell malignancies. Etk has been shown previously to provide a strong survival and metastasis signal in human prostate cancer cells, and to confer androgen independence and drug resistance. While the role of Etk in prostate carcinogenesis is well established, the functions of Btk in prostate cancer have never been investigated, likely due to the perception that Btk is a hematopoietic, but not epithelial, kinase. Herein, we found that Btk is overexpressed in prostate cancer tissues and prostate cancer cells. The level of Btk in prostate cancer tissues correlates with cancer grades. Knockdown of Btk expression selectively inhibits the growth of prostate cancer cells, but not that of the normal prostate epithelial cells, which express very little Btk. Dual inhibition of Btk and Etk has an additive inhibitory effect on prostate cancer cell growth. To explore Btk and Etk as targets for prostate cancer, we developed a small molecule dual inhibitor of Btk and Etk, CTN06. Treatment of PC3 and other prostate cancer cells, but not immortalized prostate epithelial cells with CTN06 resulted in effective cell killing, accompanied by the attenuation of Btk/Etk signals. The killing effect of CTN06 is more potent than that of commonly used inhibitors against Src, Raf/VEGFR and EGFR. CTN06 induces apoptosis as well as autophagy in human prostate cancer cells, and is a chemo-sensitizer for docetaxel (DTX), a standard of care for metastatic prostate cancer patients. CTN06 also impeded the migration of human prostate cancer cells based on a ‘wound healing' assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model. PMID:25188519

  11. Fragment-based discovery of inhibitor scaffolds targeting the metallo-β-lactamases NDM-1 and VIM-2.

    PubMed

    Christopeit, Tony; Leiros, Hanna-Kirsti S

    2016-04-15

    Metallo-β-lactamases (MBLs) render bacteria resistant to β-lactam antibiotics and are interesting drug targets to prevent the hydrolysis of β-lactam antibiotics. So far, there are no MBL inhibitors in clinical use and particularly the design of broad spectrum inhibitors targeting several MBLs has been difficult. In this study, we report four fragments inhibiting the clinically relevant New Delhi metallo-β-lactamase 1 (NDM-1) and Verona integron-encoded metallo-β-lactamase 2 (VIM-2). The fragments were identified from a library using an orthogonal screening strategy combining a surface plasmon resonance (SPR) based assay and an enzyme inhibition assay. The identified fragments showed dissociation constants (KD) ranging from 181 to 2100 μM. The binding mode of the fragments was explored using QM-polarized ligand docking. All four fragments represent interesting scaffolds for the design of broad-spectrum MBL inhibitors. PMID:26976213

  12. Chemoproteomic profiling reveals that cathepsin D off-target activity drives ocular toxicity of β-secretase inhibitors

    PubMed Central

    Zuhl, Andrea M.; Nolan, Charles E.; Brodney, Michael A.; Niessen, Sherry; Atchison, Kevin; Houle, Christopher; Karanian, David A.; Ambroise, Claude; Brulet, Jeffrey W.; Beck, Elizabeth M.; Doran, Shawn D.; O'Neill, Brian T.; am Ende, Christopher W.; Chang, Cheng; Geoghegan, Kieran F.; West, Graham M.; Judkins, Joshua C.; Hou, Xinjun; Riddell, David R.; Johnson, Douglas S.

    2016-01-01

    Inhibition of β-secretase BACE1 is considered one of the most promising approaches for treating Alzheimer's disease. Several structurally distinct BACE1 inhibitors have been withdrawn from development after inducing ocular toxicity in animal models, but the target mediating this toxicity has not been identified. Here we use a clickable photoaffinity probe to identify cathepsin D (CatD) as a principal off-target of BACE1 inhibitors in human cells. We find that several BACE1 inhibitors blocked CatD activity in cells with much greater potency than that displayed in cell-free assays with purified protein. Through a series of exploratory toxicology studies, we show that quantifying CatD target engagement in cells with the probe is predictive of ocular toxicity in vivo. Taken together, our findings designate off-target inhibition of CatD as a principal driver of ocular toxicity for BACE1 inhibitors and more generally underscore the power of chemical proteomics for discerning mechanisms of drug action. PMID:27727204

  13. Fatty Acid-binding Proteins Transport N-Acylethanolamines to Nuclear Receptors and Are Targets of Endocannabinoid Transport Inhibitors*

    PubMed Central

    Kaczocha, Martin; Vivieca, Stephanie; Sun, Jing; Glaser, Sherrye T.; Deutsch, Dale G.

    2012-01-01

    N-Acylethanolamines (NAEs) are bioactive lipids that engage diverse receptor systems. Recently, we identified fatty acid-binding proteins (FABPs) as intracellular NAE carriers. Here, we provide two new functions for FABPs in NAE signaling. We demonstrate that FABPs mediate the nuclear translocation of the NAE oleoylethanolamide, an agonist of nuclear peroxisome proliferator-activated receptor α (PPARα). Antagonism of FABP function through chemical inhibition, dominant-negative approaches, or shRNA-mediated knockdown reduced PPARα activation, confirming a requisite role for FABPs in this process. In addition, we show that NAE analogs, traditionally employed as inhibitors of the putative endocannabinoid transmembrane transporter, target FABPs. Support for the existence of the putative membrane transporter stems primarily from pharmacological inhibition of endocannabinoid uptake by such transport inhibitors, which are widely employed in endocannabinoid research despite lacking a known cellular target(s). Our approach adapted FABP-mediated PPARα signaling and employed in vitro binding, arachidonoyl-[1-14C]ethanolamide ([14C]AEA) uptake, and FABP knockdown to demonstrate that transport inhibitors exert their effects through inhibition of FABPs, thereby providing a molecular rationale for the underlying physiological effects of these compounds. Identification of FABPs as targets of transport inhibitors undermines the central pharmacological support for the existence of an endocannabinoid transmembrane transporter. PMID:22170058

  14. A transgenic zebrafish model expressing KIT-D816V recapitulates features of aggressive systemic mastocytosis.

    PubMed

    Balci, Tugce B; Prykhozhij, Sergey V; Teh, Evelyn M; Da'as, Sahar I; McBride, Eileen; Liwski, Robert; Chute, Ian C; Leger, Daniel; Lewis, Stephen M; Berman, Jason N

    2014-10-01

    Systemic mastocytosis (SM) is a rare myeloproliferative disease without curative therapy. Despite clinical variability, the majority of patients harbour a KIT-D816V mutation, but efforts to inhibit mutant KIT with tyrosine kinase inhibitors have been unsatisfactory, indicating a need for new preclinical approaches to identify alternative targets and novel therapies in this disease. Murine models to date have been limited and do not fully recapitulate the most aggressive forms of SM. We describe the generation of a transgenic zebrafish model expressing the human KIT-D816V mutation. Adult fish demonstrate a myeloproliferative disease phenotype, including features of aggressive SM in haematopoeitic tissues and high expression levels of endopeptidases, consistent with SM patients. Transgenic embryos demonstrate a cell-cycle phenotype with corresponding expression changes in genes associated with DNA maintenance and repair, such as reduced dnmt1. In addition, epcam was consistently downregulated in both transgenic adults and embryos. Decreased embryonic epcam expression was associated with reduced neuromast numbers, providing a robust in vivo phenotypic readout for chemical screening in KIT-D816V-induced disease. This study represents the first zebrafish model of a mast cell disease with an aggressive adult phenotype and embryonic markers that could be exploited to screen for novel agents in SM.

  15. The NAE inhibitor pevonedistat interacts with the HDAC inhibitor belinostat to target AML cells by disrupting the DDR.

    PubMed

    Zhou, Liang; Chen, Shuang; Zhang, Yu; Kmieciak, Maciej; Leng, Yun; Li, Lihong; Lin, Hui; Rizzo, Kathryn A; Dumur, Catherine I; Ferreira-Gonzalez, Andrea; Rahmani, Mohamed; Povirk, Lawrence; Chalasani, Sri; Berger, Allison J; Dai, Yun; Grant, Steven

    2016-05-01

    Two classes of novel agents, NEDD8-activating enzyme (NAE) and histone deacetylase (HDAC) inhibitors, have shown single-agent activity in acute myelogenous leukemia (AML)/myelodysplastic syndrome (MDS). Here we examined mechanisms underlying interactions between the NAE inhibitor pevonedistat (MLN4924) and the approved HDAC inhibitor belinostat in AML/MDS cells. MLN4924/belinostat coadministration synergistically induced AML cell apoptosis with or without p53 deficiency or FLT3-internal tandem duplication (ITD), whereas p53 short hairpin RNA (shRNA) knockdown or enforced FLT3-ITD expression significantly sensitized cells to the regimen. MLN4924 blocked belinostat-induced antiapoptotic gene expression through nuclear factor-κB inactivation. Each agent upregulated Bim, and Bim knockdown significantly attenuated apoptosis. Microarrays revealed distinct DNA damage response (DDR) genetic profiles between individual vs combined MLN4924/belinostat exposure. Whereas belinostat abrogated the MLN4924-activated intra-S checkpoint through Chk1 and Wee1 inhibition/downregulation, cotreatment downregulated multiple homologous recombination and nonhomologous end-joining repair proteins, triggering robust double-stranded breaks, chromatin pulverization, and apoptosis. Consistently, Chk1 or Wee1 shRNA knockdown significantly sensitized AML cells to MLN4924. MLN4924/belinostat displayed activity against primary AML or MDS cells, including those carrying next-generation sequencing-defined poor-prognostic cancer hotspot mutations, and CD34(+)/CD38(-)/CD123(+) populations, but not normal CD34(+) progenitors. Finally, combined treatment markedly reduced tumor burden and significantly prolonged animal survival (P < .0001) in AML xenograft models with negligible toxicity, accompanied by pharmacodynamic effects observed in vitro. Collectively, these findings argue that MLN4924 and belinostat interact synergistically by reciprocally disabling the DDR in AML/MDS cells. This strategy

  16. DYRK1B as therapeutic target in Hedgehog/GLI-dependent cancer cells with Smoothened inhibitor resistance

    PubMed Central

    Gruber, Wolfgang; Hutzinger, Martin; Elmer, Dominik Patrick; Parigger, Thomas; Sternberg, Christina; Cegielkowski, Lukasz; Zaja, Mirko; Leban, Johann; Michel, Susanne; Hamm, Svetlana; Vitt, Daniel; Aberger, Fritz

    2016-01-01

    A wide range of human malignancies displays aberrant activation of Hedgehog (HH)/GLI signaling, including cancers of the skin, brain, gastrointestinal tract and hematopoietic system. Targeting oncogenic HH/GLI signaling with small molecule inhibitors of the essential pathway effector Smoothened (SMO) has shown remarkable therapeutic effects in patients with advanced and metastatic basal cell carcinoma. However, acquired and de novo resistance to SMO inhibitors poses severe limitations to the use of SMO antagonists and urgently calls for the identification of novel targets and compounds. Here we report on the identification of the Dual-Specificity-Tyrosine-Phosphorylation-Regulated Kinase 1B (DYRK1B) as critical positive regulator of HH/GLI signaling downstream of SMO. Genetic and chemical inhibition of DYRK1B in human and mouse cancer cells resulted in marked repression of HH signaling and GLI1 expression, respectively. Importantly, DYRK1B inhibition profoundly impaired GLI1 expression in both SMO-inhibitor sensitive and resistant settings. We further introduce a novel small molecule DYRK1B inhibitor, DYRKi, with suitable pharmacologic properties to impair SMO-dependent and SMO-independent oncogenic GLI activity. The results support the use of DYRK1B antagonists for the treatment of HH/GLI-associated cancers where SMO inhibitors fail to demonstrate therapeutic efficacy. PMID:26784250

  17. PIK3CA mutations can initiate pancreatic tumorigenesis and are targetable with PI3K inhibitors

    PubMed Central

    Payne, S N; Maher, M E; Tran, N H; Van De Hey, D R; Foley, T M; Yueh, A E; Leystra, A A; Pasch, C A; Jeffrey, J J; Clipson, L; Matkowskyj, K A; Deming, D A

    2015-01-01

    Aberrations in the phosphoinositide 3-kinase (PI3K) signaling pathway have a key role in the pathogenesis of numerous cancers by altering cell growth, metabolism, proliferation and apoptosis. Interest in targeting the PI3K signaling cascade continues, as new agents are being clinically evaluated. PIK3CA mutations result in a constitutively active PI3K and are present in a subset of pancreatic cancers. Here we examine mutant PIK3CA-mediated pancreatic tumorigenesis and the response of PIK3CA mutant pancreatic cancers to dual PI3K/mammalian target of rapamycin (mTOR) inhibition. Two murine models were generated expressing a constitutively active PI3K within the pancreas. An increase in acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasms (PanINs) was identified. In one model these lesions were detected as early as 10 days of age. Invasive pancreatic ductal adenocarcinoma developed in these mice as early as 20 days of age. These cancers were highly sensitive to treatment with dual PI3K/mTOR inhibition. In the second model, PanINs and invasive cancer develop with a greater latency owing to a lesser degree of PI3K pathway activation in this murine model. In addition to PI3K pathway activation, increased ERK1/2 signaling is common in human pancreatic cancers. Phosphorylation of ERK1/2 was also investigated in these models. Phosphorylation of ERK1/2 is demonstrated in the pre-neoplastic lesions and invasive cancers. This activation of ERK1/2 is diminished with dual PI3K/mTOR inhibition. In summary, PIK3CA mutations can initiate pancreatic tumorigenesis and these cancers are particularly sensitive to dual PI3K/mTOR inhibition. Future studies of PI3K pathway inhibitors for patients with PIK3CA mutant pancreatic cancers are warranted. PMID:26436951

  18. Advances in the therapeutic use of mammalian target of rapamycin (mTOR) inhibitors in dermatology.

    PubMed

    Fogel, Alexander L; Hill, Sharleen; Teng, Joyce M C

    2015-05-01

    Significant developments in the use of mammalian target of rapamycin (mTOR) inhibitors (mTORIs) as immunosuppressant and antiproliferative agents have been made. Recent advances in the understanding of the mTOR signaling pathway and its downstream effects on tumorigenesis and vascular proliferation have broadened the clinical applications of mTORIs in many challenging disorders such as tuberous sclerosis complex, pachyonychia congenita, complex vascular anomalies, and inflammatory dermatoses. Systemic mTORI therapy has shown benefits in these areas, but is associated with significant side effects that sometimes necessitate drug holidays. To mitigate the side effects of systemic mTORIs for dermatologic applications, preliminary work to assess the potential of percutaneous therapy has been performed, and the evidence suggests that percutaneous delivery of mTORIs may allow for effective long-term therapy while avoiding systemic toxicities. Additional large placebo-controlled, double-blinded, randomized studies are needed to assess the efficacy, safety, duration, and tolerability of topical treatments. The objective of this review is to provide updated information on the novel use of mTORIs in the management of many cutaneous disorders. PMID:25769191

  19. Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection.

    PubMed

    Lawrence, Amba; Fraser, Tamieka; Gillett, Amber; Tyndall, Joel D A; Timms, Peter; Polkinghorne, Adam; Huston, Wilhelmina M

    2016-01-01

    The koala, an iconic marsupial native to Australia, is a threatened species in many parts of the country. One major factor in the decline is disease caused by infection with Chlamydia. Current therapeutic strategies to treat chlamydiosis in the koala are limited. This study examines the effectiveness of an inhibitor, JO146, which targets the HtrA serine protease for treatment of C. pecorum and C. pneumoniae in vitro and ex vivo with the aim of developing a novel therapeutic for koala Chlamydia infections. Clinical isolates from koalas were examined for their susceptibility to JO146. In vitro studies demonstrated that treatment with JO146 during the mid-replicative phase of C. pecorum or C. pneumoniae infections resulted in a significant loss of infectious progeny. Ex vivo primary koala tissue cultures were used to demonstrate the efficacy of JO146 and the non-toxic nature of this compound on peripheral blood mononuclear cells and primary cell lines established from koala tissues collected at necropsy. Our results suggest that inhibition of the serine protease HtrA could be a novel treatment strategy for chlamydiosis in koalas. PMID:27530689

  20. BRAF inhibitors suppress apoptosis through off-target inhibition of JNK signaling

    PubMed Central

    Vin, Harina; Ojeda, Sandra S; Ching, Grace; Leung, Marco L; Chitsazzadeh, Vida; Dwyer, David W; Adelmann, Charles H; Restrepo, Monica; Richards, Kristen N; Stewart, Larissa R; Du, Lili; Ferguson, Scarlett B; Chakravarti, Deepavali; Ehrenreiter, Karin; Baccarini, Manuela; Ruggieri, Rosamaria; Curry, Jonathan L; Kim, Kevin B; Ciurea, Ana M; Duvic, Madeleine; Prieto, Victor G; Ullrich, Stephen E; Dalby, Kevin N; Flores, Elsa R; Tsai, Kenneth Y

    2013-01-01

    Vemurafenib and dabrafenib selectively inhibit the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) kinase, resulting in high response rates and increased survival in melanoma. Approximately 22% of individuals treated with vemurafenib develop cutaneous squamous cell carcinoma (cSCC) during therapy. The prevailing explanation for this is drug-induced paradoxical ERK activation, resulting in hyperproliferation. Here we show an unexpected and novel effect of vemurafenib/PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases upstream of c-Jun N-terminal kinase (JNK), principally ZAK. JNK signaling is suppressed in multiple contexts, including in cSCC of vemurafenib-treated patients, as well as in mice. Expression of a mutant ZAK that cannot be inhibited reverses the suppression of JNK activation and apoptosis. Our results implicate suppression of JNK-dependent apoptosis as a significant, independent mechanism that cooperates with paradoxical ERK activation to induce cSCC, suggesting broad implications for understanding toxicities associated with BRAF inhibitors and for their use in combination therapies. DOI: http://dx.doi.org/10.7554/eLife.00969.001 PMID:24192036

  1. Amphiphilic cationic nanogels as brain-targeted carriers for activated nucleoside reverse transcriptase inhibitors

    PubMed Central

    Warren, G; Makarov, E; Lu, Y; Senanayake, T; Rivera, K; Gorantla, S; Poluektova, LY; Vinogradov, SV

    2015-01-01

    Progress in AIDS treatment shifted emphasis towards limiting adverse effects of antiviral drugs while improving the treatment of hard-to-reach viral reservoirs. Many therapeutic nucleoside reverse transcriptase inhibitors (NRTI) have a limited access to the central nervous system (CNS). Increased NRTI levels induced various complications during the therapy, including neurotoxicity, due to the NRTI toxicity to mitochondria. Here, we describe an innovative design of biodegradable cationic cholesterol-ε-polylysine nanogel carriers for delivery of triphosphorylated NRTIs that demonstrated high anti-HIV activity along with low neurotoxicity, warranting minimal side effects following systemic administration. Efficient CNS targeting was achieved by nanogel modification with brain-specific peptide vectors. Novel dual and triple-drug nanoformulations, analogous to therapeutic NRTI cocktails, displayed equal or higher antiviral activity in HIV-infected macrophages compared to free drugs. Our results suggest potential alternative approach to HIV-1 treatment focused on the effective nanodrug delivery to viral reservoirs in the CNS and reduced neurotoxicity. PMID:25559020

  2. Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection

    PubMed Central

    Lawrence, Amba; Fraser, Tamieka; Gillett, Amber; Tyndall, Joel D. A.; Timms, Peter; Polkinghorne, Adam; Huston, Wilhelmina M.

    2016-01-01

    The koala, an iconic marsupial native to Australia, is a threatened species in many parts of the country. One major factor in the decline is disease caused by infection with Chlamydia. Current therapeutic strategies to treat chlamydiosis in the koala are limited. This study examines the effectiveness of an inhibitor, JO146, which targets the HtrA serine protease for treatment of C. pecorum and C. pneumoniae in vitro and ex vivo with the aim of developing a novel therapeutic for koala Chlamydia infections. Clinical isolates from koalas were examined for their susceptibility to JO146. In vitro studies demonstrated that treatment with JO146 during the mid-replicative phase of C. pecorum or C. pneumoniae infections resulted in a significant loss of infectious progeny. Ex vivo primary koala tissue cultures were used to demonstrate the efficacy of JO146 and the non-toxic nature of this compound on peripheral blood mononuclear cells and primary cell lines established from koala tissues collected at necropsy. Our results suggest that inhibition of the serine protease HtrA could be a novel treatment strategy for chlamydiosis in koalas. PMID:27530689

  3. LLL12, a novel small inhibitor targeting STAT3 for hepatocellular carcinoma therapy

    PubMed Central

    Zuo, Mingxin; Li, Chenglong; Lin, Jiayuh; Javle, Milind

    2015-01-01

    The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently detected in clinical incidences of hepatocellular carcinoma (HCC) but not in normal human hepatocytes. STAT3 signaling plays pivotal roles in angiogenesis, survival, metastasis, and growth of HCC. Recent evidence suggests that the blockade of aberrant STAT3 pathways can be exploited as a therapeutic strategy for HCC. We have developed the novel small molecular STAT3 inhibitor LLL12 on the basis of curcumin structure using computer-aided rational design. LLL12 has shown antitumor activity in various solid tumors including breast, brain, pancreatic cancer, and glioblastoma in vitro and in vivo. In this study, we hypothesized LLL12 inhibits STAT3 phosphorylation at tyrosine 705 (Y705) in HCC and show antitumor activity in HCC in vitro and in vivo. Our results show that LLL12 selectively inhibited HCC cell proliferation and induced apoptosis in SNU387, SNU398, SNU449, and Hep3B HCC cells in vitro. Furthermore, LLL12 at 5 mg/kg/day significantly inhibited the growth of SNU398 xenografts in nude mice. Collectively, our results indicate that LLL12 could be used to target STAT3 for the effective prevention or treatment of HCC. PMID:25883212

  4. Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection.

    PubMed

    Lawrence, Amba; Fraser, Tamieka; Gillett, Amber; Tyndall, Joel D A; Timms, Peter; Polkinghorne, Adam; Huston, Wilhelmina M

    2016-01-01

    The koala, an iconic marsupial native to Australia, is a threatened species in many parts of the country. One major factor in the decline is disease caused by infection with Chlamydia. Current therapeutic strategies to treat chlamydiosis in the koala are limited. This study examines the effectiveness of an inhibitor, JO146, which targets the HtrA serine protease for treatment of C. pecorum and C. pneumoniae in vitro and ex vivo with the aim of developing a novel therapeutic for koala Chlamydia infections. Clinical isolates from koalas were examined for their susceptibility to JO146. In vitro studies demonstrated that treatment with JO146 during the mid-replicative phase of C. pecorum or C. pneumoniae infections resulted in a significant loss of infectious progeny. Ex vivo primary koala tissue cultures were used to demonstrate the efficacy of JO146 and the non-toxic nature of this compound on peripheral blood mononuclear cells and primary cell lines established from koala tissues collected at necropsy. Our results suggest that inhibition of the serine protease HtrA could be a novel treatment strategy for chlamydiosis in koalas.

  5. Structure–Activity Relationships of a Novel Capsid Targeted Inhibitor of HIV-1 Replication

    PubMed Central

    2015-01-01

    Despite the considerable successes of highly active antiretroviral therapy (HAART) for the treatment of HIV/AIDS, cumulative drug toxicities and the development of multidrug-resistant virus necessitate the search for new classes of antiretroviral agents with novel modes of action. The HIV-1 capsid (CA) protein has been structurally and functionally characterized as a druggable target. We have recently designed a novel small molecule inhibitor I-XW-053 using the hybrid structure based method to block the interface between CA N-terminal domains (NTD–NTD interface) with micromolar affinity. In an effort to optimize and improve the efficacy of I-XW-053, we have developed the structure activity relationship of I-XW-053 compound series using ligand efficiency methods. Fifty-six analogues of I-XW-053 were designed that could be subclassified into four different core domains based on their ligand efficiency values computed as the ratio of binding efficiency (BEI) and surface efficiency (SEI) indices. Compound 34 belonging to subcore-3 showed an 11-fold improvement over I-XW-053 in blocking HIV-1 replication in primary human peripheral blood mononuclear cells (PBMCs). Surface plasmon resonance experiments confirmed the binding of compound 34 to purified HIV-1 CA protein. Molecular docking studies on compound 34 and I-XW-053 to HIV-1 CA protein suggested that they both bind to NTD–NTD interface region but with different binding modes, which was further validated using site-directed mutagenesis studies. PMID:25302989

  6. Intracellular selection of peptide inhibitors that target disulphide-bridged Aβ42 oligomers

    PubMed Central

    Acerra, Nicola; Kad, Neil M; Cheruvara, Harish; Mason, Jody M

    2014-01-01

    The β-amyloid (Aβ) peptide aggregates into a number of soluble and insoluble forms, with soluble oligomers thought to be the primary factor implicated in Alzheimer's disease pathology. As a result, a wide range of potential aggregation inhibitors have been developed. However, in addition to problems with solubility and protease susceptibility, many have inadvertently raised the concentration of these soluble neurotoxic species. Sandberg et al. previously reported a β-hairpin stabilized variant of Aβ42 that results from an intramolecular disulphide bridge (A21C/A31C; Aβ42cc), which generates highly toxic oligomeric species incapable of converting into mature fibrils. Using an intracellular protein-fragment complementation (PCA) approach, we have screened peptide libraries using E. coli that harbor an oxidizing environment to permit cytoplasmic disulphide bond formation. Peptides designed to target either the first or second β-strand have been demonstrated to bind to Aβ42cc, lower amyloid cytotoxicity, and confer bacterial cell survival. Peptides have consequently been tested using wild-type Aβ42 via ThT binding assays, circular dichroism, MTT cytotoxicity assays, fluorescence microscopy, and atomic force microscopy. Results demonstrate that amyloid-PCA selected peptides function by both removing amyloid oligomers as well as inhibiting their formation. These data further support the use of semirational design combined with intracellular PCA methodology to develop Aβ antagonists as candidates for modification into drugs capable of slowing or even preventing the onset of AD. PMID:24947815

  7. A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions

    PubMed Central

    Peterson, Jeffrey R.; Lokey, R. Scott; Mitchison, Timothy J.; Kirschner, Marc W.

    2001-01-01

    Cell morphology and motility are governed largely by complex signaling networks that ultimately engage the actin cytoskeleton. Understanding how individual circuits contribute to the process of forming cellular structures would be aided greatly by the availability of specific chemical inhibitors. We have used a novel chemical screen in Xenopus cell-free extracts to identify compounds that inhibit signaling pathways regulating actin polymerization. Here we report the results of a high-throughput screen for compounds that inhibit phosphatidylinositol 4,5-bisphosphate (PIP2)-induced actin assembly and the identification of the first compound, a cyclic peptide, known to block actin assembly by inhibiting an upstream signaling component. We identify the target of this compound as N-WASP, a protein that has been investigated for its role as a node interconnecting various actin signaling networks. We show that this compound prevents activation of the Arp2/3 complex by N-WASP by allosterically stabilizing the autoinhibited conformation of N-WASP. PMID:11553809

  8. In Vitro and In Vivo Activity of Multi-Target Inhibitors Against Trypanosoma brucei

    PubMed Central

    Yang, Gyongseon; Zhu, Wei; Wang, Yang; Huang, Guozhong; Byun, Sooyoung; Choi, Gahee; Li, Kai; Huang, Zhuoli; Docampo, Roberto; Oldfield, Eric; No, Joo Hwan

    2015-01-01

    We tested a series of amidine and related compounds against Trypanosoma brucei. The most active compound was a biphenyldiamidine which had an EC50 of 7.7 nM against bloodstream form parasites. There was little toxicity against two human cell lines with CC50 > 100 μM. There was also good in vivo activity in a mouse model of infection with 100% survival at 3 mg/kg i.p. The most potent lead blocked replication of kinetoplast DNA (k-DNA), but not nuclear DNA, in the parasite. Some compounds also inhibited the enzyme farnesyl diphosphate synthase (FPPS) and some were uncouplers of oxidative phosphorylation. We developed a computational model for T. brucei cell growth inhibition (R2 = 0.76) using DNA ΔTm values for inhibitor binding, combined with T. brucei FPPS IC50 values. Overall, the results suggest that it may be possible to develop multi-target drug leads against T. brucei that act by inhibiting both k-DNA replication and isoprenoid biosynthesis. PMID:26295062

  9. Accounting for target flexibility and water molecules by docking to ensembles of target structures: the HCV NS5B palm site I inhibitors case study.

    PubMed

    Barreca, Maria Letizia; Iraci, Nunzio; Manfroni, Giuseppe; Gaetani, Rosy; Guercini, Chiara; Sabatini, Stefano; Tabarrini, Oriana; Cecchetti, Violetta

    2014-02-24

    The introduction of new anti-HCV drugs in therapy is an imperative need and is necessary with a view to develop an interferon-free therapy. Thus, the discovery and development of novel small molecule inhibitors of the viral NS5B polymerase represent an exciting area of research for many pharmaceutical companies and academic groups. This study represents a contribution to this field and relies on the identification of the best NS5B model(s) to be used in structure-based computational approaches aimed at identifying novel non-nucleoside inhibitors of one of the protein allosteric sites, namely, palm site I. First, the NS5B inhibitors at palm site I were classified as water-mediated or nonwater-mediated ligands depending on their ability to interact with or displace a specific water molecule. Then, we took advantage of the available X-ray structures of the NS5B/ligand complexes to build different models of protein/water combinations, which were used to investigate the influence on docking studies of solvent sites as well as of the influence of the protein conformations. As the overall trend, we observed improved performance in the docking results of the water-mediated inhibitors by inclusion of explicit water molecules, with an opposite behavior generally happening for the nonwater-mediated inhibitors. The best performing target structures for the two ligand sets were then used for virtual screening simulations of a library containing the known NS5B inhibitors along with related decoys to assess the best performing targets ensembles on the basis of their ability to discriminate active and inactive compounds as well as to generate the correct binding modes. The parallel use of different protein structures/water sets outperformed the use of a single target structure, with the two-protein 3H98/2W-2FVC/7W and 3HKY/NoW-3SKE/NoW models resulting in the best performing ensembles for water-mediated inhibitors and nonwater-mediated inhibitors, respectively. The information

  10. Novel reversible selective inhibitor of nuclear export shows that CRM1 is a target in colorectal cancer cells.

    PubMed

    Niu, Mingshan; Chong, Yulong; Han, Yan; Liu, Xuejiao

    2015-01-01

    Colorectal cancer arises via a multistep carcinogenic process and the deregulation of multiple pathways. Thus, the simultaneous targeting of multiple pathways may be a promising therapeutic approach for colorectal treatment. CRM1 is an attractive cancer drug target, because it can regulate multiple pathways and tumor suppressor proteins. In this study, we investigated the anti-tumor activity of a novel reversible CRM1 inhibitor S109 in colorectal cancer. Our data demonstrate that S109 inhibits proliferation and induces cell cycle arrest in colorectal cancer cells. Mechanistically, we demonstrate that the activity of S109 is associated with the nuclear retention of major tumor suppress proteins. Furthermore, the Cys528 mutation of CRM1 prevented the ability of S109 to block nuclear export and inhibit the proliferation of colorectal cancer cells. Interestingly, S109 decreased the CRM1 protein level via proteasomal pathway. We argue that reversible CRM1 inhibitors but not irreversible inhibitors can induce the degradation of CRM1, because the dissociation of reversible inhibitors of CRM1 changes the conformation of CRM1. Taken together, these findings demonstrate that CRM1 is a valid target for the treatment of colorectal cancer and provide a basis for the development of S109 therapies for colorectal cancer.

  11. MeshKit

    SciTech Connect

    2010-10-05

    MeshKit is an open-source library of mesh generation functionality. MeshKit has general mesh manipulation and generation functions such as Copoy, Move, Rotate and Extrude mesh. In addition, new quad mesh and embedded boundary Cartesian mesh algorithm (EB Mesh) are included. Interfaces to several public domain meshing algorithms (TetGen, netgen, triangle, Gmsh, camal) are also offered. This library interacts with mesh data mostly through iMesh including accessing the mesh in parallel. It also can interact with iGeom interface to provide geometry functionality such as importing solid model based geometries. iGeom and IMesh are implemented in the CGM and MOAB packages, respectively. For some non-existing function in iMesh such as tree-construction and ray-tracing, MeshKit also interacts with MOAB functions directly.

  12. MeshKit

    2010-10-05

    MeshKit is an open-source library of mesh generation functionality. MeshKit has general mesh manipulation and generation functions such as Copoy, Move, Rotate and Extrude mesh. In addition, new quad mesh and embedded boundary Cartesian mesh algorithm (EB Mesh) are included. Interfaces to several public domain meshing algorithms (TetGen, netgen, triangle, Gmsh, camal) are also offered. This library interacts with mesh data mostly through iMesh including accessing the mesh in parallel. It also can interact withmore » iGeom interface to provide geometry functionality such as importing solid model based geometries. iGeom and IMesh are implemented in the CGM and MOAB packages, respectively. For some non-existing function in iMesh such as tree-construction and ray-tracing, MeshKit also interacts with MOAB functions directly.« less

  13. Phase II trial of pazopanib (GW786034), an oral multi-targeted angiogenesis inhibitor, for adults with recurrent glioblastoma (North American Brain Tumor Consortium Study 06-02)

    PubMed Central

    Iwamoto, Fabio M.; Lamborn, Kathleen R.; Robins, H. Ian; Mehta, Minesh P.; Chang, Susan M.; Butowski, Nicholas A.; DeAngelis, Lisa M.; Abrey, Lauren E.; Zhang, Wei-Ting; Prados, Michael D.; Fine, Howard A.

    2010-01-01

    The objective of this phase II single-arm study was to evaluate the efficacy and safety of pazopanib, a multi-targeted tyrosine kinase inhibitor, against vascular endothelial growth factor receptor (VEGFR)-1, -2, and -3, platelet-derived growth factor receptor-α and -β, and c-Kit, in recurrent glioblastoma. Patients with ≤2 relapses and no prior anti-VEGF/VEGFR therapy were treated with pazopanib 800 mg daily on 4-week cycles without planned interruptions. Brain magnetic resonance imaging and clinical reassessment were made every 8 weeks. The primary endpoint was efficacy as measured by 6-month progression-free survival (PFS6). Thirty-five GBM patients with a median age of 53 years and median Karnofsky performance scale of 90 were accrued. Grade 3/4 toxicities included leukopenia (n = 1), lymphopenia (n = 2), thrombocytopenia (n = 1), ALT elevation (n = 3), AST elevation (n = 1), CNS hemorrhage (n = 1), fatigue (n = 1), and thrombotic/embolic events (n = 3); 8 patients required dose reduction. Two patients had a partial radiographic response by standard bidimensional measurements, whereas 9 patients (6 at the 8-week point and 3 only within the first month of treatment) had decreased contrast enhancement, vasogenic edema, and mass effect but <50% reduction in tumor. The median PFS was 12 weeks (95% confidence interval [CI]: 8–14 weeks) and only 1 patient had a PFS time ≥6 months (PFS6 = 3%). Thirty patients (86%) had died and median survival was 35 weeks (95% CI: 24–47 weeks). Pazopanib was reasonably well tolerated with a spectrum of toxicities similar to other anti-VEGF/VEGFR agents. Single-agent pazopanib did not prolong PFS in this patient population but showed in situ biological activity as demonstrated by radiographic responses. ClinicalTrials.gov identifier: NCT00459381. PMID:20200024

  14. Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins. The potential for selective inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tunicamycins are a heterologous family of nucleoside antibiotics that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg2+ complex established as a transition state analog for hexosamine-1-phosphate: prenol pho...

  15. Travel Medical Kit.

    PubMed

    Terry, Anne C; Haulman, N Jean

    2016-03-01

    "The traveler's medical kit is an essential tool for both the novice and expert traveler. It is designed to treat travel-related illness and injury and to ensure preexisting medical conditions are managed appropriately. Travelers are at increased risk for common gastrointestinal issues during travel. Respiratory illnesses make up approximately 8% of the ailments present in returned international travelers. Approximately 12% of travelers experience a travel-related skin condition. First aid treatment for minor injuries is essential to all travel medical kits. The complexity ranges from a small, simple case for the urban traveler to a larger, extensive case for wilderness travel." PMID:26900112

  16. Antitumor activity of pimasertib, a selective MEK 1/2 inhibitor, in combination with PI3K/mTOR inhibitors or with multi-targeted kinase inhibitors in pimasertib-resistant human lung and colorectal cancer cells.

    PubMed

    Martinelli, Erika; Troiani, Teresa; D'Aiuto, Elena; Morgillo, Floriana; Vitagliano, Donata; Capasso, Anna; Costantino, Sarah; Ciuffreda, Loreta Pia; Merolla, Francesco; Vecchione, Loredana; De Vriendt, Veerle; Tejpar, Sabine; Nappi, Anna; Sforza, Vincenzo; Martini, Giulia; Berrino, Liberato; De Palma, Raffaele; Ciardiello, Fortunato

    2013-11-01

    The RAS/RAF/MEK/MAPK and the PTEN/PI3K/AKT/mTOR pathways are key regulators of proliferation and survival in human cancer cells. Selective inhibitors of different transducer molecules in these pathways have been developed as molecular targeted anti-cancer therapies. The in vitro and in vivo anti-tumor activity of pimasertib, a selective MEK 1/2 inhibitor, alone or in combination with a PI3K inhibitor (PI3Ki), a mTOR inhibitor (everolimus), or with multi-targeted kinase inhibitors (sorafenib and regorafenib), that block also BRAF and CRAF, were tested in a panel of eight human lung and colon cancer cell lines. Following pimasertib treatment, cancer cell lines were classified as pimasertib-sensitive (IC50 for cell growth inhibition of 0.001 µM) or pimasertib-resistant. Evaluation of basal gene expression profiles by microarrays identified several genes that were up-regulated in pimasertib-resistant cancer cells and that were involved in both RAS/RAF/MEK/MAPK and PTEN/PI3K/AKT/mTOR pathways. Therefore, a series of combination experiments with pimasertib and either PI3Ki, everolimus, sorafenib or regorafenib were conducted, demonstrating a synergistic effect in cell growth inhibition and induction of apoptosis with sustained blockade in MAPK- and AKT-dependent signaling pathways in pimasertib-resistant human colon carcinoma (HCT15) and lung adenocarcinoma (H1975) cells. Finally, in nude mice bearing established HCT15 and H1975 subcutaneous tumor xenografts, the combined treatment with pimasertib and BEZ235 (a dual PI3K/mTOR inhibitor) or with sorafenib caused significant tumor growth delays and increase in mice survival as compared to single agent treatment. These results suggest that dual blockade of MAPK and PI3K pathways could overcome intrinsic resistance to MEK inhibition.

  17. Diversity-Oriented Synthesis Probe Targets Plasmodium falciparum Cytochrome b Ubiquinone Reduction Site and Synergizes With Oxidation Site Inhibitors

    PubMed Central

    Lukens, Amanda K.; Heidebrecht, Richard W.; Mulrooney, Carol; Beaudoin, Jennifer A.; Comer, Eamon; Duvall, Jeremy R.; Fitzgerald, Mark E.; Masi, Daniela; Galinsky, Kevin; Scherer, Christina A.; Palmer, Michelle; Munoz, Benito; Foley, Michael; Schreiber, Stuart L.; Wiegand, Roger C.; Wirth, Dyann F.

    2015-01-01

    Background. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways. Methods. We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds. Results. We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite. Conclusions. The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria. PMID:25336726

  18. Expanding the binding envelope of CYP51 inhibitors targeting Trypanosoma cruzi with 4-aminopyridyl-based sulfonamide derivatives

    PubMed Central

    Vieira, Debora F.; Choi, Jun Yong

    2014-01-01

    Chagas disease is a chronic infection caused by the protozoan parasite Trypanosoma cruzi, manifested in progressive cardiomyopathy and/or gastrointestinal dysfunction. Therapeutic options to prevent or treat Chagas disease are limited. CYP51, the enzyme key to the biosynthesis of eukaryotic membrane sterols, is a validated drug target in both fungi and T. cruzi. Sulfonamide derivatives of 4-aminopyridyl-based inhibitors of T. cruzi CYP51 (TcCYP51), including the sub-nanomolar compound 3, have molecular structures distinct from other validated CYP51 inhibitors. They augment the biologically relevant chemical space of molecules targeting TcCYP51. In a 2.08 Å x-ray structure, TcCYP51 is in a compound 3-induced conformation distinct from the previously characterized ground-state conformation of CYP51 drug-target complexes. That the binding site was modulated in response to an incoming inhibitor for the first time characterizes TcCYP51 as a flexible target rather than a rigid template. PMID:24771705

  19. Tumors with AKT1E17K Mutations Are Rational Targets for Single Agent or Combination Therapy with AKT Inhibitors.

    PubMed

    Davies, Barry R; Guan, Nin; Logie, Armelle; Crafter, Claire; Hanson, Lyndsey; Jacobs, Vivien; James, Neil; Dudley, Philippa; Jacques, Kelly; Ladd, Brendon; D'Cruz, Celina M; Zinda, Michael; Lindemann, Justin; Kodaira, Makoto; Tamura, Kenji; Jenkins, Emma L

    2015-11-01

    AKT1(E17K) mutations occur at low frequency in a variety of solid tumors, including those of the breast and urinary bladder. Although this mutation has been shown to transform rodent cells in culture, it was found to be less oncogenic than PIK3CA mutations in breast epithelial cells. Moreover, the therapeutic potential of AKT inhibitors in human tumors with an endogenous AKT1(E17K) mutation is not known. Expression of exogenous copies of AKT1(E17K) in MCF10A breast epithelial cells increased phosphorylation of AKT and its substrates, induced colony formation in soft agar, and formation of lesions in the mammary fat pad of immunodeficient mice. These effects were inhibited by the allosteric and catalytic AKT inhibitors MK-2206 and AZD5363, respectively. Both AKT inhibitors caused highly significant growth inhibition of breast cancer explant models with AKT1(E17K) mutation. Furthermore, in a phase I clinical study, the catalytic Akt inhibitor AZD5363 induced partial responses in patients with breast and ovarian cancer with tumors containing AKT1(E17K) mutations. In MGH-U3 bladder cancer xenografts, which contain both AKT1(E17K) and FGFR3(Y373C) mutations, AZD5363 monotherapy did not significantly reduce tumor growth, but tumor regression was observed in combination with the FGFR inhibitor AZD4547. The data show that tumors with AKT1(E17K) mutations are rational therapeutic targets for AKT inhibitors, although combinations with other targeted agents may be required where activating oncogenic mutations of other proteins are present in the same tumor.

  20. New Small Molecule Entry Inhibitors Targeting Hemagglutinin-Mediated Influenza A Virus Fusion

    PubMed Central

    Antanasijevic, Aleksandar; Wang, Minxiu; Li, Bing; Mills, Debra M.; Ames, Jessica A.; Nash, Peter J.; Williams, John D.; Peet, Norton P.; Moir, Donald T.; Prichard, Mark N.; Keith, Kathy A.; Barnard, Dale L.; Caffrey, Michael; Rong, Lijun; Bowlin, Terry L.

    2014-01-01

    Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC50] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC50] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM2 % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process. PMID:24198411

  1. Projectable Basic Electronics Kit.

    ERIC Educational Resources Information Center

    H'ng, John; And Others

    1982-01-01

    Outlines advantages derived from constructing and using a Projectable Basic Electronics Kit and provides: (1) list of components; (2) diagrams of 10 finished components (resistor; capacitor; diode; switch; bulb; transistor; meter; variable capacitor; coil; connecting terminal); and (3) diode and transistor activities. (JN)

  2. Theme Kits Made Easy.

    ERIC Educational Resources Information Center

    Eslinger, Leslie Silk

    Recognizing the long-lasting impact of young childrens learning through themes as well as the amount of teacher time spent in preparing for this type of teaching, this kit is designed to help teachers avoid the shortcomings of theme-based teaching, while capitalizing on the benefits of this approach. The book is presented in two sections. The…

  3. Ohio EPA Teachers Kit.

    ERIC Educational Resources Information Center

    Ohio State Environmental Protection Agency, Columbus.

    In an effort to provide teachers in Ohio with assistance in environmental education, the Ohio Environmental Protection Agency (EPA) has produced this teachers kit. It is designed to describe what the Ohio EPA is doing to protect Ohio's air, land, and water. The background information provides an historical account of some of the events that have…

  4. [Conference Time Kit.

    ERIC Educational Resources Information Center

    National School Public Relations Association, Washington, DC.

    This multimedia kit, for use with and by teachers from kindergarten through the upper elementary grades, consists of four components: 1) a filmstrip for teachers; 2) the 1970 edition of a handbook, "Conference Time for Teachers and Parents"; 3) a filmstrip for parents; 4) a supporting parent information leaflet "How To Confer Successfully with…

  5. User Authentication. SPEC Kit.

    ERIC Educational Resources Information Center

    Plum, Terry, Comp.; Bleiler, Richard, Comp.

    2001-01-01

    This SPEC (Systems and Procedures Exchange Center) Kit presents the results of a survey of Association of Research Libraries (ARL) member libraries designed to examine the systems research libraries use to authenticate and authorize the users of their online networked information resources. A total of 52 of 121 ARL member libraries responded to…

  6. Personalized Thematic Kits

    ERIC Educational Resources Information Center

    Bontrager, Sharon

    2010-01-01

    Teaching Spanish at the K-5 level is a passion of mine, and the author would like to share some of the practical applications that she finds most rewarding and effective. She has found enthusiastic response to the creation of detailed language learning kits that are rooted in storytelling, but expanded to include home-made board games,…

  7. Kaleo's Safe Walking Kit.

    ERIC Educational Resources Information Center

    Hawaii State Dept. of Education, Honolulu. Office of Instructional Services.

    This pedestrian safety kit for kindergarten through the third grade consists of 16 lessons encompassing many safety concepts, guidelines and skills that emphasize pedestrian safety rules, alertness, and responsible reaction to hazardous situations. Lessons include activities of varying difficulty for large group, small group or individual…

  8. Voter Education Training Kit.

    ERIC Educational Resources Information Center

    Multi-District Inst. for Political Education, Pitman, NJ.

    Guides and resources in this kit are prepared for a six week to two month secondary voter education course. The objectives are to prepare and motivate eligible students to register and vote in the presidential election, to participate in the presidential election campaigning, and to increase their overall knowledge concerning the presidential…

  9. Chat Reference. SPEC Kit.

    ERIC Educational Resources Information Center

    Ronan, Jana, Comp.; Turner, Carol, Comp.

    2002-01-01

    This SPEC (Systems and Procedures Exchange Center) Kit presents the results of a survey of Association of Research Libraries (ARL) member libraries designed to gather data on chat reference service. A total of 66 of 124 ARL member libraries responded to the survey. A copy of the questionnaire with tabulated results is presented. Representative…

  10. World Disarmament Kit.

    ERIC Educational Resources Information Center

    Woito, Robert, Ed.

    This kit presents a comprehensive introduction for students to arms control and disarmament issues. Included are copies of published and unpublished articles for each topic. Section I provides a self-survey to enable students to assess their own attitudes, values, and knowledge. The survey poses questions for which students select one of several…

  11. Balloons and Science Kit.

    ERIC Educational Resources Information Center

    Balloon Council, Washington, DC.

    This document provides background information on balloons including: (1) the history of balloons; (2) balloon manufacturing; (3) biodegradability; (4) the fate of latex balloons; and (5) the effect of balloons on the rainforest and sea mammals. Also included as part of this instructional kit are four fun experiments that allow students to…

  12. The ESL Starter Kit.

    ERIC Educational Resources Information Center

    Virginia Commonwealth Univ., Richmond. Virginia Adult Education and Literacy Resource Center.

    The kit is intended for teachers beginning to teach English as a Second Language (ESL). The first part offers some ideas for testing, registering, and placing students according to their needs and goals. A sample registration form, placement test, list of commercially-available tests, and sample needs assessments are included here. The second…

  13. Early Childhood Kits.

    ERIC Educational Resources Information Center

    National Center on Educational Media and Materials for the Handicapped, Columbus, OH.

    Selected from the National Instructional Materials Information System (NIMIS)--a computer based on-line interactive retrieval system on special education materials, the bibliography covers 80 kits for developing skills at the early childhood level. Entries are presented in order of NIMIS accession number and include the following information:…

  14. Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase

    PubMed Central

    Palencia, Andrés; Li, Xianfeng; Bu, Wei; Choi, Wai; Ding, Charles Z.; Easom, Eric E.; Feng, Lisa; Hernandez, Vincent; Houston, Paul; Liu, Liang; Meewan, Maliwan; Mohan, Manisha; Rock, Fernando L.; Sexton, Holly; Zhang, Suoming; Zhou, Yasheen; Wan, Baojie; Wang, Yuehong; Franzblau, Scott G.; Woolhiser, Lisa; Gruppo, Veronica; Lenaerts, Anne J.; O'Malley, Theresa; Parish, Tanya; Cooper, Christopher B.; Waters, M. Gerard; Ma, Zhenkun; Ioerger, Thomas R.; Sacchettini, James C.; Rullas, Joaquín; Angulo-Barturen, Iñigo; Pérez-Herrán, Esther; Mendoza, Alfonso; Barros, David; Cusack, Stephen; Plattner, Jacob J.

    2016-01-01

    The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid. PMID:27503647

  15. A novel p38 MAPK docking groove-targeted compound is a potent inhibitor of inflammatory hyperalgesia

    PubMed Central

    Willemen, Hanneke L.D.M.; Campos, Pedro M.; Lucas, Elisa; Morreale, Antonio; Gil-Redondo, Rubén; Agut, Juan; González, Florenci V.; Ramos, Paula; Heijnen, Cobi; Mayor, Federico; Kavelaars, Annemieke; Murga, Cristina

    2014-01-01

    Synopsis The mitogen activated protein kinase (MAPK) p38 is an important mediator of inflammation and of inflammatory and neuropathic pain. We recently described that docking-groove dependent interactions are important for p38 MAPK-mediated signal transduction. Thus, virtual screening was performed to identify putative docking groove-targeted p38 MAPK inhibitors. Several compounds of the benzooxadiazol family were identified with low micromolar inhibitory activity both in a p38 MAPK activity assay, and in THP-1 human monocytes acting as inhibitors of LPS-induced TNFα secretion. Positions 2 and 5 in the phenyl ring are essential for the described inhibitory activity with a chloride in position 5 and a methyl-group in position 2 yielding the best results with an IC50 of 1.8 μM (FGA-19 compound). Notably, FGA-19 exerted a potent and long-lasting analgesic effect in vivo when tested in a mouse model of inflammatory hyperalgesia. A single intrathecal injection of FGA-19 completely resolved hyperalgesia, being ten times as potent and displaying longer lasting effects than the established p38 MAPK inhibitor SB239063. FGA-19 also reversed persistent pain in a model of post-inflammatory hyperalgesia (in LysM-GRK2+/− mice). These potent in vivo effects put forward p38 MAPK docking-site targeted inhibitors as a potential novel strategy for the treatment of inflammatory pain. PMID:24517375

  16. A FluoPol-ABPP PAD2 High-Throughput Screen Identifies the First Calcium Site Inhibitor Targeting the PADs

    PubMed Central

    2015-01-01

    The protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process termed deimination or citrullination. PADs likely play a role in the progression of a range of disease states because dysregulated PAD activity is observed in a host of inflammatory diseases and cancer. For example, recent studies have shown that PAD2 activates ERα target gene expression in breast cancer cells by citrullinating histone H3 at ER target promoters. To date, all known PAD inhibitors bind directly to the enzyme active site. PADs, however, also require calcium ions to drive a conformational change between the inactive apo-state and the fully active calcium bound holoenzyme, suggesting that it would be possible to identify inhibitors that bind the apoenzyme and prevent this conformational change. As such, we set out to develop a screen that can identify PAD2 inhibitors that bind to either the apo or calcium bound form of PAD2. Herein, we provide definitive proof of concept for this approach and report the first PAD inhibitor, ruthenium red (Ki of 17 μM), to preferentially bind the apoenzyme. PMID:24467619

  17. In Silico Design and Biological Evaluation of a Dual Specificity Kinase Inhibitor Targeting Cell Cycle Progression and Angiogenesis

    PubMed Central

    Latham, Antony M.; Kankanala, Jayakanth; Fearnley, Gareth W.; Gage, Matthew C.; Kearney, Mark T.; Homer-Vanniasinkam, Shervanthi; Wheatcroft, Stephen B.; Fishwick, Colin W. G.; Ponnambalam, Sreenivasan

    2014-01-01

    Background Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues. Methodology We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis. Conclusions We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery. PMID:25393739

  18. Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase.

    PubMed

    Palencia, Andrés; Li, Xianfeng; Bu, Wei; Choi, Wai; Ding, Charles Z; Easom, Eric E; Feng, Lisa; Hernandez, Vincent; Houston, Paul; Liu, Liang; Meewan, Maliwan; Mohan, Manisha; Rock, Fernando L; Sexton, Holly; Zhang, Suoming; Zhou, Yasheen; Wan, Baojie; Wang, Yuehong; Franzblau, Scott G; Woolhiser, Lisa; Gruppo, Veronica; Lenaerts, Anne J; O'Malley, Theresa; Parish, Tanya; Cooper, Christopher B; Waters, M Gerard; Ma, Zhenkun; Ioerger, Thomas R; Sacchettini, James C; Rullas, Joaquín; Angulo-Barturen, Iñigo; Pérez-Herrán, Esther; Mendoza, Alfonso; Barros, David; Cusack, Stephen; Plattner, Jacob J; Alley, M R K

    2016-10-01

    The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid. PMID:27503647

  19. miR-21 Reduces Hydrogen Peroxide-Induced Apoptosis in c-kit+ Cardiac Stem Cells In Vitro through PTEN/PI3K/Akt Signaling

    PubMed Central

    Wang, Yan; Long, Xianping; Zhao, Ranzun; Wang, Zhenglong; Liu, Zhijiang

    2016-01-01

    The low survival rate of cardiac stem cells (CSCs) in the infarcted myocardium hampers cell therapy for ischemic cardiomyopathy. MicroRNA-21 (miR-21) and one of its target proteins, PTEN, contribute to the survival and proliferation of many cell types, but their prosurvival effects in c-kit+ CSC remain unclear. Thus, we hypothesized that miR-21 reduces hydrogen peroxide- (H2O2-) induced apoptosis in c-kit+ CSC and estimated the contribution of PTEN/PI3K/Akt signaling to this oxidative circumstance. miR-21 mimics efficiently reduced H2O2-induced apoptosis in c-kit+ CSC, as evidenced by the downregulation of the proapoptosis proteins caspase-3 and Bax and upregulation of the antiapoptotic Bcl-2. In addition, the gain of function of miR-21 in c-kit+ CSC downregulated the protein level of PTEN although its mRNA level changed slightly; in the meantime, miR-21 overexpression also increased phospho-Akt (p-Akt). The antiapoptotic effects of miR-21 were comparable with Phen (bpV), the selective inhibitor of PTEN, while miR-21 inhibitor or PI3K's inhibitor LY294002 efficiently attenuated the antiapoptotic effect of miR-21. Taken together, these results indicate that the anti-H2O2-induced apoptosis effect of miR-21 in c-kit+ CSC is contributed by PTEN/PI3K/Akt signaling. miR-21 could be a potential molecule to facilitate the c-kit+ CSC therapy in ischemic myocardium. PMID:27803763

  20. A new screening method to identify inhibitors of the Lol (localization of lipoproteins) system, a novel antibacterial target.

    PubMed

    Ito, Hideaki; Ura, Atsushi; Oyamada, Yoshihiro; Yoshida, Hiroaki; Yamagishi, Jun-Ichi; Narita, Shin-Ichiro; Matsuyama, Shin-Ichi; Tokuda, Hajime

    2007-01-01

    As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram-negative bacteria, it is considered to be a promising target for the development of anti-gram-negative bacterial agents. However, no high-throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA-dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system. Using this new screening method, we screened 23,600 in-house chemical compounds and found 2 Lol system inhibitors. We therefore conclude that our new screening method can efficiently identify new antibacterial agents that target the Lol system.

  1. Evolution of NADPH Oxidase Inhibitors: Selectivity and Mechanisms for Target Engagement

    PubMed Central

    Altenhöfer, Sebastian; Radermacher, Kim A.; Kleikers, Pamela W.M.; Wingler, Kirstin

    2015-01-01

    Abstract Significance: Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. Recent Advances: Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. Critical Issues: Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. Future Directions: The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition. Antioxid. Redox Signal. 23, 406–427. PMID:24383718

  2. Design of HIV Protease Inhibitors Targeting Protein Backbone: An Effective Strategy for Combating Drug Resistance

    SciTech Connect

    Ghosh, Arun K.; Chapsal, Bruno D.; Weber, Irene T.; Mitsuya, Hiroaki

    2008-06-03

    The discovery of human immunodeficiency virus (HIV) protease inhibitors (PIs) and their utilization in highly active antiretroviral therapy (HAART) have been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS). However, despite the successes in disease management and the decrease of HIV/AIDS-related mortality, several drawbacks continue to hamper first-generation protease inhibitor therapies. The rapid emergence of drug resistance has become the most urgent concern because it renders current treatments ineffective and therefore compels the scientific community to continue efforts in the design of inhibitors that can efficiently combat drug resistance.

  3. Phase II Study of Nilotinib in Melanoma Harboring KIT Alterations Following Progression to Prior KIT Inhibition

    PubMed Central

    Carvajal, Richard D.; Lawrence, Donald P.; Weber, Jeffrey S.; Gajewski, Thomas F.; Gonzalez, Rene; Lutzky, Jose; O’Day, Steven J.; Hamid, Omid; Wolchok, Jedd D.; Chapman, Paul B.; Sullivan, Ryan J.; Teitcher, Jerrold B.; Ramaiya, Nikhil; Giobbie-Hurder, Anita; Antonescu, Cristina R.; Heinrich, Michael C.; Bastian, Boris C.; Corless, Christopher L.; Fletcher, Jonathan A.; Hodi, F. Stephen

    2016-01-01

    Purpose Although durable responses can be achieved with tyrosine kinase inhibitors such as imatinib in melanomas harboring KIT mutations, the efficacy of alternative inhibitors after progression to imatinib and the activity of these agents on brain metastases is unknown. Experimental Design We conducted a phase II study of nilotinib 400 mg BID in two cohorts of patients with melanomas harboring KIT mutations or amplification: A) those refractory or intolerant to a prior KIT inhibitor; and B) those with brain metastases. The primary endpoint was 4-month disease control rate. Secondary endpoints included response rate, time-to-progression and overall survival. A Simon two-stage and a single-stage design was planned to assess for the primary endpoint in Cohorts A and B, respectively. Results Twenty patients were enrolled and 19 treated (11-Cohort A; 8-Cohort B). Three patients on Cohort A (27%; 95% CI, 8% – 56%) and 1 on Cohort B (12.5%; 90% CI, 0.6% – 47%) achieved the primary endpoint. Two partial responses were observed in Cohort A (18.2%, 90% CI, 3% – 47%); none were observed in Cohort B. The median time-to-progression and overall survival was 3·3 (90% CI, 2.1 – 3.9 months) and 9.1 months (90% CI, 4.3 – 14.2 months), respectively, in all treated patients. Conclusion Nilotinib may achieve disease control in patients with melanoma harboring KIT alterations and whose disease progressed after imatinib therapy. The efficacy of this agent in KIT altered melanoma with brain metastasis is limited. PMID:25695690

  4. Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin.

    PubMed

    Yu, Ker; Toral-Barza, Lourdes; Shi, Celine; Zhang, Wei-Guo; Lucas, Judy; Shor, Boris; Kim, Jamie; Verheijen, Jeroen; Curran, Kevin; Malwitz, David J; Cole, Derek C; Ellingboe, John; Ayral-Kaloustian, Semiramis; Mansour, Tarek S; Gibbons, James J; Abraham, Robert T; Nowak, Pawel; Zask, Arie

    2009-08-01

    The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC(50), 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G(1) cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTEN-null tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy.

  5. The class I HDAC inhibitor Romidepsin targets inflammatory breast cancer tumor emboli and synergizes with paclitaxel to inhibit metastasis.

    PubMed

    Robertson, Fredika M; Chu, Khoi; Boley, Kimberly M; Ye, Zaiming; Liu, Hui; Wright, Moishia C; Moraes, Ricardo; Zhang, Xuejun; Green, Tessa L; Barsky, Sanford H; Heise, Carla; Cristofanilli, Massimo

    2013-01-01

    Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. IBC has distinctive characteristics including invasion of tumor emboli into the skin and rapid disease progression. Given our previous studies suggesting that HDAC inhibitors have promise in targeting IBC, the present study revealed that the class I HDAC inhibitor Romidepsin (FK-288, Istodax; Celgene Corporation, Summit, NJ) potently induced destruction of IBC tumor emboli and lymphatic vascular architecture. associated with inhibition of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha, (HIF1alpha) proteins in the Mary-X pre-clinical model of IBC. Romidepsin treatment induced clinically relevant biomarkers in including induction of acetylated Histone 3 (Ac-H3) proteins, apoptosis, and increased p21WAF1/CIP1. Romidepsin, alone and synergistically when combined with Paclitaxel, effectively eliminated both primary tumors and metastatic lesions at multiple sites formed by the SUM149 IBC cell line. This is the first report of the ability of an HDAC inhibitor to eradicate IBC tumor emboli, to destroy the integrity of lymphatic vessel architecture and to target metastasis. Furthermore, Romidepsin, in combination with a taxane, warrants evaluation as a therapeutic strategy that may effectively target the skin involvement and rapid metastasis that are hallmarks of IBC.

  6. Supplementary Kits for Individualized Instruction.

    ERIC Educational Resources Information Center

    Hopkins, Roberta

    The purpose of the kits is to facilitate the teaching of basic science skills. The kits can be used in the regular classroom for which they were designed or as instruments for teaching of students in learning disability classes. The kits are designed in the areas of plants, animals, metric measurement, chemistry, geology, and space study. Each kit…

  7. Immunomodulatory effects of deacetylase inhibitors: therapeutic targeting of FOXP3+ regulatory T cells

    PubMed Central

    Wang, Liqing; de Zoeten, Edwin F.; Greene, Mark I.; Hancock, Wayne W.

    2010-01-01

    Classical zinc-dependent histone deacetylases (HDACs) catalyse the removal of acetyl groups from histone tails and also from many non-histone proteins, including the transcription factor FOXP3, a key regulator of the development and function of regulatory T cells. Many HDAC inhibitors are in cancer clinical trials, but a subset of HDAC inhibitors has important anti-inflammatory or immunosuppressive effects that might be of therapeutic benefit in immuno-inflammatory disorders or post-transplantation. At least some of these effects result from the ability of HDAC inhibitors to enhance the production and suppressive functions of FOXP3+ regulatory T cells. Understanding which HDACs contribute to the regulation of the functions of regulatory T cells may further stimulate the development of new class- or subclass-specific HDAC inhibitors with applications beyond oncology. PMID:19855427

  8. Targeting FASN in Breast Cancer and the Discovery of Promising Inhibitors from Natural Products Derived from Traditional Chinese Medicine

    PubMed Central

    Cheng, Chien-shan; Wang, Zhiyu; Chen, Jianping

    2014-01-01

    Molecular targeted therapy has been developed for cancer chemoprevention and treatment. Cancer cells process a fundamental change in its bioenergetic metabolism from normal cells on an altered lipid metabolism, also known as the de novo fatty acid synthesis, for sustaining their high proliferation rates. Fatty acid synthesis is now associated with clinically aggressive tumor behavior and tumor cell growth and has become a novel target pathway for chemotherapy development. Although the underlying mechanisms of the altered de novo fatty acid synthesis still remains unclear, recent progress has shown that by targeting Fatty acid synthase (FASN), a key enzyme that catalyzes the synthesis of endogenous long chain fatty acid could be a critical target for drug discovery. However, relatively few FASN inhibitors have been discovered. With the long history of clinical practices and numerous histological case study reports, traditional Chinese medicine enjoys an important role in seeking bioactive anticancer natural compounds. Herein, we will give an overall picture of the current progress of molecular targeted therapy in cancer fatty acid synthesis, describe the advances in the research on natural products-derived FASN inhibitors and their potential for enhancing our understanding of fatty acids in tumor biology, and may provide new therapeutic moieties for breast cancer patient care. PMID:24778702

  9. Chemoproteomics demonstrates target engagement and exquisite selectivity of the clinical phosphodiesterase 10A inhibitor MP-10 in its native environment.

    PubMed

    Schülke, Jan-Philip; McAllister, Laura A; Geoghegan, Kieran F; Parikh, Vinod; Chappie, Thomas A; Verhoest, Patrick R; Schmidt, Christopher J; Johnson, Douglas S; Brandon, Nicholas J

    2014-12-19

    Phosphodiesterases (PDEs) regulate the levels of the second messengers cAMP and cGMP and are important drug targets. PDE10A is highly enriched in medium spiny neurons of the striatum and is an attractive drug target for the treatment of basal ganglia diseases like schizophrenia, Parkinson's disease, or Huntington's disease. Here we describe the design, synthesis, and application of a variety of chemical biology probes, based on the first clinically tested PDE10A inhibitor MP-10, which were used to characterize the chemoproteomic profile of the clinical candidate in its native environment. A clickable photoaffinity probe was used to measure target engagement of MP-10 and revealed differences between whole cell and membrane preparations. Moreover, our results illustrate the importance of the linker design in the creation of functional probes. Biotinylated affinity probes allowed identification of drug-interaction partners in rodent and human tissue and quantitative mass spectrometry analysis revealed highly specific binding of MP-10 to PDE10A with virtually no off-target binding. The profiling of PDE10A chemical biology probes described herein illustrates a strategy by which high affinity inhibitors can be converted into probes for determining selectivity and target engagement of drug candidates in complex biological matrices from native sources.

  10. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

    PubMed Central

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

    2015-01-01

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  11. 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.

  12. 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

  13. Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase

    PubMed Central

    Xu, Hong-Tao; Colby-Germinario, Susan P.; Hassounah, Said; Quashie, Peter K.; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R.

    2015-01-01

    The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. PMID:26574011

  14. Rational combination of targeted therapies as a strategy to overcome the mechanisms of resistance to inhibitors of EGFR signaling.

    PubMed

    Bianco, Roberto; Damiano, Vincenzo; Gelardi, Teresa; Daniele, Gennaro; Ciardiello, Fortunato; Tortora, Giampaolo

    2007-01-01

    The epidermal growth factor receptor (EGFR) has been widely used as a target for novel anticancer agents, such as blocking antibodies and small molecular weight tyrosine kinase compounds. In spite of recent advances in cancer cell biology, leading to the introduction of clinically active new drugs, such as cetuximab, panitumumab and erlotinib, unfortunately disease control remains unsuccessful due to the presence of constitutive resistance to EGFR inhibitors in most patients and the development of acquired resistance in the responders. A large number of molecular abnormalities in tumor cells seem to partly contribute to their resistance to anti-EGFR therapy: increased angiogenesis, constitutive activation of downstream mediators, overexpression of other tyrosine kinase receptors. Moreover, some mutations in the EGFR receptor kinase domain seem to play a crucial role in determining the sensitivity of cancer cells to specific inhibitors by altering the conformation of the receptor and its activity. The development of rational combinations of anticancer agents and EGFR inhibitors, able to exert synergistic cytotoxic interactions, has been widely accepted and used in both preclinical and clinical studies. Although the failure of large clinical trial based on empirical combination of anti-EGFR and classic chemotherapeutic agents, several preclinical data seems to support the hypothesis that combining EGFR inhibitors and other novel agents could efficiently inhibit tumor growth and overcome intrinsic resistance to a single-agent based therapy. This review focuses on the role of complementary signalling pathways in the development of resistance to EGFR targeting agents and the rationale to combine novel inhibitors as anticancer therapy.

  15. Oral mucosal injury caused by mammalian target of rapamycin inhibitors: emerging perspectives on pathobiology and impact on clinical practice.

    PubMed

    Peterson, Douglas E; O'Shaughnessy, Joyce A; Rugo, Hope S; Elad, Sharon; Schubert, Mark M; Viet, Chi T; Campbell-Baird, Cynthia; Hronek, Jan; Seery, Virginia; Divers, Josephine; Glaspy, John; Schmidt, Brian L; Meiller, Timothy F

    2016-08-01

    In recent years oral mucosal injury has been increasingly recognized as an important toxicity associated with mammalian target of rapamycin (mTOR) inhibitors, including in patients with breast cancer who are receiving everolimus. This review addresses the state-of-the-science regarding mTOR inhibitor-associated stomatitis (mIAS), and delineates its clinical characteristics and management. Given the clinically impactful pain associated with mIAS, this review also specifically highlights new research focusing on the study of the molecular basis of pain. The incidence of mIAS varies widely (2-78%). As reported across multiple mTOR inhibitor clinical trials, grade 3/4 toxicity occurs in up to 9% of patients. Managing mTOR-associated oral lesions with topical oral, intralesional, and/or systemic steroids can be beneficial, in contrast to the lack of evidence supporting steroid treatment of oral mucositis caused by high-dose chemotherapy or radiation. However, steroid management is not uniformly efficacious in all patients receiving mTOR inhibitors. Furthermore, technology does not presently exist to permit clinicians to predict a priori which of their patients will develop these lesions. There thus remains a strategic need to define the pathobiology of mIAS, the molecular basis of pain, and risk prediction relative to development of the clinical lesion. This knowledge could lead to novel future interventions designed to more effectively prevent mIAS and improve pain management if clinically significant mIAS lesions develop. PMID:27334013

  16. Identification of a new dengue virus inhibitor that targets the viral NS4B protein and restricts genomic RNA replication.

    PubMed

    van Cleef, Koen W R; Overheul, Gijs J; Thomassen, Michael C; Kaptein, Suzanne J F; Davidson, Andrew D; Jacobs, Michael; Neyts, Johan; van Kuppeveld, Frank J M; van Rij, Ronald P

    2013-08-01

    Dengue virus (DENV) is an important human arthropod-borne virus with a major impact on public health. Nevertheless, a licensed vaccine or specific treatment is still lacking. We therefore screened the NIH Clinical Collection (NCC), a library of drug-like small molecules, for inhibitors of DENV replication using a cell line that contains a stably replicating DENV serotype 2 (DENV2) subgenomic replicon. The most potent DENV inhibitor in the NCC was δ opioid receptor antagonist SDM25N. This compound showed antiviral activity against wild-type DENV2 in both Hela and BHK-21 cells, but not in the C6/36 cell line derived from the mosquito Aedes albopictus. The structurally related compound naltrindole also inhibited DENV replication, albeit less potently. Using a transient subgenomic replicon, we demonstrate that SDM25N restricts genomic RNA replication rather than translation of the viral genome. We identified a single amino acid substitution (F164L) in the NS4B protein that confers resistance to SDM25N. Remarkably, an NS4B amino acid substitution (P104L), which was previously shown to confer resistance to the DENV inhibitor NITD-618, also provided resistance to SDM25N. In conclusion, we have identified a new DENV inhibitor, SDM25N, which restricts genomic RNA replication by - directly or indirectly - targeting the viral NS4B protein. PMID:23735301

  17. Targeted delivery of the hydroxylase inhibitor DMOG provides enhanced efficacy with reduced systemic exposure in a murine model of colitis.

    PubMed

    Tambuwala, Murtaza M; Manresa, Mario C; Cummins, Eoin P; Aversa, Vincenzo; Coulter, Ivan S; Taylor, Cormac T

    2015-11-10

    Targeting hypoxia-sensitive pathways has recently been proposed as a new therapeutic approach to the treatment of intestinal inflammation. HIF-hydroxylases are enzymes which confer hypoxic-sensitivity upon the hypoxia-inducible factor (HIF), a major regulator of the adaptive response to hypoxia. Previous studies have shown that systemic (intraperitoneal) administration of hydroxylase inhibitors such as dimethyloxalylglycine (DMOG) is profoundly protective in multiple models of colitis, however the therapeutic potential of this approach is limited due to potential side-effects associated with systemic drug exposure and the fact that orally delivered DMOG is ineffective (likely due to drug inactivation by gastric acid). In order to overcome these issues, we formulated DMOG in a liquid emulsion drug delivery system which, when coated with specific polymer coatings, permits oral delivery of a reduced dose which is released locally throughout the colon. This colon-targeted DMOG formulation demonstrated increased relative colonic bioactivity with reduced systemic exposure and provided a similar degree of protection to systemic (intraperitoneal) administration at a 40-fold lower dose in DSS-induced colitis. In summary, targeted delivery of DMOG to the colon provides local protection resulting in enhanced efficacy with reduced systemic exposure in the treatment of colitis. This novel approach to targeting hydroxylase inhibitors to specific diseased regions of the GI tract may improve it's potential as a new therapeutic in inflammatory bowel diseases such as ulcerative colitis.

  18. Targeting TORC1/2 enhances sensitivity to EGFR inhibitors in head and neck cancer preclinical models.

    PubMed

    Cassell, Andre; Freilino, Maria L; Lee, Jessica; Barr, Sharon; Wang, Lin; Panahandeh, Mary C; Thomas, Sufi M; Grandis, Jennifer R

    2012-11-01

    Head and neck squamous cell carcinoma (HNSCC) is characterized by overexpression of the epidermal growth factor receptor (EGFR) where treatments targeting EGFR have met with limited clinical success. Elucidation of the key downstream-pathways that remain activated in the setting of EGFR blockade may reveal new therapeutic targets. The present study was undertaken to test the hypothesis that inhibition of the mammalian target of rapamycin (mTOR) complex would enhance the effects of EGFR blockade in HNSCC preclinical models. Treatment of HNSCC cell lines with the newly developed TORC1/TORC2 inhibitor OSI-027/ASP4876 resulted in dose-dependent inhibition of proliferation with abrogation of phosphorylation of known downstream targets including phospho-AKT (Ser473), phospho-4E-BP1, phospho-p70s6K, and phospho-PRAS40. Furthermore, combined treatment with OSI-027 and erlotinib resulted in enhanced biochemical effects and synergistic growth inhibition in vitro. Treatment of mice bearing HNSCC xenografts with a combination of the Food and Drug Administration (FDA)-approved EGFR inhibitor cetuximab and OSI-027 demonstrated a significant reduction of tumor volumes compared with either treatment alone. These findings suggest that TORC1/TORC2 inhibition in conjunction with EGFR blockade represents a plausible therapeutic strategy for HNSCC. PMID:23226094

  19. PET imaging demonstrates histone deacetylase target engagement and clarifies brain penetrance of known and novel small molecule inhibitors in rat.

    PubMed

    Schroeder, F A; Wang, C; Van de Bittner, G C; Neelamegam, R; Takakura, W R; Karunakaran, A; Wey, H Y; Reis, S A; Gale, J; Zhang, Y L; Holson, E B; Haggarty, S J; Hooker, J M

    2014-10-15

    Histone deacetylase (HDAC) enzymes have been demonstrated as critical components in maintaining chromatin homeostasis, CNS development, and normal brain function. Evidence in mouse models links HDAC expression to learning, memory, and mood-related behaviors; small molecule HDAC inhibitor tool compounds have been used to demonstrate the importance of specific HDAC subtypes in modulating CNS-disease-related behaviors in rodents. So far, no direct evidence exists to understand the quantitative changes in HDAC target engagement that are necessary to alter biochemistry and behavior in a living animal. Understanding the relationship between target engagement and in vivo effect is essential in refining new ways to alleviate disease. We describe here, using positron emission tomography (PET) imaging of rat brain, the in vivo target engagement of a subset of class I/IIb HDAC enzymes implicated in CNS-disease (HDAC subtypes 1, 2, 3, and 6). We found marked differences in the brain penetrance of tool compounds from the hydroxamate and benzamide HDAC inhibitor classes and resolved a novel, highly brain penetrant benzamide, CN147, chronic treatment with which resulted in an antidepressant-like effect in a rat behavioral test. Our work highlights a new translational path for understanding the molecular and behavioral consequences of HDAC target engagement. PMID:25188794

  20. Multiplex Imaging and Cellular Target Identification of Kinase Inhibitors via an Affinity-Based Proteome Profiling Approach

    PubMed Central

    Su, Ying; Pan, Sijun; Li, Zhengqiu; Li, Lin; Wu, Xiaoyuan; Hao, Piliang; Sze, Siu Kwan; Yao, Shao Q.

    2015-01-01

    MLN8237 is a highly potent and presumably selective inhibitor of Aurora kinase A (AKA) and has shown promising antitumor activities. Like other kinase inhibitors which target the ATP-binding site of kinases, MLN8237 might be expected to have potential cellular off-targets. Herein, we report the first photoaffinity-based, small molecule AKA probe capable of both live-cell imaging of AKA activities and in situ proteome profiling of potential off-targets of MLN8237 (including AKA-associating proteins). By using two mutually compatible, bioorthogonal reactions (copper-catalyzed azide-alkyne cycloaddition chemistry and TCO-tetrazine ligation), we demostrate small molecule-based multiplex bioimaging for simultaneous in situ monitoring of two important cell-cycle regulating kinases (AKA and CDK1). A broad range of proteins, as potential off-targets of MLN8237 and AKA's-interacting partners, is subsequently identified by affinity-based proteome profiling coupled with large-scale LC-MS/MS analysis. From these studies, we discover novel AKA interactions which were further validated by cell-based immunoprecipitation (IP) experiments. PMID:25579846

  1. A proteomics approach to identifying key protein targets involved in VEGF inhibitor mediated attenuation of bleomycin-induced pulmonary fibrosis.

    PubMed

    Kulkarni, Yogesh M; Dutta, Sucharita; Iyer, Anand Krishnan V; Venkatadri, Rajkumar; Kaushik, Vivek; Ramesh, Vani; Wright, Clayton A; Semmes, Oliver John; Yakisich, Juan S; Azad, Neelam

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a life expectancy of less than 5 years post diagnosis for most patients. Poor molecular characterization of IPF has led to insufficient understanding of the pathogenesis of the disease, resulting in lack of effective therapies. In this study, we have integrated a label-free LC-MS based approach with systems biology to identify signaling pathways and regulatory nodes within protein interaction networks that govern phenotypic changes that may lead to IPF. Ingenuity Pathway Analysis of proteins modulated in response to bleomycin treatment identified PI3K/Akt and Wnt signaling as the most significant profibrotic pathways. Similar analysis of proteins modulated in response to vascular endothelial growth factor (VEGF) inhibitor (CBO-P11) treatment identified natural killer cell signaling and PTEN signaling as the most significant antifibrotic pathways. Mechanistic/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) were identified to be key mediators of pro- and antifibrotic response, where bleomycin (BLM) treatment resulted in increased expression and VEGF inhibitor treatment attenuated expression of mTOR and ERK. Using a BLM mouse model of pulmonary fibrosis and VEGF inhibitor CBO-P11 as a therapeutic measure, we identified a comprehensive set of signaling pathways and proteins that contribute to the pathogenesis of pulmonary fibrosis that can be targeted for therapy against this fatal disease.

  2. Presumed LRP1-targeting transport peptide delivers β-secretase inhibitor to neurons in vitro with limited efficiency

    PubMed Central

    Kim, Jong Ah; Casalini, Tommaso; Brambilla, Davide; Leroux, Jean-Christophe

    2016-01-01

    Interfering with the activity of β-secretase to reduce the production of Aβ peptides is a conceivable therapeutic strategy for Alzheimer’s disease. However, the development of efficient yet safe inhibitors is hampered by secondary effects, usually linked to the indiscriminate inhibition of other substrates’ processing by the targeted enzyme. Based on the spatial compartmentalization of the cleavage of the amyloid precursor protein by β-secretase, we hypothesized that by exploiting the endocytosis receptor low-density lipoprotein receptor-related protein it would be possible to direct an otherwise cell-impermeable inhibitor to the endosomes of neurons, boosting the drug’s efficacy and importantly, sparing the off-target effects. We used the transport peptide Angiopep to build an endocytosis-competent conjugate and found that although the peptide facilitated the inhibitor’s internalization into neurons and delivered it to the endosomes, the delivery was not efficient enough to potently reduce β-secretase activity at the cellular level. This is likely connected to the finding that in the cell lines we used, Angiopep’s internalization was not mediated by its presumed receptor to a significant extent. Additionally, Angiopep exploited different internalization mechanisms when applied alone or when conjugated to the inhibitor, highlighting the impact that drug conjugation can have on transport peptides. PMID:27682851

  3. In Silico Designing and Analysis of Inhibitors against Target Protein Identified through Host-Pathogen Protein Interactions in Malaria

    PubMed Central

    Samant, Monika; Chadha, Nidhi; Tiwari, Anjani K.; Hasija, Yasha

    2016-01-01

    Malaria, a life-threatening blood disease, has been a major concern in the field of healthcare. One of the severe forms of malaria is caused by the parasite Plasmodium falciparum which is initiated through protein interactions of pathogen with the host proteins. It is essential to analyse the protein-protein interactions among the host and pathogen for better understanding of the process and characterizing specific molecular mechanisms involved in pathogen persistence and survival. In this study, a complete protein-protein interaction network of human host and Plasmodium falciparum has been generated by integration of the experimental data and computationally predicting interactions using the interolog method. The interacting proteins were filtered according to their biological significance and functional roles. α-tubulin was identified as a potential protein target and inhibitors were designed against it by modification of amiprophos methyl. Docking and binding affinity analysis showed two modified inhibitors exhibiting better docking scores of −10.5 kcal/mol and −10.43 kcal/mol and an improved binding affinity of −83.80 kJ/mol and −98.16 kJ/mol with the target. These inhibitors can further be tested and validated in vivo for their properties as an antimalarial drug. PMID:27057354

  4. In silico identification of novel kinase inhibitors by targeting B-Raf(v660e) from natural products database.

    PubMed

    Wang, Zi-jie; Wan, Zhi-ning; Chen, Xu-dong; Wu, Chuan-fang; Gao, Guo-long; Liu, Rong; Shi, Zheng; Bao, Jin-ku

    2015-04-01

    The Ras/Raf/MEK/ERK (MAPK) signaling pathway has gained much attention from scientific community for therapeutic intervention in the past decades, specifically in oncology. Notably, a most prevalent B-Raf(v600e) mutant in Raf kinase family exhibits elevated kinase activity and results in constitutive activation of the MAPK pathway, thus making it a promising drug target for cancer therapy. Herein, virtual screening is applied to identify its potential inhibitors. Following the 25 ns molecular dynamic (MD) simulations, ZINC38541768, ZINC38541767, and ZINC12496469 are identified as B-Raf(v600e) potential inhibitors in a DFG-in conformation. Furthermore, according to the molecular mechanics/generalized born surface area (MM/GBSA) method, these three small molecules exhibit similar and good binding affinity toward B-Raf(v600e) (-38.76 kcal mol(-1), -42.60 kcal mol(-1), and -39.04 kcal mol(-1)). At the same time, several critical residues, such as I463, V471 in the P-loop, and DFG motif residue D594 within the A-loop, are also well clarified. All these results may not only indicate some future applications of inhibitors targeting B-Raf(v600e), but also benefit B-Raf(v600e) harboring cancer patients.

  5. Cathepsin K inhibitors: a novel target but promising approach in the treatment of osteoporosis.

    PubMed

    Helali, Asadul Mazid; Iti, Farhana Matin; Mohamed, Isa Naina

    2013-12-01

    Osteoporosis is a pathologic process characterized by low bone mass with skeletal fragility and an increased risk of fracture. It occurs due to an imbalance between bone resorption and formation. Although current antiresorptive therapy halts bone loss, it does not cure the condition as it also inhibits bone formation. Recent preclinical and clinical trials suggest that the inhibition of resorption by cathepsin K inhibitors increases bone formation. Cathepsin K is a papainlike cysteine protease with high potent collagenase activity and predominantly expressed in osteoclasts. While allowing demineralization, cathepsin K inhibitors suppress the degradation of type I collagen (the major organic matrix of bone) and thus enhancing bone formation. Many of these inhibitors have passed preclinical studies and are presently in clinical trials at different stages of advancement. This review explores the promising role of cathepsin K as a novel antiresorptive for the treatment of osteoporosis.

  6. Design, Synthesis, and Structure–Activity Relationship Studies of Fluorescent Inhibitors of Cycloxygenase-2 as Targeted Optical Imaging Agents

    PubMed Central

    2013-01-01

    Cycloxygenase-2 (COX-2) is an attractive target for molecular imaging because it is an inducible enzyme that is expressed in response to inflammatory and proliferative stimuli. Recently, we reported that conjugation of indomethacin with carboxy-X-rhodamine dyes results in the formation of effective, targeted, optical imaging agents able to detect COX-2 in inflammatory tissues and premalignant and malignant tumors (Uddin et al. Cancer Res. 2010, 70, 3618–3627). The present paper summarizes the details of the structure–activity relationship (SAR) studies performed for lead optimization of these dyes. A wide range of fluorescent conjugates were designed and synthesized, and each of them was tested for the ability to selectively inhibit COX-2 as the purified protein and in human cancer cells. The SAR study revealed that indomethacin conjugates are the best COX-2-targeted agents compared to the other carboxylic acid-containing nonsteroidal anti-inflammatory drugs (NSAIDs) or COX-2-selective inhibitors (COXIBs). An n-butyldiamide linker is optimal for tethering bulky fluorescent functionalities onto the NSAID or COXIB cores. The activity of conjugates also depends on the size, shape, and electronic properties of the organic fluorophores. These reagents are taken up by COX-2-expressing cells in culture, and the uptake is blocked by pretreatment with a COX inhibitor. In in vivo settings, these reagents become highly enriched in COX-2-expressing tumors compared to surrounding normal tissue, and they accumulate selectively in COX-2-expressing tumors as compared with COX-2-negative tumors grown in mice. Thus, COX-2-targeted fluorescent inhibitors are useful for preclinical and clinical detection of lesions containing elevated levels of COX-2. PMID:23488616

  7. Targeting the Motion of Shikimate Kinase: Development of Competitive Inhibitors that Stabilize an Inactive Open Conformation of the Enzyme.

    PubMed

    Prado, Verónica; Lence, Emilio; Maneiro, María; Vázquez-Ucha, Juan C; Beceiro, Alejandro; Thompson, Paul; Hawkins, Alastair R; González-Bello, Concepción

    2016-06-01

    The large conformational changes observed by Molecular Dynamics simulation studies on the product release in the LID and shikimic acid binding (SB) domains of the shikimate kinase (SK) enzyme have been exploited in the development of reversible competitive inhibitors against SK from Mycobacterium tuberculosis and Helicobacter pylori. This enzyme is a recognized target for antibiotic drug discovery. The reported C5-substituted shikimic acid analogues interact with the dynamic apolar pocket that surrounds the C4 and C5 hydroxyl groups of the natural substrate, cause the opening of the LID and SB domains, and capture the essential arginine far from the ATP binding site as required for catalysis. The 3-nitrobenzyl 3e and 5-benzothiophenyl derivatives 3i proved to be the most potent inhibitors. An ester prodrug of 3i was the most efficient derivative in achieving good in vitro activity against H. pylori, having a MIC value of 4 μg/mL. PMID:27191386

  8. Using AlphaScreen(®) to Identify Small-Molecule Inhibitors Targeting a Conserved Host-Pathogen Interaction.

    PubMed

    Austin, Sisley; Taouji, Saïd; Chevet, Eric; Wodrich, Harald; Rayne, Fabienne

    2016-01-01

    AlphaScreen(®) is a technology particularly suitable for bi-molecular inhibitor screening assays, e.g. using protein-protein interactions with purified recombinant proteins. Each binding partner of the bi-molecular interaction is coupled either to donor or to acceptor beads. The technology is based on the quantifiable transfer of oxygen singlets from donor to acceptor microbeads brought together by a specific interaction between the partners. We identified the conserved interaction between WW domains of cellular ubiquitin ligases of the Nedd4 family and a short peptide motif (PPxY) present in several structural and non-structural viral proteins as a potential drug target. Using an AlphaScreen assay recapitulating the interaction between Nedd4.2 and the PPxY motif of the adenoviral capsid protein VI, we screened a library of small molecules and identified specific inhibitors of this interaction. PMID:27613056

  9. Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of Rapamycin

    SciTech Connect

    Knight, Steven D.; Adams, Nicholas D.; Burgess, Joelle L.; Chaudhari, Amita M.; Darcy, Michael G.; Donatelli, Carla A.; Luengo, Juan I.; Newlander, Ken A.; Parrish, Cynthia A.; Ridgers, Lance H.; Sarpong, Martha A.; Schmidt, Stanley J.; Aller, Glenn S.Van; Carson, Jeffrey D.; Diamond, Melody A.; Elkins, Patricia A.; Gardiner, Christine M.; Garver, Eric; Gilbert, Seth A.; Gontarek, Richard R.; Jackson, Jeffrey R.; Kershner, Kevin L.; Luo, Lusong; Raha, Kaushik; Sherk, Christian S.; Sung, Chiu-Mei; Sutton, David; Tummino, Peter J.; Wegrzyn, Ronald J.; Auger, Kurt R.; Dhanak, Dashyant

    2010-09-30

    Phosphoinositide 3-kinase {alpha} (PI3K{alpha}) is a critical regulator of cell growth and transformation, and its signaling pathway is the most commonly mutated pathway in human cancers. The mammalian target of rapamycin (mTOR), a class IV PI3K protein kinase, is also a central regulator of cell growth, and mTOR inhibitors are believed to augment the antiproliferative efficacy of PI3K/AKT pathway inhibition. 2,4-Difluoro-N-{l_brace}2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl{r_brace}benzenesulfonamide (GSK2126458, 1) has been identified as a highly potent, orally bioavailable inhibitor of PI3K{alpha} and mTOR with in vivo activity in both pharmacodynamic and tumor growth efficacy models. Compound 1 is currently being evaluated in human clinical trials for the treatment of cancer.

  10. Inhibitors of Foot and Mouth Disease Virus Targeting a Novel Pocket of the RNA-Dependent RNA Polymerase

    PubMed Central

    Cornelison, Ceili A.; Rai, Devendra K.; Matzek, Kayla B.; Leslie, Maxwell D.; Schafer, Elizabeth; Marchand, Bruno; Adedeji, Adeyemi; Michailidis, Eleftherios; Dorst, Christopher A.; Moran, Jennifer; Pautler, Christie; Rodriguez, Luis L.; McIntosh, Mark A.; Rieder, Elizabeth; Sarafianos, Stefan G.

    2010-01-01

    Background Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections. Methodology/Principal Findings Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC50s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC50 = 12 µM and EC90 = 20 µM). Significance We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD. PMID:21203539

  11. Virtual Screening and Biological Evaluation of Inhibitors Targeting the XPA-ERCC1 Interaction

    PubMed Central

    Perez-Pineiro, Rolando; Wishart, David; Dumontet, Charles; Tuszynski, Jack A.

    2012-01-01

    Background Nucleotide excision repair (NER) removes many types of DNA lesions including those induced by UV radiation and platinum-based therapy. Resistance to platinum-based therapy correlates with high expression of ERCC1, a major element of the NER machinery. The interaction between ERCC1 and XPA is essential for a successful NER function. Therefore, one way to regulate NER is by inhibiting the activity of ERCC1 and XPA. Methodology/Principal Findings Here we continued our earlier efforts aimed at the identification and characterization of novel inhibitors of the ERCC1-XPA interaction. We used a refined virtual screening approach combined with a biochemical and biological evaluation of the compounds for their ability to interact with ERCC1 and to sensitize cells to UV radiation. Our findings reveal a new validated ERCC1-XPA inhibitor that significantly sensitized colon cancer cells to UV radiation indicating a strong inhibition of the ERCC1-XPA interaction. Conclusions NER is a major factor in acquiring resistance to platinum-based therapy. Regulating the NER pathway has the potential of improving the efficacy of platinum treatments. One approach that we followed is to inhibit the essential interaction between the two NER elements, ERCC1 and XPA. Here, we performed virtual screening against the ERCC1-XPA interaction and identified novel inhibitors that block the XPA-ERCC1 binding. The identified inhibitors significantly sensitized colon cancer cells to UV radiation indicating a strong inhibition of the ERCC1-XPA interaction. PMID:23272099

  12. Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE.

    PubMed

    Van de Wouwer, Dorien; Vanholme, Ruben; Decou, Raphaël; Goeminne, Geert; Audenaert, Dominique; Nguyen, Long; Höfer, René; Pesquet, Edouard; Vanholme, Bartel; Boerjan, Wout

    2016-09-01

    Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization. PMID:27485881

  13. Avenaciolides: potential MurA-targeted inhibitors against peptidoglycan biosynthesis in methicillin-resistant Staphylococcus aureus (MRSA).

    PubMed

    Chang, Ching-Ming; Chern, Jeffy; Chen, Ming-Yi; Huang, Kai-Fa; Chen, Chein-Hung; Yang, Yu-Liang; Wu, Shih-Hsiung

    2015-01-14

    Discovery of new antibiotics for combating methicillin-resistant Staphylococcus aureus (MRSA) is of vital importance in the post-antibiotic era. Here, we report four avenaciolide derivatives (1-4) isolated from Neosartorya fischeri, three of which had significant antimicrobial activity against MRSA. The morphology of avenaciolide-treated cells was protoplast-like, which indicated that cell wall biosynthesis was interrupted. Comparing the structures and minimum inhibitory concentrations of 1-4, the α,β-unsaturated carbonyl group seems to be an indispensable moiety for antimicrobial activity. Based on a structural similarity survey of other inhibitors with the same moiety, we revealed that MurA was the drug target. This conclusion was validated by (31)P NMR spectroscopy and MS/MS analysis. Although fosfomycin, which is the only clinically used MurA-targeted antibiotic, is ineffective for treating bacteria harboring the catalytically important Cys-to-Asp mutation, avenaciolides 1 and 2 inhibited not only wild-type but also fosfomycin-resistant MurA in an unprecedented way. Molecular simulation revealed that 2 competitively perturbs the formation of the tetrahedral intermediate in MurA. Our findings demonstrated that 2 is a potent inhibitor of MRSA and fosfomycin-resistant MurA, laying the foundation for the development of new scaffolds for MurA-targeted antibiotics.

  14. BAY 11-7082 is a broad-spectrum inhibitor with anti-inflammatory activity against multiple targets.

    PubMed

    Lee, Jaehwi; Rhee, Man Hee; Kim, Eunji; Cho, Jae Youl

    2012-01-01

    BAY 11-7082 (BAY) is an inhibitor of κB kinase (IKK) that has pharmacological activities that include anticancer, neuroprotective, and anti-inflammatory effects. In this study, BAY-pharmacological target pathways were further characterized to determine how this compound simultaneously suppresses various responses. Primary and cancerous (RAW264.7 cells) macrophages were activated by lipopolysaccharide, a ligand of toll-like receptor 4. As reported previously, BAY strongly suppressed the production of nitric oxide, prostaglandin E(2), and tumor necrosis factor-α and reduced the translocation of p65, major subunit of nuclear factor-κB, and its upstream signaling events such as phosphorylation of IκBα, IKK, and Akt. In addition, BAY also suppressed the translocation and activation of activator protein-1, interferon regulatory factor-3, and signal transducer and activator of transcription-1 by inhibiting the phosphorylation or activation of extracellular signal-related kinase, p38, TANK-binding protein, and Janus kinase-2. These data strongly suggest that BAY is an inhibitor with multiple targets and could serve as a lead compound in developing strong anti-inflammatory drugs with multiple targets in inflammatory responses.

  15. Recombinant Buckwheat Trypsin Inhibitor Induces Mitophagy by Directly Targeting Mitochondria and Causes Mitochondrial Dysfunction in Hep G2 Cells.

    PubMed

    Wang, Zhuanhua; Li, Shanshan; Ren, Rong; Li, Jiao; Cui, Xiaodong

    2015-09-01

    Mitochondria are essential targets for cancer chemotherapy and other disease treatments. Recombinant buckwheat trypsin inhibitor (rBTI), a member of the potato type I proteinase inhibitor family, was derived from tartary buckwheat extracts. Our results showed that rBTI directly targeted mitochondria and induced mitochondrial fragmentation and mitophagy. This occurs through enhanced depolarization of the mitochondrial membrane potential, increasing reactive oxygen species (ROS) generation associated with the rise of the superoxide dismutase and catalase activity and glutathione peroxidase (GSH) content, and changes in the GSH/oxidized glutathione ratio. Mild and transient ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 mitophagy to remove dysfunctional mitochondria. Furthermore, rBTI could directly induce mitochondrial fragmentation. It was also noted that rBTI highly increased colocalization of mitochondria in treated cells compared to nontreated cells. Tom 20, a subunit of the translocase of the mitochondrial outer membrane complex responsible for recognizing mitochondrial presequences, may be the direct target of rBTI.

  16. Recombinant Buckwheat Trypsin Inhibitor Induces Mitophagy by Directly Targeting Mitochondria and Causes Mitochondrial Dysfunction in Hep G2 Cells.

    PubMed

    Wang, Zhuanhua; Li, Shanshan; Ren, Rong; Li, Jiao; Cui, Xiaodong

    2015-09-01

    Mitochondria are essential targets for cancer chemotherapy and other disease treatments. Recombinant buckwheat trypsin inhibitor (rBTI), a member of the potato type I proteinase inhibitor family, was derived from tartary buckwheat extracts. Our results showed that rBTI directly targeted mitochondria and induced mitochondrial fragmentation and mitophagy. This occurs through enhanced depolarization of the mitochondrial membrane potential, increasing reactive oxygen species (ROS) generation associated with the rise of the superoxide dismutase and catalase activity and glutathione peroxidase (GSH) content, and changes in the GSH/oxidized glutathione ratio. Mild and transient ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 mitophagy to remove dysfunctional mitochondria. Furthermore, rBTI could directly induce mitochondrial fragmentation. It was also noted that rBTI highly increased colocalization of mitochondria in treated cells compared to nontreated cells. Tom 20, a subunit of the translocase of the mitochondrial outer membrane complex responsible for recognizing mitochondrial presequences, may be the direct target of rBTI. PMID:26301894

  17. BAY 11-7082 Is a Broad-Spectrum Inhibitor with Anti-Inflammatory Activity against Multiple Targets

    PubMed Central

    Lee, Jaehwi; Rhee, Man Hee; Kim, Eunji; Cho, Jae Youl

    2012-01-01

    BAY 11-7082 (BAY) is an inhibitor of κB kinase (IKK) that has pharmacological activities that include anticancer, neuroprotective, and anti-inflammatory effects. In this study, BAY-pharmacological target pathways were further characterized to determine how this compound simultaneously suppresses various responses. Primary and cancerous (RAW264.7 cells) macrophages were activated by lipopolysaccharide, a ligand of toll-like receptor 4. As reported previously, BAY strongly suppressed the production of nitric oxide, prostaglandin E2, and tumor necrosis factor-α and reduced the translocation of p65, major subunit of nuclear factor-κB, and its upstream signaling events such as phosphorylation of IκBα, IKK, and Akt. In addition, BAY also suppressed the translocation and activation of activator protein-1, interferon regulatory factor-3, and signal transducer and activator of transcription-1 by inhibiting the phosphorylation or activation of extracellular signal-related kinase, p38, TANK-binding protein, and Janus kinase-2. These data strongly suggest that BAY is an inhibitor with multiple targets and could serve as a lead compound in developing strong anti-inflammatory drugs with multiple targets in inflammatory responses. PMID:22745523

  18. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2007-10-02

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  19. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  20. Telescience Resource Kit

    NASA Technical Reports Server (NTRS)

    Schneider, Michelle; Lippincott, Jeff; Chubb, Steve; Whitaker, Jimmy; Rice, Jim; Gillis, Robert; Sims, Chris; Sellers, Donna; Bailey, Darrell (Technical Monitor)

    2002-01-01

    The Telescience Resource Kit (TReK) is a PC based ground control system. It can be used by a single individual or in a group environment to monitor and control spacecraft systems and payloads. Capabilities include data receipt, data processing, data storage, data management, and data transmission. Commercial-Off-The-Shelf (COTS) hardware and software have been employed to reduce development costs, operations and maintenance costs, and to effectively take advantage of new commercial products as they become available. The TReK system is currently being used to monitor and control payloads aboard the International Space Station. It is located at sites around the world.

  1. A novel targeted inhibitor of the alternative pathway of complement and its therapeutic application in ischemia/reperfusion injury.

    PubMed

    Huang, Yuxiang; Qiao, Fei; Atkinson, Carl; Holers, V Michael; Tomlinson, Stephen

    2008-12-01

    Bioavailability and therapeutic efficacy of soluble Crry, a mouse inhibitor of all complement activation pathways, is significantly enhanced when linked to a fragment of complement receptor 2 (CR2), a receptor that targets C3 activation products. In this study, we characterize alternative pathway-specific inhibitors consisting of a single or dimeric N-terminal region of mouse factor H (fH; short consensus repeats 1-5) linked to the same CR2 fragment (CR2-fH and CR2-fHfH). Both CR2-fH and CR2-fHfH were highly effective at inhibiting the alternative pathway in vitro and demonstrated a higher specific activity than CR2-Crry. CR2-fH was also more effective than endogenous serum fH in blocking target deposition of C3. Target binding and complement inhibitory activity of CR2-fH/CR2-fHfH was dependent on CR2- and C3-mediated interactions. The alternative pathway of complement plays a role in intestine ischemia/reperfusion injury. However, serum fH fails to provide protection against intestine ischemia/reperfusion injury although it can bind to and provide cell surfaces with protection from complement and is present in plasma at a high concentration. In a mouse model, CR2-fH and CR2-fHfH provided complete protection from local (intestine) and remote (lung) injury. CR2-fH targeted to the site of local injury and greatly reduced levels of tissue C3 deposition. Thus, the targeting mechanism significantly enhances alternative pathway-specific complement inhibitory activity of the N-terminal domain of fH and has the potential to reduce side effects that may be associated with systemic complement blockade. The data further indicate alternative pathway dependence for local and remote injury following intestinal ischemia/reperfusion in a clinically relevant therapeutic paradigm.

  2. PACE4-based molecular targeting of prostate cancer using an engineered ⁶⁴Cu-radiolabeled peptide inhibitor.

    PubMed

    Couture, Frédéric; Levesque, Christine; Dumulon-Perreault, Véronique; Ait-Mohand, Samia; D'Anjou, François; Day, Robert; Guérin, Brigitte

    2014-08-01

    The potential of PACE4 as a pharmacological target in prostate cancer has been demonstrated as this proprotein convertase is strongly overexpressed in human prostate cancer tissues and its inhibition, using molecular or pharmacological approaches, results in reduced cell proliferation and tumor progression in mouse tumor xenograft models. We developed a PACE4 high-affinity peptide inhibitor, namely, the multi-leucine (ML), and sought to determine whether this peptide could be exploited for the targeting of prostate cancer for diagnostic or molecular imaging purposes. We conjugated a bifunctional chelator 1,4,7-triazacyclononane-1,4,7- triacetic acid (NOTA) to the ML peptide for copper-64 ((64)Cu) labeling and positron emission tomography (PET)- based prostate cancer detection. Enzyme kinetic assays against recombinant PACE4 showed that the NOTA-modified ML peptide displays identical inhibitory properties compared to the unmodified peptide. In vivo biodistribution of the (64)Cu/NOTA-ML peptide evaluated in athymic nude mice bearing xenografts of two human prostate carcinoma cell lines showed a rapid and high uptake in PACE4-expressing LNCaP tumor at an early time point and in PACE4-rich organs. Co-injection of unlabeled peptide confirmed that tumor uptake was target-specific. PACE4-negative tumors displayed no tracer uptake 15 minutes after injection, while the kidneys, demonstrated high uptake due to rapid renal clearance of the peptide. The present study supports the feasibility of using a (64)Cu/NOTA-ML peptide for PACE4-targeted prostate cancer detection and PACE4 status determination by PET imaging but also provides evidence that ML inhibitor-based drugs would readily reach tumor sites under in vivo conditions for pharmacological intervention or targeted radiation therapy.

  3. A novel targeted inhibitor of the alternative pathway of complement and its therapeutic application in ischemia/reperfusion injury.

    PubMed

    Huang, Yuxiang; Qiao, Fei; Atkinson, Carl; Holers, V Michael; Tomlinson, Stephen

    2008-12-01

    Bioavailability and therapeutic efficacy of soluble Crry, a mouse inhibitor of all complement activation pathways, is significantly enhanced when linked to a fragment of complement receptor 2 (CR2), a receptor that targets C3 activation products. In this study, we characterize alternative pathway-specific inhibitors consisting of a single or dimeric N-terminal region of mouse factor H (fH; short consensus repeats 1-5) linked to the same CR2 fragment (CR2-fH and CR2-fHfH). Both CR2-fH and CR2-fHfH were highly effective at inhibiting the alternative pathway in vitro and demonstrated a higher specific activity than CR2-Crry. CR2-fH was also more effective than endogenous serum fH in blocking target deposition of C3. Target binding and complement inhibitory activity of CR2-fH/CR2-fHfH was dependent on CR2- and C3-mediated interactions. The alternative pathway of complement plays a role in intestine ischemia/reperfusion injury. However, serum fH fails to provide protection against intestine ischemia/reperfusion injury although it can bind to and provide cell surfaces with protection from complement and is present in plasma at a high concentration. In a mouse model, CR2-fH and CR2-fHfH provided complete protection from local (intestine) and remote (lung) injury. CR2-fH targeted to the site of local injury and greatly reduced levels of tissue C3 deposition. Thus, the targeting mechanism significantly enhances alternative pathway-specific complement inhibitory activity of the N-terminal domain of fH and has the potential to reduce side effects that may be associated with systemic complement blockade. The data further indicate alternative pathway dependence for local and remote injury following intestinal ischemia/reperfusion in a clinically relevant therapeutic paradigm. PMID:19017999

  4. Targeted therapies and immune checkpoint inhibitors in the treatment of metastatic melanoma patients: a guide and update for pathologists.

    PubMed

    Kakavand, Hojabr; Wilmott, James S; Long, Georgina V; Scolyer, Richard A

    2016-02-01

    The previously dismal prospects for patients with advanced stage metastatic melanoma have greatly improved in recent years. Enhanced understanding of both the pathogenesis of melanoma and its molecular drivers, as well as the importance and regulation of anti-tumour immune responses, have provided new therapeutic opportunities for melanoma patients. There are two major distinct categories of systemic treatments with activity for patients with metastatic melanoma: (1) targeted therapies, which act to inhibit the oncogenes that drive the aberrant growth and dissemination of the tumour; and (2) immune checkpoint inhibitor therapies, which act to enhance anti-tumour immune responses by blocking negative regulators of immunity. Pathologists play a critical and expanding role in the selection of the most appropriate treatment for individual metastatic melanoma patients in the modern era of personalised/precision medicine. The molecular pathology testing of melanoma tumour tissue for the presence of targetable oncogenic mutations is already part of routine practice in many institutions. In addition, other potential oncogenic therapeutic targets continue to be identified and pathology testing techniques must readily adapt to this rapidly changing field. Recent research findings suggest that pathological assessment of tumour associated immune cells and immunosuppressive ligand expression of the tumour are likely to be important in identifying patients most likely to benefit from immune checkpoint inhibitors. Similarly, pathological and molecular observations of on-treatment tumour tissue biopsies taken from patients on targeted therapies have provided new insights into the mechanisms of action of targeted molecular therapies, have contributed to the identification of resistance mechanisms to these novel therapies and may be of higher value for selecting patients most likely to benefit from therapies. These data have already provided a rational biological basis for the

  5. Selective serotonin reuptake inhibitor fluoxetine inhibits replication of human enteroviruses B and D by targeting viral protein 2C.

    PubMed

    Ulferts, Rachel; van der Linden, Lonneke; Thibaut, Hendrik Jan; Lanke, Kjerstin H W; Leyssen, Pieter; Coutard, Bruno; De Palma, Armando M; Canard, Bruno; Neyts, Johan; van Kuppeveld, Frank J M

    2013-04-01

    Although the genus Enterovirus contains many important human pathogens, there is no licensed drug for either the treatment or the prophylaxis of enterovirus infections. We report that fluoxetine (Prozac)--a selective serotonin reuptake inhibitor--inhibits the replication of human enterovirus B (HEV-B) and HEV-D but does not affect the replication of HEV-A and HEV-C or human rhinovirus A or B. We show that fluoxetine interferes with viral RNA replication, and we identified viral protein 2C as the target of this compound. PMID:23335743

  6. KIT — EDRN Public Portal

    Cancer.gov

    SCF-sR, also known as KIT, is the human homolog of the proto-oncogene c-kit. C-kit was first identified as the cellular homolog of the feline sarcoma viral oncogene v-kit. Human KIT is a tyrosine-protein kinase that acts as cell-surface receptor for the cytokine KITLG/SCF and plays an essential role in the regulation of cell survival and proliferation, hematopoiesis, stem cell maintenance, gametogenesis, mast cell development, migration and function, and in melanogenesis. KIT is a type 3 transmembrane receptor for MGF (mast cell growth factor, also known as stem cell factor). Mutations in this gene are associated with gastrointestinal stromal tumors, mast cell disease, acute myelogenous lukemia, and piebaldism. Multiple transcript variants encoding different isoforms have been found for this gene.

  7. Benzodiazepines and benzotriazepines as protein interaction inhibitors targeting bromodomains of the BET family

    PubMed Central

    Filippakopoulos, Panagis; Picaud, Sarah; Fedorov, Oleg; Keller, Marco; Wrobel, Matthias; Morgenstern, Olaf; Bracher, Franz; Knapp, Stefan

    2012-01-01

    Benzodiazepines are psychoactive drugs with anxiolytic, sedative, skeletal muscle relaxant and amnestic properties. Recently triazolo-benzodiazepines have been also described as potent and highly selective protein interaction inhibitors of bromodomain and extra-terminal (BET) proteins, a family of transcriptional co-regulators that play a key role in cancer cell survival and proliferation, but the requirements for high affinity interaction of this compound class with bromodomains has not been described. Here we provide insight into the structure–activity relationship (SAR) and selectivity of this versatile scaffold. In addition, using high resolution crystal structures we compared the binding mode of a series of benzodiazepine (BzD) and related triazolo-benzotriazepines (BzT) derivatives including clinically approved drugs such as alprazolam and midazolam. Our analysis revealed the importance of the 1-methyl triazolo ring system for BET binding and suggests modifications for the development of further high affinity bromodomain inhibitors. PMID:22137933

  8. Benzodiazepines and benzotriazepines as protein interaction inhibitors targeting bromodomains of the BET family.

    PubMed

    Filippakopoulos, Panagis; Picaud, Sarah; Fedorov, Oleg; Keller, Marco; Wrobel, Matthias; Morgenstern, Olaf; Bracher, Franz; Knapp, Stefan

    2012-03-15

    Benzodiazepines are psychoactive drugs with anxiolytic, sedative, skeletal muscle relaxant and amnestic properties. Recently triazolo-benzodiazepines have been also described as potent and highly selective protein interaction inhibitors of bromodomain and extra-terminal (BET) proteins, a family of transcriptional co-regulators that play a key role in cancer cell survival and proliferation, but the requirements for high affinity interaction of this compound class with bromodomains has not been described. Here we provide insight into the structure-activity relationship (SAR) and selectivity of this versatile scaffold. In addition, using high resolution crystal structures we compared the binding mode of a series of benzodiazepine (BzD) and related triazolo-benzotriazepines (BzT) derivatives including clinically approved drugs such as alprazolam and midazolam. Our analysis revealed the importance of the 1-methyl triazolo ring system for BET binding and suggests modifications for the development of further high affinity bromodomain inhibitors.

  9. Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor.

    PubMed

    Martínez-Hoyos, María; Perez-Herran, Esther; Gulten, Gulcin; Encinas, Lourdes; Álvarez-Gómez, Daniel; Alvarez, Emilio; Ferrer-Bazaga, Santiago; García-Pérez, Adolfo; Ortega, Fátima; Angulo-Barturen, Iñigo; Rullas-Trincado, Joaquin; Blanco Ruano, Delia; Torres, Pedro; Castañeda, Pablo; Huss, Sophie; Fernández Menéndez, Raquel; González Del Valle, Silvia; Ballell, Lluis; Barros, David; Modha, Sundip; Dhar, Neeraj; Signorino-Gelo, François; McKinney, John D; García-Bustos, Jose Francisco; Lavandera, Jose Luis; Sacchettini, James C; Jimenez, M Soledad; Martín-Casabona, Nuria; Castro-Pichel, Julia; Mendoza-Losana, Alfonso

    2016-06-01

    Despite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment. PMID:27428438

  10. Optics learning through affordable kit

    NASA Astrophysics Data System (ADS)

    P, Anusha N.; Shaji, Chitra; Sharan, Alok

    2014-10-01

    An affordable kit which helps to understand some of the optical phenomena qualitatively and quantitatively is presented in this paper. It supplements optics taught in classes. The kit consists of equipments which are available in the market at nominal cost such as laser pointer, lenses, glass plates, razor blades, coins, ball bearing etc. Experiments which come under wave optics (interference and diffraction) and ray optics (reflection and refraction) are explained using this kit.

  11. Education Payload Operation - Kit D

    NASA Technical Reports Server (NTRS)

    Keil, Matthew

    2009-01-01

    Education Payload Operation - Kit D (EPO-Kit D) includes education items that will be used to support the live International Space Station (ISS) education downlinks and Education Payload Operation (EPO) demonstrations onboard the ISS. The main objective of EPO-Kit D supports the National Aeronautics and Space Administration (NASA) goal of attracting students to study and seek careers in science, technology, engineering, and mathematics.

  12. Optics learning through affordable kit

    SciTech Connect

    P, Anusha N E-mail: chitrashaji@gmail.com Shaji, Chitra E-mail: chitrashaji@gmail.com Sharan, Alok E-mail: chitrashaji@gmail.com

    2014-10-15

    An affordable kit which helps to understand some of the optical phenomena qualitatively and quantitatively is presented in this paper. It supplements optics taught in classes. The kit consists of equipments which are available in the market at nominal cost such as laser pointer, lenses, glass plates, razor blades, coins, ball bearing etc. Experiments which come under wave optics (interference and diffraction) and ray optics (reflection and refraction) are explained using this kit.

  13. Defining balanced conditions for inhibitor screening assays that target bisubstrate enzymes.

    PubMed

    Yang, Jingsong; Copeland, Robert A; Lai, Zhihong

    2009-02-01

    High-throughput screening (HTS) is a common mechanism for identifying lead compounds for drug discovery efforts. Small molecules can inhibit enzymes by a variety of mechanisms, such as competitive, noncompetitive, and uncompetitive with respect to the substrate(s) of the catalytic reaction. To optimize the chances of finding the broadest diversity of inhibitor modalities during screening, one must run assays under ;;balanced'' conditions where the potency of inhibitors with various modes of action falls within a similar range. When an enzyme reaction involves more than one substrate, the definition and assessment of the apparent potency of inhibitors (IC(50)), in relation to their true potency (K(i)), can be nontrivial. This article provides a theoretical analysis, on the basis of the Cheng-Prusoff derivation, of the IC(50)/K( i) relationship of bisubstrate enzyme reactions following various sequential kinetic mechanisms, as well as the application and limitations of this information for defining optimal screening conditions for such enzymes. PMID:19196704

  14. In Silico Screening Identifies a Novel Potential PARP1 Inhibitor Targeting Synthetic Lethality in Cancer Treatment

    PubMed Central

    Li, Jian; Zhou, Nan; Cai, Peiling; Bao, Jinku

    2016-01-01

    Synthetic lethality describes situations in which defects in two different genes or pathways together result in cell death. This concept has been applied to drug development for cancer treatment, as represented by Poly (ADP-ribose) polymerase (PARPs) inhibitors. In the current study, we performed a computational screening to discover new PARP inhibitors. Among the 11,247 compounds analyzed, one natural product, ZINC67913374, stood out by its superior performance in the simulation analyses. Compared with the FDA approved PARP1 inhibitor, olaparib, our results demonstrated that the ZINC67913374 compound achieved a better grid score (−86.8) and amber score (−51.42). Molecular dynamics simulations suggested that the PARP1-ZINC67913374 complex was more stable than olaparib. The binding free energy for ZINC67913374 was −177.28 kJ/mol while that of olaparib was −159.16 kJ/mol. These results indicated ZINC67913374 bound to PARP1 with a higher affinity, which suggest ZINC67913374 has promising potential for cancer drug development. PMID:26907257

  15. Inhibitors of glutamate release from breast cancer cells; new targets for cancer-induced bone-pain.

    PubMed

    Fazzari, Jennifer; Lin, Hanxin; Murphy, Cecilia; Ungard, Robert; Singh, Gurmit

    2015-02-11

    Glutamate is an important signaling molecule in a wide variety of tissues. Aberrant glutamatergic signaling disrupts normal tissue homeostasis and induces several disruptive pathological conditions including pain. Breast cancer cells secrete high levels of glutamate and often metastasize to bone. Exogenous glutamate can disrupt normal bone turnover and may be responsible for cancer-induced bone pain (CIBP). CIBP is a significant co-morbidity that affects quality of life for many advanced-stage breast cancer patients. Current treatment options are commonly accompanied by serious side-effects that negatively impact patient care. Identifying small molecule inhibitors of glutamate release from aggressive breast cancer cells advances a novel, mechanistic approach to targeting CIBP that could advance treatment for several pathological conditions. Using high-throughput screening, we investigated the ability of approximately 30,000 compounds from the Canadian Compound Collection to reduce glutamate release from MDA-MB-231 breast cancer cells. This line is known to secrete high levels of glutamate and has been demonstrated to induce CIBP by this mechanism. Positive chemical hits were based on the potency of each molecule relative to a known pharmacological inhibitor of glutamate release, sulfasalazine. Efficacy was confirmed and drug-like molecules were identified as potent inhibitors of glutamate secretion from MDA-MB-231, MCF-7 and Mat-Ly-Lu cells.

  16. Mammalian target of rapamycin inhibitors induce tumor cell apoptosis in vivo primarily by inhibiting VEGF expression and angiogenesis.

    PubMed

    Frost, Patrick; Berlanger, Eileen; Mysore, Veena; Hoang, Bao; Shi, Yijiang; Gera, Joseph; Lichtenstein, Alan

    2013-01-01

    We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES)-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

  17. New Insight into the Anti-liver Fibrosis Effect of Multitargeted Tyrosine Kinase Inhibitors: From Molecular Target to Clinical Trials.

    PubMed

    Qu, Kai; Huang, Zichao; Lin, Ting; Liu, Sinan; Chang, Hulin; Yan, Zhaoyong; Zhang, Hongxin; Liu, Chang

    2015-01-01

    Tyrosine kinases (TKs) is a family of tyrosine protein kinases with important functions in the regulation of a broad variety of physiological cell processes. Overactivity of TK disturbs cellular homeostasis and has been linked to the development of certain diseases, including various fibrotic diseases. In regard to liver fibrosis, several TKs, such as vascular endothelial growth factor receptor, platelet-derived growth factor receptor, fibroblast growth factor receptor, and epidermal growth factor receptor kinases, have been identified as central mediators in collagen production and potential targets for anti-liver fibrosis therapies. Given the essential role of TKs during liver fibrogenesis, multitargeted inhibitors of aberrant TK activity, including sorafenib, erlotinib, imatinib, sunitinib, nilotinib, brivanib and vatalanib, have been shown to have potential for treating liver fibrosis. Beneficial effects are observed by researchers of this field using these multitargeted TK inhibitors in preclinical animal models and in patients with liver fibrosis. The present review will briefly summarize the anti-liver fibrosis effects of multitargeted TK inhibitors and molecular mechanisms. PMID:26834633

  18. [Establishment of double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers].

    PubMed

    Xie, Tao; Qin, Zhi-Zhen; Zhou, Rui; Zhao, Ying; Du, Guan-hua

    2015-04-01

    A double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers was established. In the reaction system of xanthine oxidase, WST-1 works as the probe for the ultra oxygen anion generation, and product uric acid works as xanthine oxidase activity indicator. By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector, the changes of these indicators' concentration were observed and the influence factors of this reaction system to establish the high throughput screening model were studied. And the model is confirmed by positive drugs. In the reaction system, the final volume of reaction system is 50 μL and the concentrations of xanthine oxidase is 4 mU x mL(-1), xanthine 250 μmol x L(-1) and WST-1 100 μmol x L(-1), separately. The Z'-factor of model for xanthine oxidase inhibitors is 0.537 4, S/N is 47.519 9; the Z'-factor of model for superoxide anion scavengers is 0.507 4, S/N is 5.388 9. This model for xanthine oxidase inhibitors and superoxide anion scavengers has more common characteristics of the good stability, the fewer reagent types and quantity, the good repeatability, and so on. And it can be widely applied in high-throughput screening research.

  19. New Insight into the Anti-liver Fibrosis Effect of Multitargeted Tyrosine Kinase Inhibitors: From Molecular Target to Clinical Trials

    PubMed Central

    Qu, Kai; Huang, Zichao; Lin, Ting; Liu, Sinan; Chang, Hulin; Yan, Zhaoyong; Zhang, Hongxin; Liu, Chang

    2016-01-01

    Tyrosine kinases (TKs) is a family of tyrosine protein kinases with important functions in the regulation of a broad variety of physiological cell processes. Overactivity of TK disturbs cellular homeostasis and has been linked to the development of certain diseases, including various fibrotic diseases. In regard to liver fibrosis, several TKs, such as vascular endothelial growth factor receptor, platelet-derived growth factor receptor, fibroblast growth factor receptor, and epidermal growth factor receptor kinases, have been identified as central mediators in collagen production and potential targets for anti-liver fibrosis therapies. Given the essential role of TKs during liver fibrogenesis, multitargeted inhibitors of aberrant TK activity, including sorafenib, erlotinib, imatinib, sunitinib, nilotinib, brivanib and vatalanib, have been shown to have potential for treating liver fibrosis. Beneficial effects are observed by researchers of this field using these multitargeted TK inhibitors in preclinical animal models and in patients with liver fibrosis. The present review will briefly summarize the anti-liver fibrosis effects of multitargeted TK inhibitors and molecular mechanisms. PMID:26834633

  20. A New Class of Molecular Targeted Radioprotectors: GSK-3beta Inhibitors

    SciTech Connect

    Thotala, Dinesh K.; Geng Ling; Dickey, Amy K.; Hallahan, Dennis E.; Yazlovitskaya, Eugenia M.

    2010-02-01

    Purpose: Development of new treatments is critical to effective protection against radiation-induced injury. We investigate the potential of developing small-molecule inhibitors of glycogen synthase kinase 3beta (GSK-3beta)-SB216763 or SB415286-as radioprotective agents to attenuate intestinal injury. Methods and Materials: A survival study was done by use of C57BL/6J mice to evaluate the radioprotective effect of GSK-3beta inhibitors. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and immunohistochemical staining for Bax and Bcl-2 were used to assess apoptosis in the small intestines of the treated mice. A clonogenic survival study, apoptosis assays (staining with annexin V or 4',6-diamidino-2-phenylindole), and immunoblot analysis of beta-catenin, Bcl-2, Bax, and caspase 3 were done by use of Rat intestinal epithelial cell line IEC-6 cells. Results: Pretreatment with SB415286 significantly improved survival of mice irradiated with 8 and 12 Gy. Mice pretreated with SB216763 or SB415286 showed a significant reduction in TUNEL- and Bax-positive cells and an increase in Bcl-2-positive cells in intestinal crypts at 4 and/or 12 h after radiation with 4 and/or 8 Gy compared with radiation alone. Pretreatment of irradiated IEC-6 cells with GSK-3beta inhibitors significantly increased clonogenic survival compared with cells treated with radiation alone. This increase was due to the attenuation of radiation-induced apoptosis, as shown by annexin V and 4',6-diamidino-2-phenylindole assays, as well as immunoblot analysis of Bcl-2, Bax, and caspase 3. Conclusions: Glycogen synthase kinase 3beta small-molecule inhibitors protect mouse intestine from radiation-induced damage in cell culture and in vivo and improve survival of mice. Molecular mechanisms of this protection involve attenuated radiation-induced apoptosis regulated by Bcl-2, Bax, and caspase 3. Therefore GSK-3beta inhibitors reduce deleterious consequences of intestinal irradiation and

  1. Targeted molecular therapy of head and neck squamous cell carcinoma with the tyrosine kinase inhibitor vandetanib in a mouse model

    PubMed Central

    Sano, Daisuke; Fooshee, David R.; Zhao, Mei; Andrews, Genevieve A.; Frederick, Mitchell J.; Galer, Chad; Milas, Zvonimir L.; Morrow, Phuong Khanh H.; Myers, Jeffrey N.

    2010-01-01

    Background We investigated the effects of vandetanib, an inhibitor of vascular endothelial growth factor receptor 2 (VEGFR-2) and epidermal growth factor receptor (EGFR), alone and in combination with paclitaxel in an orthotopic mouse model of human head and neck squamous cell carcinoma (HNSCC). Methods The in vitro effects of vandetanib (ZACTIMA™) were assessed in two HNSCC cell lines on cell growth, apoptosis, and receptor and downstream signaling morecule expression and phosphorylation levels. We assessed in vivo effects of vandetanib and/or paclitaxel by measuring tumor cell apoptosis, endothelial cell apoptosis, microvessel density, tumor size, and animal survival. Results In vitro, vandetanib inhibited the phosphorylation of EGFR and its downstream targets in HNSCC cells and inhibited proliferation and induced apoptosis of HNSCC cells and extended survival and inhibited tumor growth in nude mice orthotopically injected with human HNSCC. Conclusion Vandetanib has the potential to be a novel molecular targeted therapy for HNSCC. PMID:20629091

  2. Neuropathy target esterase inhibitors: enantiomeric separation and stereospecificity of 2-substituted-4H-1,3,2-benzodioxaphosphorin 2-oxides.

    PubMed

    Wu, S Y; Casida, J E

    1994-01-01

    2-Substituted-4H-1,3,2-benzodioxaphosphorin 2-oxides (2-substituted-BDPOs) are known to be potent neuropathy target esterase (NTE) inhibitors (I50s for the racemates of 0.2-3 nM) when the 2-substituents are n-alkyl (C5-C12), N-alkoxy (C7-C10), or p-n-alkylbenzyl (C3 and C4). The list of potent inhibitors (I50s < 3 nM) is expanded by the new n-alkylamino (C9) and n-alkylthio (C5, C7, and C9) analogs reported here. The optimal chain length of the 2-substituent is about 10 atoms in the alkylamino and alkylthio series as in our previous study on alkyl and alkoxy moieties. In contrast, an I50 of 60 nM is reported for o-methylphenoxy-BDPO, the neuropathic metabolite of tri-o-cresyl phosphate (TOCP). In addition to substituent effects, each of these compounds contains two enantiomers of unknown stereospecificity as NTE inhibitors. Separation by chiral HPLC with the CHIRALCEL OC column and hexane-2-propanol eluent gives individual enantiomers of > 98% e.e. and a stereospecificity for NTE inhibition depending on the type and chain length of the 2-substituent; e.g., the ratio for inhibitory potency of the individual enantiomers is 1.7-fold for nonylthio, 1255-fold for nonylamino, and 9-fold for the TOCP metabolite. In comparing enantiomeric pairs of BDPOs with alkyl, alkoxy, alkylamino, alkylthio, benzyl, p-butylbenzyl, o-methylphenoxy, or phenyl as the 2-substituent, the more retained enantiomer in HPLC is always the better NTE inhibitor (in a series of twenty-two pairs) and housefly toxicant (based on two pairs) than the less retained one.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8155829

  3. Synthesis and antitumor evaluation of novel 5-substituted-4-hydroxy-8-nitroquinazolines as EGFR signaling-targeted inhibitors.

    PubMed

    Jin, Yi; Li, Hui-Yuan; Lin, Li-Ping; Tan, Jinzhi; Ding, Jian; Luo, Xiaomin; Long, Ya-Qiu

    2005-10-01

    The synthesis and biological activity of a series of novel 5-substituted-4-hydroxy-8-nitroquinazolines that may function as inhibitors of EGFR- and/or ErbB-2-related oncogenic signaling are described. These compounds were prepared by S(N)Ar reaction of 5-chloro-4-hydroxy-8-nitroquinazoline with alkyl or aryl amines, or alkyl alcohol as nucleophiles. Although the enzyme assay showed a weak inhibition effect against both EGFR and ErbB-2 tyrosine kinases, the cell-based antitumor activity turned out promising. Compounds having 5-anilino substituent exhibit high potency with 5-(4-methoxy)anilino-4-hydroxy-8-nitroquinazoline (1h) being the best dual EGFR/ErbB-2 inhibitors, which effectively inhibited the growth of both EGFR (MDA-MB-468, IC(50)<0.01microM) and ErbB-2 (SK-BR-3, IC(50)=13microM) overexpressing human tumor cell lines in vitro. More interestingly, the variation of the substituent(s) at the 3- and/or 4-position of the 5-anilino portion was found to modulate the selectivity and potency dramatically. However, compounds having an alkylamino or alkyloxy group at the 5-position of 4-hydroxy-8-nitroquinazolines are essentially inactive. These results are consistent with molecular modeling observations. This study was the first attempt to identify new structural types of dual EGFR/ErbB-2-related signaling inhibitors by incorporation of the anilino group at the 5-position of 4-hydroxy-8-nitroquinazolines' core structure, providing promising new templates for further development of potent inhibitors targeting both EGFR and ErbB-2 tyrosine kinases.

  4. Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease.

    PubMed

    Korkmaz, Brice; Lesner, Adam; Guarino, Carla; Wysocka, Magdalena; Kellenberger, Christine; Watier, Hervé; Specks, Ulrich; Gauthier, Francis; Jenne, Dieter E

    2016-07-01

    Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future. PMID:27329045

  5. Inhibitors and Antibody Fragments as Potential Anti-Inflammatory Therapeutics Targeting Neutrophil Proteinase 3 in Human Disease.

    PubMed

    Korkmaz, Brice; Lesner, Adam; Guarino, Carla; Wysocka, Magdalena; Kellenberger, Christine; Watier, Hervé; Specks, Ulrich; Gauthier, Francis; Jenne, Dieter E

    2016-07-01

    Proteinase 3 (PR3) has received great scientific attention after its identification as the essential antigenic target of antineutrophil cytoplasm antibodies in Wegener's granulomatosis (now called granulomatosis with polyangiitis). Despite many structural and functional similarities between neutrophil elastase (NE) and PR3 during biosynthesis, storage, and extracellular release, unique properties and pathobiological functions have emerged from detailed studies in recent years. The development of highly sensitive substrates and inhibitors of human PR3 and the creation of PR3-selective single knockout mice led to the identification of nonredundant roles of PR3 in cell death induction via procaspase-3 activation in cell cultures and in mouse models. According to a study in knockout mice, PR3 shortens the lifespan of infiltrating neutrophils in tissues and accelerates the clearance of aged neutrophils in mice. Membrane exposure of active human PR3 on apoptotic neutrophils reprograms the response of macrophages to phagocytosed neutrophils, triggers secretion of proinflammatory cytokines, and undermines immune silencing and tissue regeneration. PR3-induced disruption of the anti-inflammatory effect of efferocytosis may be relevant for not only granulomatosis with polyangiitis but also for other autoimmune diseases with high neutrophil turnover. Inhibition of membrane-bound PR3 by endogenous inhibitors such as the α-1-protease inhibitor is comparatively weaker than that of NE, suggesting that the adverse effects of unopposed PR3 activity resurface earlier than those of NE in individuals with α-1-protease inhibitor deficiency. Effective coverage of PR3 by anti-inflammatory tools and simultaneous inhibition of both PR3 and NE should be most promising in the future.

  6. Discovery of Inhibitors of 4′-Phosphopantetheine Adenylyltransferase (PPAT) To Validate PPAT as a Target for Antibacterial Therapy

    PubMed Central

    Walkup, Grant K.; Lahiri, Sushmita D.; Huynh, Hoan; Neckermann, Georg; Utley, Luke; Nash, Tory J.; Brock, Jesse; San Martin, Maryann; Kutschke, Amy; Johnstone, Michele; Laganas, Valerie; Hajec, Laurel; Gu, Rong-Fang; Ni, Haihong; Chen, Brendan; Hutchings, Kim; Holt, Elise; McKinney, David; Gao, Ning; Livchak, Stephania; Thresher, Jason

    2013-01-01

    Inhibitors of 4′-phosphopantetheine adenylyltransferase (PPAT) were identified through high-throughput screening of the AstraZeneca compound library. One series, cycloalkyl pyrimidines, showed inhibition of PPAT isozymes from several species, with the most potent inhibition of enzymes from Gram-positive species. Mode-of-inhibition studies with Streptococcus pneumoniae and Staphylococcus aureus PPAT demonstrated representatives of this series to be reversible inhibitors competitive with phosphopantetheine and uncompetitive with ATP, binding to the enzyme-ATP complex. The potency of this series was optimized using structure-based design, and inhibition of cell growth of Gram-positive species was achieved. Mode-of-action studies, using generation of resistant mutants with targeted sequencing as well as constructs that overexpress PPAT, demonstrated that growth suppression was due to inhibition of PPAT. An effect on bacterial burden was demonstrated in mouse lung and thigh infection models, but further optimization of dosing requirements and compound properties is needed before these compounds can be considered for progress into clinical development. These studies validated PPAT as a novel target for antibacterial therapy. PMID:24041904

  7. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases.

    PubMed

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  8. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases

    PubMed Central

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  9. KE108-conjugated unimolecular micelles loaded with a novel HDAC inhibitor thailandepsin-A for targeted neuroendocrine cancer therapy.

    PubMed

    Chen, Guojun; Jaskula-Sztul, Renata; Harrison, April; Dammalapati, Ajitha; Xu, Wenjin; Cheng, Yiqiang; Chen, Herbert; Gong, Shaoqin

    2016-08-01

    Neuroendocrine (NE) cancers can cause significant patient morbidity. Besides surgery, there are no curative treatments for NE cancers and their metastases, emphasizing the need for the development of other forms of therapy. In this study, multifunctional unimolecular micelles were developed for targeted NE cancer therapy. The unimolecular micelles were formed by multi-arm star amphiphilic block copolymer poly(amidoamine)-poly(valerolactone)-poly(ethylene glycol) conjugated with KE108 peptide and Cy5 dye (abbreviated as PAMAM-PVL-PEG-KE108/Cy5). The unimolecular micelles with a spherical core-shell structure exhibited a uniform size distribution and excellent stability. The hydrophobic drug thailandepsin-A (TDP-A), a recently discovered HDAC inhibitor, was physically encapsulated into the hydrophobic core of the micelles. KE108 peptide, a somatostatin analog possessing high affinity for all five subtypes of somatostatin receptors (SSTR 1-5), commonly overexpressed in NE cancer cells, was used for the first time as an NE cancer targeting ligand. KE108 exhibited superior targeting abilities compared to other common somatostatin analogs, such as octreotide, in NE cancer cell lines. The in vitro assays demonstrated that the TDP-A-loaded, KE108-targeted micelles exhibited the best capabilities in suppressing NE cancer cell growth. Moreover, the in vivo near-infrared fluorescence imaging on NE-tumor-bearing nude mice showed that KE108-conjugated micelles exhibited the greatest tumor accumulation due to their passive targeting and active targeting capabilities. Finally, TDP-A-loaded and KE108-conjugated micelles possessed the best anticancer efficacy without detectable systemic toxicity. Thus, these novel TDP-A-loaded and KE108-conjugated unimolecular micelles offer a promising approach for targeted NE cancer therapy. PMID:27156249

  10. Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments.

    PubMed

    Fonfria, Elena; Donald, Sarah; Cadd, Verity A

    2016-01-01

    Botulinum neurotoxins (BoNTs) are highly potent multi-domain proteins, responsible for botulism in animals and humans. The modular structural organization of BoNTs has led to the development of novel engineered bio-therapeutic proteins called targeted secretion inhibitors (TSIs). We report here that botulinum neurotoxin A (BoNT/A) and a TSI/A in which the neuronal binding domain of BoNT/A has been substituted by an epidermal growth factor (EGF) ligand, named EGFR-targeted TSI/A, exploit different routes to gain entry in the same in vitro neuroblastoma cell system, SiMa cells. We found that the EGF ligand conferred the affinity to the EGFR-targeted TSI/A at the EGF receptor when compared to an untargeted TSI/A and also the ability to internalize into the cells and cleave its cytosolic target protein SNAP-25. Using high content analysis we found that both BoNT/A and the EGFR-targeted TSI/A enter the cell in a concentration-dependent manner and in compartments which are able to translocate the proteins into the cytosol within 4 h. The EGFR-targeted TSI/A internalized into a compartment which gave a punctate staining pattern by immunofluorescence and partially overlapped with structures positive for the early endosomal marker EAA1; whereas BoNT/A did not internalize into a punctate compartment but did so in an acidifying compartment consistent with local synaptic vesicle recycling. These findings show that the BoNT/A translocation domain, common to both BoNT/A and the EGFR-targeted TSI/A, is a versatile tool for cytosolic delivery from distinct intracellular vesicular compartments. PMID:26329879

  11. Transition-state inhibitors of purine salvage and other prospective enzyme targets in malaria.

    PubMed

    Ducati, Rodrigo G; Namanja-Magliano, Hilda A; Schramm, Vern L

    2013-07-01

    Malaria is a leading cause of human death within the tropics. The gradual generation of drug resistance imposes an urgent need for the development of new and selective antimalarial agents. Kinetic isotope effects coupled to computational chemistry have provided the relevant details on geometry and charge of enzymatic transition states to facilitate the design of transition-state analogs. These features have been reproduced into chemically stable mimics through synthetic chemistry, generating inhibitors with dissociation constants in the pico- to femto-molar range. Transition-state analogs are expected to contribute to the control of malaria.

  12. Targets for dioxin: Genes for plasminogen activator inhibitor-2 and interleukin-1. beta

    SciTech Connect

    Sutter, T.R.; Guzman, K.; Dold, K.M. ); Greenlee, W.F. )

    1991-10-18

    Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), a widespread environmental contaminant, may elicit its effects by altering gene expression in susceptible cells. Five TCDD-responsive complementary DNA clones were isolated from a human keratinocyte cell line. One of these clones encodes plasminogen activator inhibitor-2, a factor that influences growth and differentiation by regulating proteolysis of the extracellular matrix. Another encodes the cytokine interleukin-1{beta}. Thus, TCDD alters the expression of growth regulator genes and has effects similar to those of other tumor-promoting agents that affect both inflammation and differentiation.

  13. Benzimidazole analogs as WTA biosynthesis inhibitors targeting methicillin resistant Staphylococcus aureus.

    PubMed

    Yang, Shu-Wei; Pan, Jianping; Yang, Christine; Labroli, Marc; Pan, Weidong; Caldwell, John; Ha, Sookhee; Koseoglu, Sandra; Xiao, Jing C; Mayhood, Todd; Sheth, Payal R; Garlisi, Charles G; Wu, Jin; Lee, Sang Ho; Wang, Hao; Tan, Christopher M; Roemer, Terry; Su, Jing

    2016-10-01

    A series of benzimidazole analogs have been synthesized to improve the profile of the previous lead compounds tarocin B and 1. The syntheses, structure-activity relationships, and selected biochemical data of these analogs are described. The optimization efforts allowed the identification of 21, a fluoro-substituted benzimidazole, exhibiting potent TarO inhibitory activity and typical profile for a wall teichoic acid (WTA) biosynthesis inhibitor. Compound 21 displayed a potent synergistic and bactericidal effect in combination with imipenem against diverse methicillin-resistant Staphylococci. PMID:27575474

  14. Transition-state inhibitors of purine salvage and other prospective enzyme targets in malaria

    PubMed Central

    Ducati, Rodrigo G.; Namanja-Magliano, Hilda A.; Schramm, Vern L.

    2013-01-01

    Malaria is a leading cause of human death within the tropics. The gradual generation of drug resistance imposes an urgent need for the development of new and selective antimalarial agents. Kinetic isotope effects coupled to computational chemistry have provided the relevant details on geometry and charge of enzymatic transition states to facilitate the design of transition-state analogs. These features have been reproduced into chemically stable mimics through synthetic chemistry, generating inhibitors with dissociation constants in the pico- to femto-molar range. Transition-state analogs are expected to contribute to the control of malaria. PMID:23859211

  15. Dynamic combinatorial chemistry: a tool to facilitate the identification of inhibitors for protein targets.

    PubMed

    Mondal, Milon; Hirsch, Anna K H

    2015-04-21

    Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify ligands for biological targets given that it enables the target to direct the synthesis and amplification of its strongest binder(s) from the library of interconverting compounds. Since the first report of DCC applied to the discovery of binders for a protein, this elegant tool has been employed on a range of protein targets at various stages of medicinal-chemistry projects. A series of suitable, reversible reactions that are biocompatible have been established and the portfolio of analytical techniques is growing. Despite progress, in most cases, the libraries employed remain of moderate size. We present here the most recent advances in the field of DCC applied to protein targets, paying particular attention to the experimental conditions and analytical methods chosen.

  16. PDGFRA Regulates Proliferation of Gastrointestinal Stromal Tumor Cells with Mutations in KIT by Stabilizing ETV1

    PubMed Central

    Hayashi, Yujiro; Bardsley, Michael R.; Toyomasu, Yoshitaka; Milosavljevic, Srdjan; Gajdos, Gabriella B.; Choi, Kyoung Moo; Reid-Lombardo, KMarie; Kendrick, Michael L.; Bingener-Casey, Juliane; Tang, Chih-Min; Sicklick, Jason K.; Gibbons, Simon J.; Farrugia, Gianrico; Taguchi, Takahiro; Gupta, Anu; Rubin, Brian P.; Fletcher, Jonathan A.; Ramachandran, Abhijit; Ordog, Tamas

    2015-01-01

    Background & Aims In gastrointestinal muscles, KIT is predominantly expressed by interstitial cells of Cajal (ICC) and PDGFRA is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be co-expressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1. Methods We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA+ subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit+/copGFP, Pdgfra+/eGFP and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation. Results KIT and PDGFRA were co-expressed in 3%–5% of mouse ICC, 35%−44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT+ETV1+PDGFRA+ GIST cells (half-maximal inhibitory concentration: IC50=5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (IC50>230 n

  17. Changes in c-Kit expression levels during the course of radiation therapy for nasopharyngeal carcinoma

    PubMed Central

    Jiang, Feng; Hu, Wei; Zhang, Bicheng; Xu, Jing; Shui, Yongjie; Zhou, Xiaofeng; Ren, Xiaoqiu; Chen, Xiaozhong; Shen, Li; Wei, Qichun

    2016-01-01

    In the era of intensity-modulated radiotherapy, distant metastasis is currently the main cause of treatment failure for nasopharyngeal carcinoma (NPC). Additional therapeutic strategies are required to control the metastasis and improve survival. One strategy is targeted therapy, for example against c-Kit. In the current study, the frequency of c-Kit expression was determined immunohistochemically in 106 NPC patients. c-Kit expression changes during the course of radiation therapy were detected in 41 cases via weekly biopsy. Twelve cases (11.3%) had c-Kit expression scores of 3+ and 16 (15.1%) had scores of 2+. Thus, c-Kit overexpression (2+ or 3+) was observed in 28 (26.4%) patients. There were 35 (33.0%) and 43 (40.6%) patients with c-Kit expression scores of 1+ and 0, respectively. Furthermore, a trend of decreased c-Kit expression was observed after commencing radiotherapy according to the 41 NPC patients who were biopsied weekly. Therefore, c-Kit overexpression was identified to be common in NPC, and evaluating c-Kit as a therapeutic target for metastatic NPC via c-Kit overexpression subsequent to first line treatment may be of interest. To the best of our knowledge, the present study is the first to demonstrate a trend of decreased c-Kit expression during the course of radiotherapy.

  18. Targets for AD treatment: conflicting messages from γ-secretase inhibitors

    PubMed Central

    Sambamurti, Kumar; Greig, Nigel H.; Utsuki, Tadanobu; Barnwell, Eliza L.; Sharma, Ekta; Mazell, Cheryl; Bhat, Narayan R.; Kindy, Mark S.; Lahiri, Debomoy K.; Pappolla, Miguel A

    2011-01-01

    Current evidence suggests that Alzheimer’s disease (AD) is a multi-factorial disease that starts with accumulation of multiple proteins. We have previously proposed that inhibition of γ-secretase may impair membrane recycling causing neurodegeneration starting at synapses (Sambamurti et al., 2006). We also proposed familal AD (FAD) mutations increase Aβ42 by inhibiting γ-secretase. Herein, we discuss the failure of Eli Lilly’s γ-secretase inhibitor, semagacestat, in clinical trials in the light of our hypothesis, which extends the problem beyond toxicity of Aβ aggregates. We elaborate that γ-secretase inhibitors lead to accumulation of amyloid precursor protein (APP) C-terminal fragments (CTFs) that can later be processed by γ-secretase to yields bursts of Aβ to facilitate aggregation. Although we do not exclude a role for toxic Aβ aggregates, inhibition of γ-secretase can affect numerous substrates other than APP to affect multiple pathways and the combined accumulation of multiple peptides in the membrane may impair its function and turnover. Taken together, protein processing and turnover pathways play an important role in maintaining cellular homeostasis and unless we clearly see consistent disease-related increase in their levels or activity, we need to focus on preserving their function rather than inhibiting them for treatment of AD and similar diseases. PMID:21320126

  19. Restriction endonuclease inhibitor IPI* of bacteriophage T4: a novel structure for a dedicated target.

    PubMed

    Rifat, Dalin; Wright, Nathan T; Varney, Kristen M; Weber, David J; Black, Lindsay W

    2008-01-18

    Phage T4 protects its DNA from the two-gene-encoded gmrS/gmrD (glucose-modified hydroxymethylcytosine restriction endonuclease) CT of pathogenic Escherichia coli, CT596, by injecting several hundred copies of the 76-amino-acid-residue nuclease inhibitor, IPI*, into the infected host. Here, the three-dimensional solution structure of mature IPI* is reported as determined by nuclear magnetic resonance techniques using 1290 experimental nuclear Overhauser effect and dipolar coupling constraints ( approximately 17 constraints per residue). Close examination of this oblate-shaped protein structure reveals a novel fold consisting of two small beta-sheets (beta1: B1 and B2; beta2: B3-B5) flanked at the N- and C-termini by alpha-helices (H1 and H2). Such a fold is very compact in shape and allows ejection of IPI* through the narrow 30-A portal and tail tube apertures of the virion without unfolding. Structural and dynamic measurements identify an exposed hydrophobic knob that is a putative gmrS/gmrD-binding site. A single gene from the uropathogenic E. coli UT189, which codes for a gmrS/gmrD-like UT fusion enzyme (with approximately 90% identity to the heterodimeric CT enzyme), has evolved IPI* inhibitor immunity. Analysis of the gmrS/gmrD restriction endonuclease enzyme family and its IPI* family phage antagonists reveals an evolutionary pathway that has elaborated a surprisingly diverse and specifically fitted set of coevolving attack and defense structures.

  20. International Literacy Day Tool Kit.

    ERIC Educational Resources Information Center

    2002

    This tool kit suggests various International Literacy Day activities to raise awareness of the issues of adult literacy and language learning, to connect local literacy programs with national programs, and to help achieve the National Literacy Summit goal by 2010. The kit is intended for individuals, programs, and organizations that want to call…

  1. Look Around You, Kit I.

    ERIC Educational Resources Information Center

    1971

    Developing pupils' awareness of their environment, learning to distinguish between what is pleasant and unpleasant, and examining acts of man to determine which are destructive and which are in harmony with nature are the purposes of Scholastic's Earth Corps Environmental Study Kits for grades 1-6. This kit is designed to help the child develop…

  2. Cosmic Light EDU kit

    NASA Astrophysics Data System (ADS)

    Doran, Rosa

    2015-08-01

    In 2015 we celebrate the International Year of Light, a great opportunity to promote awareness about the importance of light coming from the Cosmos and what messages it is bringing to mankind. In parallel a unique moment to attract the attention of stakeholders on the dangers of light pollution and its impact in our lives and our pursuit of more knowledge. In this presentation I want to present one of the conrnerstones of IYL2015, a partnership between the Galileo Teacher Training Program, Universe Awareness and Globe at Night, the Cosmic Light EDU kit. The aim of this project is to assemble a core set of tools and resources representing our basic knowledge pilars about the Universe and simple means to preserve our night sky.

  3. 14 CFR Appendix A to Part 121 - First Aid Kits and Emergency Medical Kits

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false First Aid Kits and Emergency Medical Kits A... REQUIREMENTS: DOMESTIC, FLAG, AND SUPPLEMENTAL OPERATIONS Pt. 121, App. A Appendix A to Part 121—First Aid Kits and Emergency Medical Kits Approved first-aid kits, at least one approved emergency medical kit,...

  4. A Novel Dimeric Inhibitor Targeting Beta2GPI in Beta2GPI/Antibody Complexes Implicated in Antiphospholipid Syndrome

    SciTech Connect

    A Kolyada; C Lee; A De Biasio; N Beglova

    2011-12-31

    {beta}2GPI is a major antigen for autoantibodies associated with antiphospholipid syndrome (APS), an autoimmune disease characterized by thrombosis and recurrent pregnancy loss. Only the dimeric form of {beta}2GPI generated by anti-{beta}2GPI antibodies is pathologically important, in contrast to monomeric {beta}2GPI which is abundant in plasma. We created a dimeric inhibitor, A1-A1, to selectively target {beta}2GPI in {beta}2GPI/antibody complexes. To make this inhibitor, we isolated the first ligand-binding module from ApoER2 (A1) and connected two A1 modules with a flexible linker. A1-A1 interferes with two pathologically important interactions in APS, the binding of {beta}2GPI/antibody complexes with anionic phospholipids and ApoER2. We compared the efficiency of A1-A1 to monomeric A1 for inhibition of the binding of {beta}2GPI/antibody complexes to anionic phospholipids. We tested the inhibition of {beta}2GPI present in human serum, {beta}2GPI purified from human plasma and the individual domain V of {beta}2GPI. We demonstrated that when {beta}2GPI/antibody complexes are formed, A1-A1 is much more effective than A1 in inhibition of the binding of {beta}2GPI to cardiolipin, regardless of the source of {beta}2GPI. Similarly, A1-A1 strongly inhibits the binding of dimerized domain V of {beta}2GPI to cardiolipin compared to the monomeric A1 inhibitor. In the absence of anti-{beta}2GPI antibodies, both A1-A1 and A1 only weakly inhibit the binding of pathologically inactive monomeric {beta}2GPI to cardiolipin. Our results suggest that the approach of using a dimeric inhibitor to block {beta}2GPI in the pathological multivalent {beta}2GPI/antibody complexes holds significant promise. The novel inhibitor A1-A1 may be a starting point in the development of an effective therapeutic for antiphospholipid syndrome.

  5. Synthetic and Crystallographic Studies of a New Inhibitor Series Targeting Bacillus anthracis Dihydrofolate Reductase

    PubMed Central

    Beierlein, Jennifer M.; Frey, Kathleen M.; Bolstad, David B.; Pelphrey, Phillip M.; Joska, Tammy M.; Smith, Adrienne E.; Priestley, Nigel D.; Wright, Dennis L.; Anderson, Amy C.

    2008-01-01

    Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structure of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 Å resolution. The structure reveals several features that can be exploited for further development of this lead series. PMID:19007108

  6. Angiotensin receptor neprilysin inhibitor LCZ696: a novel targeted therapy for arterial hypertension?

    PubMed

    Katsi, Vasiliki; Skalis, Georgios; Pavlidis, Antonis N; Makris, Thomas; Nihoyannopoulos, Petros; Tousoulis, Dimitris; Kallikazaros, Ioannis

    2015-10-01

    The need for novel antihypertensive therapies represents a continuous challenge. LCZ696 is a first-in-class angiotensin receptor neprilysin inhibitor that has been shown to enhance endogenous natriuretic peptide (NP) actions on neurohormonal activation. This effect seems to be additive to that of the renin-angiotensin-aldosterone system (RAAS) suppression, as impressively suggested in the PARADIGM HF study. LCZ696 has been shown to be effective in reducing blood pressure in several small studies; however, its effectiveness and safety remain to be proved in larger studies. This review summarizes the role of RAAS and NP system in the pathophysiology of hypertension and reviews the current data on the antihypertensive effects of LCZ696. PMID:27532450

  7. Development of dual targeting inhibitors against aggregations of amyloid-β and tau protein.

    PubMed

    Fuse, Shinichiro; Matsumura, Keisuke; Fujita, Yuki; Sugimoto, Hachiro; Takahashi, Takashi

    2014-10-01

    Aggregations of both amyloid-β (Aβ) and hyper-phosphorylated tau proteins are recognized as key pathological manifestations of Alzheimer's disease (AD). Agents that inhibit both those forms of aggregation show promise as drug candidates. Seventeen oligo heteroaromatic compounds were rapidly synthesized via a one-pot, 3- or 4-component coupling procedure. Evaluations showed that compounds E16 and E18 were the most potent inhibitors of Aβ and tau aggregations (E16: IC50s = 0.38, 0.29 μM against Aβ, tau, respectively, E18: IC50s = 0.55, 0.30 μM against Aβ, tau, respectively).

  8. Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors

    PubMed Central

    Qvit, Nir; Schechtman, Deborah; Pena, Darlene Aparecida; Berti, Denise Aparecida; Soares, Chrislaine Oliveira; Miao, Qianqian; Liang, Liying (Annie); Baron, Lauren A.; Teh-Poot, Christian; Martínez-Vega, Pedro; Ramirez-Sierra, Maria Jesus; Churchill, Eric; Cunningham, Anna D.; Malkovskiy, Andrey V.; Federspiel, Nancy A.; Gozzo, Fabio Cesar; Torrecilhas, Ana Claudia; Manso Alves, Maria Julia; Jardim, Armando; Momar, Ndao; Dumonteil, Eric; Mochly-Rosen, Daria

    2016-01-01

    Parasitic diseases cause ∼500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase) and TRACK (Trypanosomareceptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs. PMID:27054066

  9. Synthetic and Crystallographic Studies of a New Inhibitor Series Targeting Bacillus anthracis Dihydrofolate Reductase

    SciTech Connect

    Beierlein, J.; Frey, K; Bolstad, D; Pelphrey, P; Joska, T; Smith, A; Priestley, N; Wright, D; Anderson, A

    2008-01-01

    Bacillus anthracis, the causative agent of anthrax, poses a significant biodefense danger. Serious limitations in approved therapeutics and the generation of resistance have produced a compelling need for new therapeutic agents against this organism. Bacillus anthracis is known to be insensitive to the clinically used antifolate, trimethoprim, because of a lack of potency against the dihydrofolate reductase enzyme. Herein, we describe a novel lead series of B. anthracis dihydrofolate reductase inhibitors characterized by an extended trimethoprim-like scaffold. The best lead compound adds only 22 Da to the molecular weight and is 82-fold more potent than trimethoprim. An X-ray crystal structure of this lead compound bound to B. anthracis dihydrofolate reductase in the presence of NADPH was determined to 2.25 A resolution. The structure reveals several features that can be exploited for further development of this lead series.

  10. A Targeted Inhibitor of the Alternative Complement Pathway Accelerates Recovery From Smoke-Induced Ocular Injury

    PubMed Central

    Woodell, Alex; Jones, Bryan W.; Williamson, Tucker; Schnabolk, Gloriane; Tomlinson, Stephen; Atkinson, Carl; Rohrer, Bärbel

    2016-01-01

    Purpose Morphologic and genetic evidence exists that an overactive complement system driven by the complement alternative pathway (AP) is involved in pathogenesis of age-related macular degeneration (AMD). Smoking is the only modifiable risk factor for AMD. As we have shown that smoke-related ocular pathology can be prevented in mice that lack an essential activator of AP, we ask here whether this pathology can be reversed by increasing inhibition in AP. Methods Mice were exposed to either cigarette smoke (CS) or filtered air (6 hours/day, 5 days/week, 6 months). Smoke-exposed animals were then treated with the AP inhibitor (CR2-fH) or vehicle control (PBS) for the following 3 months. Spatial frequency and contrast sensitivity were assessed by optokinetic response paradigms at 6 and 9 months; additional readouts included assessment of retinal morphology by electron microscopy (EM) and gene expression analysis by quantitative RT-PCR. Results The CS mice treated with CR2-fH showed significant improvement in contrast threshold compared to PBS-treated mice, whereas spatial frequency was unaffected by CS or pharmacologic intervention. Treatment with CR2-fH in CS animals reversed thinning of the retina observed in PBS-treated mice as analyzed by spectral-domain optical coherence tomography, and reversed most morphologic changes in RPE and Bruch's membrane seen in CS animals by EM. Conclusions Taken together, these findings suggest that AP inhibitors not only prevent, but have the potential to accelerate the clearance of complement-mediated ocular injury. Improving our understanding of the regulation of the AP is paramount to developing novel treatment approaches for AMD. PMID:27064393

  11. Discovery of Potent VEGFR-2 Inhibitors based on Furopyrimidine and Thienopyrimidne Scaffolds as Cancer Targeting Agents

    PubMed Central

    Aziz, Marwa A.; Serya, Rabah A. T.; Lasheen, Deena S.; Abdel-Aziz, Amal Kamal; Esmat, Ahmed; Mansour, Ahmed M.; Singab, Abdel Nasser B.; Abouzid, Khaled A. M.

    2016-01-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In this study, a series of novel furo[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine based-derivatives were designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The synthesized compounds were evaluated for their ability to in vitro inhibit VEGFR-2 kinase enzyme. Seven compounds (15b, 16c, 16e, 21a, 21b, 21c and 21e) demonstrated highly potent dose-related VEGFR-2 inhibition with IC50 values in nanomolar range, of which the thieno[2,3-d]pyrimidine based-derivatives (21b, 21c and 21e) exhibited IC50 values of 33.4, 47.0 and 21 nM respectively. Moreover, furo[2,3-d]pyrimidine-based derivative (15b) showed the strongest inhibition of human umbilical vein endothelial cells (HUVEC) proliferation with 99.5% inhibition at 10 μM concentration. Consistent with our in vitro findings, compounds (21b and 21e) orally administered at 5 and 10 mg/kg/day for 8 consecutive days demonstrated potent anticancer activity in Erhlich ascites carcinoma (EAC) solid tumor murine model. Such compounds blunted angiogenesis in EAC as evidenced by reduced percent microvessel via decreasing VEGFR-2 phosphorylation with subsequent induction of apoptotic machinery. Furthermore, Miles vascular permeability assay confirmed their antiangiogenic effects in vivo. Intriguingly, such compounds showed no obvious toxicity. PMID:27080011

  12. Discovery of Potent VEGFR-2 Inhibitors based on Furopyrimidine and Thienopyrimidne Scaffolds as Cancer Targeting Agents

    NASA Astrophysics Data System (ADS)

    Aziz, Marwa A.; Serya, Rabah A. T.; Lasheen, Deena S.; Abdel-Aziz, Amal Kamal; Esmat, Ahmed; Mansour, Ahmed M.; Singab, Abdel Nasser B.; Abouzid, Khaled A. M.

    2016-04-01

    Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In this study, a series of novel furo[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine based-derivatives were designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The synthesized compounds were evaluated for their ability to in vitro inhibit VEGFR-2 kinase enzyme. Seven compounds (15b, 16c, 16e, 21a, 21b, 21c and 21e) demonstrated highly potent dose-related VEGFR-2 inhibition with IC50 values in nanomolar range, of which the thieno[2,3-d]pyrimidine based-derivatives (21b, 21c and 21e) exhibited IC50 values of 33.4, 47.0 and 21 nM respectively. Moreover, furo[2,3-d]pyrimidine-based derivative (15b) showed the strongest inhibition of human umbilical vein endothelial cells (HUVEC) proliferation with 99.5% inhibition at 10 μM concentration. Consistent with our in vitro findings, compounds (21b and 21e) orally administered at 5 and 10 mg/kg/day for 8 consecutive days demonstrated potent anticancer activity in Erhlich ascites carcinoma (EAC) solid tumor murine model. Such compounds blunted angiogenesis in EAC as evidenced by reduced percent microvessel via decreasing VEGFR-2 phosphorylation with subsequent induction of apoptotic machinery. Furthermore, Miles vascular permeability assay confirmed their antiangiogenic effects in vivo. Intriguingly, such compounds showed no obvious toxicity.

  13. Slowing down fat digestion and absorption by an oxadiazolone inhibitor targeting selectively gastric lipolysis.

    PubMed

    Point, Vanessa; Bénarouche, Anais; Zarrillo, Julie; Guy, Alexandre; Magnez, Romain; Fonseca, Laurence; Raux, Brigitt; Leclaire, Julien; Buono, Gérard; Fotiadu, Frédéric; Durand, Thierry; Carrière, Frédéric; Vaysse, Carole; Couëdelo, Leslie; Cavalier, Jean-François

    2016-11-10

    Based on a previous study and in silico molecular docking experiments, we have designed and synthesized a new series of ten 5-Alkoxy-N-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one derivatives (RmPPOX). These molecules were further evaluated as selective and potent inhibitors of mammalian digestive lipases: purified dog gastric lipase (DGL) and guinea pig pancreatic lipase related protein 2 (GPLRP2), as well as porcine (PPL) and human (HPL) pancreatic lipases contained in porcine pancreatic extracts (PPE) and human pancreatic juices (HPJ), respectively. These compounds were found to strongly discriminate classical pancreatic lipases (poorly inhibited) from gastric lipase (fully inhibited). Among them, the 5-(2-(Benzyloxy)ethoxy)-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one (BemPPOX) was identified as the most potent inhibitor of DGL, even more active than the FDA-approved drug Orlistat. BemPPOX and Orlistat were further compared in vitro in the course of test meal digestion, and in vivo with a mesenteric lymph duct cannulated rat model to evaluate their respective impacts on fat absorption. While Orlistat inhibited both gastric and duodenal lipolysis and drastically reduced fat absorption in rats, BemPPOX showed a specific action on gastric lipolysis that slowed down the overall lipolysis process and led to a subsequent reduction of around 55% of the intestinal absorption of fatty acids compared to controls. All these data promote BemPPOX as a potent candidate to efficiently regulate the gastrointestinal lipolysis, and to investigate its link with satiety mechanisms and therefore develop new strategies to "fight against obesity".

  14. Slowing down fat digestion and absorption by an oxadiazolone inhibitor targeting selectively gastric lipolysis.

    PubMed

    Point, Vanessa; Bénarouche, Anais; Zarrillo, Julie; Guy, Alexandre; Magnez, Romain; Fonseca, Laurence; Raux, Brigitt; Leclaire, Julien; Buono, Gérard; Fotiadu, Frédéric; Durand, Thierry; Carrière, Frédéric; Vaysse, Carole; Couëdelo, Leslie; Cavalier, Jean-François

    2016-11-10

    Based on a previous study and in silico molecular docking experiments, we have designed and synthesized a new series of ten 5-Alkoxy-N-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one derivatives (RmPPOX). These molecules were further evaluated as selective and potent inhibitors of mammalian digestive lipases: purified dog gastric lipase (DGL) and guinea pig pancreatic lipase related protein 2 (GPLRP2), as well as porcine (PPL) and human (HPL) pancreatic lipases contained in porcine pancreatic extracts (PPE) and human pancreatic juices (HPJ), respectively. These compounds were found to strongly discriminate classical pancreatic lipases (poorly inhibited) from gastric lipase (fully inhibited). Among them, the 5-(2-(Benzyloxy)ethoxy)-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one (BemPPOX) was identified as the most potent inhibitor of DGL, even more active than the FDA-approved drug Orlistat. BemPPOX and Orlistat were further compared in vitro in the course of test meal digestion, and in vivo with a mesenteric lymph duct cannulated rat model to evaluate their respective impacts on fat absorption. While Orlistat inhibited both gastric and duodenal lipolysis and drastically reduced fat absorption in rats, BemPPOX showed a specific action on gastric lipolysis that slowed down the overall lipolysis process and led to a subsequent reduction of around 55% of the intestinal absorption of fatty acids compared to controls. All these data promote BemPPOX as a potent candidate to efficiently regulate the gastrointestinal lipolysis, and to investigate its link with satiety mechanisms and therefore develop new strategies to "fight against obesity". PMID:27543878

  15. Design of novel hypoxia-targeting IDO hybrid inhibitors conjugated with an unsubstituted L-TRP as an IDO affinity moiety.

    PubMed

    Nakashima, Hitomi; Ikkyu, Kazuhiro; Nakashima, Kouichiro; Sano, Keiichiro; Uto, Yoshihiro; Nakata, Eiji; Nagasawa, Hideko; Sugimoto, Hiroshi; Shiro, Yoshitsugu; Nakagawa, Yoshinori; Hori, Hitoshi

    2010-01-01

    We presented here design, syntheses and inhibitory activities of novel hypoxia-targeting IDO hybrid inhibitors conjugated with an unsubstituted L-Trp as an IDO affinity moiety without inhibitor 1MT, such as L-Trp-TPZ hybrids 1 (TX-2274), 2 (UTX-3), 3 (UTX-4), and 4 (UTX-2). TPZ-monoxide hybrids 1 and 3 were good competitive IDO inhibitors, while TPZ hybrids 2 and 4 were uncompetitive IDO inhibitors. Among them TPZ-monoxide hybrid 1 have the strongest IDO inhibitory activity. It suggests that TPZ-monoxide hybrids 1 and 3 are able to bind the active site of IDO, TPZ hybrids 2 and 4 are able to bind the enzyme-substrate complex. We proposed the possible mechanism of action of TPZ hybrid 2 that may first affect as a hypoxic cytotoxin, and then metabolized to TPZ-monoxide hybrid 1, which may do as an IDO inhibitor more effectively than its parent TPZ hybrid 2.

  16. A phosphorescent rhenium(I) histone deacetylase inhibitor: mitochondrial targeting and paraptosis induction.

    PubMed

    Ye, Rui-Rong; Tan, Cai-Ping; Lin, Yan-Nan; Ji, Liang-Nian; Mao, Zong-Wan

    2015-05-14

    In this report, we designed a histone deacetylase-targeted phosphorescent Re(I) complex ReLMito. Colocalization studies suggested that ReLMito could specially localize to mitochondria. We also demonstrated that ReLMito could induce paraptosis in cancer cells. These features endowed the complex with potential to induce and monitor mitochondrial morphological changes during the paraptosis simultaneously.

  17. A novel protocol to accelerate dynamic combinatorial chemistry via isolation of ligand-target adducts from dynamic combinatorial libraries: a case study identifying competitive inhibitors of lysozyme.

    PubMed

    Fang, Zheng; He, Wei; Li, Xin; Li, Zhengjiang; Chen, Beining; Ouyang, Pingkai; Guo, Kai

    2013-09-15

    A novel protocol based on size-exclusion chromatography (SEC) and MS was established to accelerate dynamic combinatorial chemistry (DCC) in this study. By isolating ligand-target adducts from the dynamic combinatorial library (DCL), ligands could be identified directly by MS after denaturation. Three new inhibitors for lysozyme were discovered by this SEC-MS protocol in a case study. Km Data for these new inhibitors was also determined.

  18. 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

  19. Small-Molecule Inhibitors of the Pseudaminic Acid Biosynthetic Pathway: Targeting Motility as a Key Bacterial Virulence Factor

    PubMed Central

    Ménard, Robert; Schoenhofen, Ian C.; Tao, Limei; Aubry, Annie; Bouchard, Patrice; Reid, Christopher W.; Lachance, Paule; Twine, Susan M.; Fulton, Kelly M.; Cui, Qizhi; Hogues, Hervé; Purisima, Enrico O.

    2014-01-01

    Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 μM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 μM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 μM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella. PMID:25267679

  20. Structure-Based Identification of a Potent Inhibitor Targeting Stp1-Mediated Virulence Regulation in Staphylococcus aureus.

    PubMed

    Zheng, Weihao; Cai, Xiaodan; Xie, Mingsheng; Liang, Yujie; Wang, Tao; Li, Zigang

    2016-08-18

    The increasing threats of antibiotic resistance urge the need for developing new strategies against bacterial infections. Targeting eukaryotic-like Ser/Thr phosphatase Stp1-mediated virulence regulation represents a promising approach for combating staphylococcal infection yet to be explored. Here, we report the 2.32-Å resolution crystal structure of Stp1. Stp1 binds an unexpected fourth metal ion, which is important for Stp1's enzymatic activity as demonstrated by amino acid substitution studies. Inspired by the structural details of Stp1, we identified a potent and selective Stp1 inhibitor, aurintricarboxylic acid (ATA). Transcriptome analysis and biochemical studies supported Stp1 as the target of ATA inhibition within the pathogen, preventing upregulation of virulence genes. Notably, ATA did not affect in vitro growth of Staphylococcus aureus, while simultaneously attenuating staphylococcal virulence in mice. Our findings demonstrate that ATA is a potent anti-virulence compound against staphylococcal infection, laying the foundation for further developing new scaffolds for Stp1-targeted small molecules. PMID:27499528

  1. A novel angiogenesis inhibitor impairs lovo cell survival via targeting against human VEGFR and its signaling pathway of phosphorylation.

    PubMed

    Zhang, Y M; Dai, B L; Zheng, L; Zhan, Y Z; Zhang, J; Smith, W W; Wang, X L; Chen, Y N; He, L C

    2012-10-11

    Colorectal cancer represents the fourth commonest malignancy, and constitutes a major cause of significant morbidity and mortality among other diseases. However, the chemical therapy is still under development. Angiogenesis plays an important role in colon cancer development. We developed HMQ18-22 (a novel analog of taspine) with the aim to target angiogenesis. We found that HMQ18-22 significantly reduced angiogenesis of chicken chorioallantoic membrane (CAM) and mouse colon tissue, and inhibited cell migration and tube formation as well. Then, we verified the interaction between HMQ18-22 and VEGFR2 by AlphaScreen P-VEGFR assay, screened the targets on angiogenesis by VEGF Phospho Antibody Array, validated the target by western blot and RNAi in lovo cells. We found HMQ18-22 could decrease phosphorylation of VEGFR2(Tyr(1214)), VEGFR1(Tyr(1333)), Akt(Tyr(326)), protein kinase Cα (PKCα) (Tyr(657)) and phospholipase-Cγ-1 (PLCγ-1) (Tyr(771)). Most importantly, HMQ18-22 inhibited proliferation of lovo cell and tumor growth in a human colon tumor xenografted model of athymic mice. Compared with normal lovo cells proliferation, the inhibition on proliferation of knockdown cells (VEGFR2, VEGFR1, Akt, PKCα and PLCγ-1) by HMQ18-22 decreased. These results suggested that HMQ18-22 is a novel angiogenesis inhibitor and can be a useful therapeutic candidate for colon cancer intervention.

  2. Chemoproteomics Reveals Novel Protein and Lipid Kinase Targets of Clinical CDK4/6 Inhibitors in Lung Cancer.

    PubMed

    Sumi, Natalia J; Kuenzi, Brent M; Knezevic, Claire E; Remsing Rix, Lily L; Rix, Uwe

    2015-12-18

    Several selective CDK4/6 inhibitors are in clinical trials for non-small cell lung cancer (NSCLC). Palbociclib (PD0332991) is included in the phase II/III Lung-MAP trial for squamous cell lung carcinoma (LUSQ). We noted differential cellular activity between palbociclib and the structurally related ribociclib (LEE011) in LUSQ cells. Applying an unbiased mass spectrometry-based chemoproteomics approach in H157 cells and primary tumor samples, we here report distinct proteome-wide target profiles of these two drug candidates in LUSQ, which encompass novel protein and, for palbociclib only, lipid kinases. In addition to CDK4 and 6, we observed CDK9 as a potent target of both drugs. Palbociclib interacted with several kinases not targeted by ribociclib, such as casein kinase 2 and PIK3R4, which regulate autophagy. Furthermore, palbociclib engaged several lipid kinases, most notably, PIK3CD and PIP4K2A/B/C. Accordingly, we observed modulation of autophagy and inhibition of AKT signaling by palbociclib but not ribociclib.

  3. A Java commodity grid kit.

    SciTech Connect

    von Laszewski, G.; Foster, I.; Gawor, J.; Lane, P.; Mathematics and Computer Science

    2001-07-01

    In this paper we report on the features of the Java Commodity Grid Kit. The Java CoG Kit provides middleware for accessing Grid functionality from the Java framework. Java CoG Kit middleware is general enough to design a variety of advanced Grid applications with quite different user requirements. Access to the Grid is established via Globus protocols, allowing the Java CoG Kit to communicate also with the C Globus reference implementation. Thus, the Java CoG Kit provides Grid developers with the ability to utilize the Grid, as well as numerous additional libraries and frameworks developed by the Java community to enable network, Internet, enterprise, and peer-to peer computing. A variety of projects have successfully used the client libraries of the Java CoG Kit to access Grids driven by the C Globus software. In this paper we also report on the efforts to develop server side Java CoG Kit components. As part of this research we have implemented a prototype pure Java resource management system that enables one to run Globus jobs on platforms on which a Java virtual machine is supported, including Windows NT machines.

  4. Optimizing Medical Kits for Spaceflight

    NASA Technical Reports Server (NTRS)

    Keenan, A. B,; Foy, Millennia; Myers, G.

    2014-01-01

    The Integrated Medical Model (IMM) is a probabilistic model that estimates medical event occurrences and mission outcomes for different mission profiles. IMM simulation outcomes describing the impact of medical events on the mission may be used to optimize the allocation of resources in medical kits. Efficient allocation of medical resources, subject to certain mass and volume constraints, is crucial to ensuring the best outcomes of in-flight medical events. We implement a new approach to this medical kit optimization problem. METHODS We frame medical kit optimization as a modified knapsack problem and implement an algorithm utilizing a dynamic programming technique. Using this algorithm, optimized medical kits were generated for 3 different mission scenarios with the goal of minimizing the probability of evacuation and maximizing the Crew Health Index (CHI) for each mission subject to mass and volume constraints. Simulation outcomes using these kits were also compared to outcomes using kits optimized..RESULTS The optimized medical kits generated by the algorithm described here resulted in predicted mission outcomes more closely approached the unlimited-resource scenario for Crew Health Index (CHI) than the implementation in under all optimization priorities. Furthermore, the approach described here improves upon in reducing evacuation when the optimization priority is minimizing the probability of evacuation. CONCLUSIONS This algorithm provides an efficient, effective means to objectively allocate medical resources for spaceflight missions using the Integrated Medical Model.

  5. A Novel Assay for Screening Inhibitors Targeting HIV Integrase LEDGF/p75 Interaction Based on Ni2+ Coated Magnetic Agarose Beads

    PubMed Central

    Dawei, Zhang; Hongqiu, He; Mengmeng, Liu; Zhixia, Meng; Shunxing, Guo

    2016-01-01

    HIV-1 integrase (IN) plays an essential role in viral replication and thus serves as an important target for chemotherapeutic intervention against HIV-1 infection. However, the current three clinical IN inhibitors, raltegravir, elvitegravir and dolutegravir share the same inhibitory mechanism, resulting in a common clinical resistance profile which have emerged in infected patients receiving treatment. Therefore, it is important to develop small molecule inhibitors that impair IN function with distinct mechanisms of action. In this work, a magnetic-beads based biochemical assay targeting the protein-protein interaction (PPI) between HIV IN and the cellular cofactor LEDGF/p75 was developed for identification of HIV-1 IN inhibitors. Furthermore, a library containing 1000 US. Food and Drug Administration (FDA)-approved drugs currently used for human medication was screened to identify inhibitors targeting the PPI. The assay was proved to be quite robust and with the novel assay we successfully identified dexlansoprazole (IC50 of 4.8 μM), a FDA-approved proton pump inhibitor, as a potential inhibitor for the PPI between IN and LEDGF/p75, which bound to the LEDGF/p75 partner with a kinetic dissociation (Kd) constant of 330 nM ± 2.6 nM. PMID:27633629

  6. A Novel Assay for Screening Inhibitors Targeting HIV Integrase LEDGF/p75 Interaction Based on Ni(2+) Coated Magnetic Agarose Beads.

    PubMed

    Dawei, Zhang; Hongqiu, He; Mengmeng, Liu; Zhixia, Meng; Shunxing, Guo

    2016-01-01

    HIV-1 integrase (IN) plays an essential role in viral replication and thus serves as an important target for chemotherapeutic intervention against HIV-1 infection. However, the current three clinical IN inhibitors, raltegravir, elvitegravir and dolutegravir share the same inhibitory mechanism, resulting in a common clinical resistance profile which have emerged in infected patients receiving treatment. Therefore, it is important to develop small molecule inhibitors that impair IN function with distinct mechanisms of action. In this work, a magnetic-beads based biochemical assay targeting the protein-protein interaction (PPI) between HIV IN and the cellular cofactor LEDGF/p75 was developed for identification of HIV-1 IN inhibitors. Furthermore, a library containing 1000 US. Food and Drug Administration (FDA)-approved drugs currently used for human medication was screened to identify inhibitors targeting the PPI. The assay was proved to be quite robust and with the novel assay we successfully identified dexlansoprazole (IC50 of 4.8 μM), a FDA-approved proton pump inhibitor, as a potential inhibitor for the PPI between IN and LEDGF/p75, which bound to the LEDGF/p75 partner with a kinetic dissociation (Kd) constant of 330 nM ± 2.6 nM. PMID:27633629

  7. A Novel Assay for Screening Inhibitors Targeting HIV Integrase LEDGF/p75 Interaction Based on Ni(2+) Coated Magnetic Agarose Beads.

    PubMed

    Dawei, Zhang; Hongqiu, He; Mengmeng, Liu; Zhixia, Meng; Shunxing, Guo

    2016-09-16

    HIV-1 integrase (IN) plays an essential role in viral replication and thus serves as an important target for chemotherapeutic intervention against HIV-1 infection. However, the current three clinical IN inhibitors, raltegravir, elvitegravir and dolutegravir share the same inhibitory mechanism, resulting in a common clinical resistance profile which have emerged in infected patients receiving treatment. Therefore, it is important to develop small molecule inhibitors that impair IN function with distinct mechanisms of action. In this work, a magnetic-beads based biochemical assay targeting the protein-protein interaction (PPI) between HIV IN and the cellular cofactor LEDGF/p75 was developed for identification of HIV-1 IN inhibitors. Furthermore, a library containing 1000 US. Food and Drug Administration (FDA)-approved drugs currently used for human medication was screened to identify inhibitors targeting the PPI. The assay was proved to be quite robust and with the novel assay we successfully identified dexlansoprazole (IC50 of 4.8 μM), a FDA-approved proton pump inhibitor, as a potential inhibitor for the PPI between IN and LEDGF/p75, which bound to the LEDGF/p75 partner with a kinetic dissociation (Kd) constant of 330 nM ± 2.6 nM.

  8. Intra-airway administration of small interfering RNA targeting plasminogen activator inhibitor-1 attenuates allergic asthma in mice.

    PubMed

    Miyamoto, Shintaro; Hattori, Noboru; Senoo, Tadashi; Onari, Yojiro; Iwamoto, Hiroshi; Kanehara, Masashi; Ishikawa, Nobuhisa; Fujitaka, Kazunori; Haruta, Yoshinori; Murai, Hiroshi; Yokoyama, Akihito; Kohno, Nobuoki

    2011-12-01

    Recent studies suggest that plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of the fibrinolytic system, may promote the development of asthma. To further investigate the significance of PAI-1 in the pathogenesis of asthma and determine the possibility that PAI-1 could be a therapeutic target for asthma, this study was conducted. First, PAI-1 levels in induced sputum (IS) from asthmatic subjects and healthy controls were measured. In asthmatic subjects, IS PAI-1 levels were elevated, compared with that of healthy controls, and were significantly higher in patients with long-duration asthma compared with short-duration asthma. PAI-1 levels were also found to correlate with IS transforming growth factor-β levels. Then, acute and chronic asthma models induced by ovalbumin were established in PAI-1-deficient mice and wild-type mice that received intra-airway administrations of small interfering RNA against PAI-1 (PAI-1-siRNA). We could demonstrate that eosinophilic airway inflammation and airway hyperresponsiveness were reduced in an acute asthma model, and airway remodeling was suppressed in a chronic asthma model in both PAI-1-deficient mice and wild-type mice that received intra-airway administration of PAI-1-siRNA. These results indicate that PAI-1 is strongly involved in the pathogenesis of asthma, and intra-airway administration of PAI-1-siRNA may be able to become a new therapeutic approach for asthma.

  9. Overcoming inherent resistance to histone deacetylase inhibitors in multiple myeloma cells by targeting pathways integral to the actin cytoskeleton

    PubMed Central

    Mithraprabhu, S; Khong, T; Spencer, A

    2014-01-01

    Histone deacetylase inhibitors (HDACi) are novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with multiple myeloma (MM). Although HDACi have demonstrable synergy when combined with proteasome inhibitors (PIs), recent evidence indicates that combination of HDACi and PI is beneficial only in a subset of patients with advanced MM, clearly indicating that other rational combinations should be explored. In this context we hypothesized that understanding the molecular signature associated with inherent resistance to HDACi would provide a basis for the identification of therapeutic combinations with improved clinical efficacy. Using human myeloma cell lines (HMCL) categorized as sensitive, intermediate or resistant to HDACi, gene expression profiling (GEP) and gene ontology enrichment analyses were performed to determine if a genetic signature associated with inherent resistance to HDACi-resistance could be identified. Correlation of GEP to increasing or decreasing sensitivity to HDACi indicated a unique 35-gene signature that was significantly enriched for two pathways – regulation of actin cytoskeleton and protein processing in endoplasmic reticulum. When HMCL and primary MM samples were treated with a combination of HDACi and agents targeting the signaling pathways integral to the actin cytoskeleton, synergistic cell death was observed in all instances, thus providing a rationale for combining these agents with HDACi for the treatment of MM to overcome resistance. This report validates a molecular approach for the identification of HDACi partner drugs and provides an experimental framework for the identification of novel therapeutic combinations for anti-MM treatment. PMID:24651437

  10. KDM4B as a Target for Prostate Cancer: Structural Analysis and Selective Inhibition by a Novel Inhibitor

    PubMed Central

    2015-01-01

    The KDM4/JMJD2 Jumonji C-containing histone lysine demethylases (KDM4A–KDM4D), which selectively remove the methyl group(s) from tri/dimethylated lysine 9/36 of H3, modulate transcriptional activation and genome stability. The overexpression of KDM4A/KDM4B in prostate cancer and their association with androgen receptor suggest that KDM4A/KDM4B are potential progression factors for prostate cancer. Here, we report the crystal structure of the KDM4B·pyridine 2,4-dicarboxylic acid·H3K9me3 ternary complex, revealing the core active-site region and a selective K9/K36 site. A selective KDM4A/KDM4B inhibitor, 4, that occupies three subsites in the binding pocket is identified by virtual screening. Pharmacological and genetic inhibition of KDM4A/KDM4B significantly blocks the viability of cultured prostate cancer cells, which is accompanied by increased H3K9me3 staining and transcriptional silencing of growth-related genes. Significantly, a substantial portion of differentially expressed genes are AR-responsive, consistent with the roles of KDM4s as critical AR activators. Our results point to KDM4 as a useful therapeutic target and identify a new inhibitor scaffold. PMID:24971742

  11. BET inhibitor OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cells

    PubMed Central

    Berrou, Jeannig; Dupont, Mélanie; Bertrand, Sibyl; Masse, Aline; Raffoux, Emmanuel; Itzykson, Raphaël; Delord, Marc; Riveiro, Maria E.; Herait, Patrice; Baruchel, André; Dombret, Hervé; Gardin, Claude

    2015-01-01

    The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients. PMID:25989842

  12. Novel Therapeutic Targets for Phosphodiesterase 5 Inhibitors: current state-of-the-art on systemic arterial hypertension and atherosclerosis.

    PubMed

    Vasquez, Elisardo C; Gava, Agata L; Graceli, Jones B; Balarini, Camille M; Campagnaro, Bianca P; Pereira, Thiago Melo C; Meyrelles, Silvana S

    2016-01-01

    The usefulness of selective inhibitors of phosphodiesterase 5 (PDE5) is well known, first for the treatment of male erectile dysfunction and more recently for pulmonary hypertension. The discovery that PDE5 is present in the systemic artery endothelium and smooth muscle cells led investigators to test the extra sexual effects of sildenafil, the first and most investigated PDE5 inhibitor, in diseases affecting the systemic arteries. Cumulative data from experimental and clinical studies have revealed beneficial effects of sildenafil on systemic arterial hypertension and its target organs, such as the heart, kidneys and vasculature. An important effect of sildenafil is reduction of hypertension and improvement of endothelial function in experimental models of hypertension and hypertensive subjects. Interestingly, in angiotensin-dependent hypertension, its beneficial effects on endothelial and kidney dysfunctions seem to at least in part be caused by its ability to decrease the levels of angiotensin II and increase angiotensin 1-7, in addition to improving nitric oxide bioavailability and diminishing reactive oxygen species. Another remarkable finding on the effects of sildenafil comes from studies in apolipoprotein E knockout mice, a model of atherosclerosis that closely resembles human atherosclerotic disease. In this review, we focus on the promising beneficial effects of sildenafil for treating systemic high blood pressure, especially resistant hypertension, and the endothelial dysfunction that is present in hypertension and atherosclerosis.

  13. Rational development of 4-aminopyridyl-based inhibitors targeting Trypanosoma cruzi CYP51 as anti-Chagas agents

    PubMed Central

    Choi, Jun Yong; Calvet, Claudia M.; Gunatilleke, Shamila S.; Ruiz, Claudia; Cameron, Michael D.; McKerrow, James H.; Podust, Larissa M.; Roush, William R.

    2013-01-01

    A new series of 4-aminopyridyl-based lead inhibitors targeting Trypanosoma cruzi CYP51 (TcCYP51) has been developed using structure-based drug design as well as structure-property relationship (SPR) analyses. The screening hit starting point, LP10 (KD ≤ 42 nM; EC50 of 0.65 µM), has been optimized to give the potential leads 14t, 27i, 27q, 27r, and 27t, that have low nanomolar binding affinity to TcCYP51 and significant activity against T. cruzi amastigotes cultured in human myoblasts (EC50 = 14–18 nM for 27i and 27r). Many of the optimized compounds have improved microsome stability, and most are selective against human CYPs 1A2, 2D6 and 3A4 (<50% inhibition at 1 µM). A rationale for the improvement of microsome stability and selectivity of inhibitors against human metabolic CYP enzymes is presented. In addition, the binding mode of 14t with the T. brucei CYP51 (TbCYP51) ortholog has been characterized by x-ray structure analysis. PMID:24079662

  14. Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

    PubMed Central

    Chang, Chien-Yi; Krishnan, Thiba; Wang, Hao; Chen, Ye; Yin, Wai-Fong; Chong, Yee-Meng; Tan, Li Ying; Chong, Teik Min; Chan, Kok-Gan

    2014-01-01

    N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. PMID:25430794

  15. Targeting angiogenesis with multitargeted tyrosine kinase inhibitors in the treatment of non-small cell lung cancer.

    PubMed

    Scagliotti, Giorgio; Govindan, Ramaswamy

    2010-01-01

    It has been >35 years since the link between angiogenesis and the growth of tumors was first reported. Targeting angiogenesis became feasible with the availability of bevacizumab, an anti-vascular endothelial growth factor monoclonal antibody. Initial studies revealed that the combination of bevacizumab and chemotherapy led to longer overall survival times than with chemotherapy alone in patients with advanced colorectal cancer. Since then, drug development strategies have added small molecule tyrosine kinase inhibitors to the panel of antiangiogenic agents under evaluation; data from numerous trials are now available. The challenge now is to identify the optimal antiangiogenic agent for specific patient groups and to understand not only the mechanistic differences between agents, but also the variability in their antitumor activity across different tumor types and their differing side-effect profiles. As in other solid tumors, angiogenesis contributes to the development of non-small cell lung cancer (NSCLC), and this review summarizes the role of angiogenesis in this disease. We review the current developmental status of antiangiogenic tyrosine kinase inhibitors (including vandetanib, sunitinib, axitinib, sorafenib, vatalanib, and pazopanib) in NSCLC and conclude by briefly discussing the need for optimal patient selection and potential future directions. PMID:20427383

  16. Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C

    PubMed Central

    Ulferts, Rachel; de Boer, S. Matthijn; van der Linden, Lonneke; Bauer, Lisa; Lyoo, Hey Rhyoung; Maté, Maria J.; Lichière, Julie; Canard, Bruno; Lelieveld, Daphne; Omta, Wienand; Egan, David; Coutard, Bruno

    2016-01-01

    Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired. PMID:26856848

  17. Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target.

    PubMed

    Chang, Chien-Yi; Krishnan, Thiba; Wang, Hao; Chen, Ye; Yin, Wai-Fong; Chong, Yee-Meng; Tan, Li Ying; Chong, Teik Min; Chan, Kok-Gan

    2014-01-01

    N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography-mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. PMID:25430794

  18. Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C.

    PubMed

    Ulferts, Rachel; de Boer, S Matthijn; van der Linden, Lonneke; Bauer, Lisa; Lyoo, Hey Rhyoung; Maté, Maria J; Lichière, Julie; Canard, Bruno; Lelieveld, Daphne; Omta, Wienand; Egan, David; Coutard, Bruno; van Kuppeveld, Frank J M

    2016-05-01

    Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired. PMID:26856848

  19. Design, synthesis, and biological evaluation of novel FAK scaffold inhibitors targeting the FAK-VEGFR3 protein-protein interaction.

    PubMed

    Gogate, Priyanka N; Ethirajan, Manivannan; Kurenova, Elena V; Magis, Andrew T; Pandey, Ravindra K; Cance, William G

    2014-06-10

    Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine kinases, which function as key modulators of survival and metastasis signals in cancer cells. Previously, we reported that small molecule chlorpyramine hydrochloride (C4) specifically targets the interaction between FAK and VEGFR3 and exhibits anti-tumor efficacy. In this study, we designed and synthesized a series of 1 (C4) analogs on the basis of structure activity relationship and molecular modeling. The resulting new compounds were evaluated for their binding to the FAT domain of FAK and anti-cancer activity. Amongst all tested analogs, compound 29 augmented anti-proliferative activity in multiple cancer cell lines with stronger binding to the FAT domain of FAK and disrupted the FAK-VEGFR3 interaction. In conclusion, we hope that this work will contribute to further studies of more potent and selective FAK-VEGFR3 protein-protein interaction inhibitors.

  20. A platinum-based hybrid drug design approach to circumvent acquired resistance to molecular targeted tyrosine kinase inhibitors

    NASA Astrophysics Data System (ADS)

    Wei, Yuming; Poon, Daniel C.; Fei, Rong; Lam, Amy S. M.; Au-Yeung, Steve C. F.; To, Kenneth K. W.

    2016-05-01

    Three molecular targeted tyrosine kinase inhibitors (TKI) were conjugated to classical platinum-based drugs with an aim to circumvent TKI resistance, predominately mediated by the emergence of secondary mutations on oncogenic kinases. The hybrids were found to maintain specificity towards the same oncogenic kinases as the original TKI. Importantly, they are remarkably less affected by TKI resistance, presumably due to their unique structure and the observed dual mechanism of anticancer activity (kinase inhibition and DNA damage). The study is also the first to report the application of a hybrid drug approach to switch TKIs from being efflux transporter substrates into non-substrates. TKIs cannot penetrate into the brain for treating metastases because of efflux transporters at the blood brain barrier. The hybrids were found to escape drug efflux and they accumulate more than the original TKI in the brain in BALB/c mice. Further development of the hybrid compounds is warranted.

  1. Targeting the mitotic checkpoint for cancer therapy with NMS-P715, an inhibitor of MPS1 kinase.

    PubMed

    Colombo, Riccardo; Caldarelli, Marina; Mennecozzi, Milena; Giorgini, Maria Laura; Sola, Francesco; Cappella, Paolo; Perrera, Claudia; Depaolini, Stefania Re; Rusconi, Luisa; Cucchi, Ulisse; Avanzi, Nilla; Bertrand, Jay Aaron; Bossi, Roberto Tiberio; Pesenti, Enrico; Galvani, Arturo; Isacchi, Antonella; Colotta, Francesco; Donati, Daniele; Moll, Jürgen

    2010-12-15

    MPS1 kinase is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. It has been found aberrantly overexpressed in a wide range of human tumors and is necessary for tumoral cell proliferation. Here we report the identification and characterization of NMS-P715, a selective and orally bioavailable MPS1 small-molecule inhibitor, which selectively reduces cancer cell proliferation, leaving normal cells almost unaffected. NMS-P715 accelerates mitosis and affects kinetochore components localization causing massive aneuploidy and cell death in a variety of tumoral cell lines and inhibits tumor growth in preclinical cancer models. Inhibiting the SAC could represent a promising new approach to selectively target cancer cells.

  2. A platinum-based hybrid drug design approach to circumvent acquired resistance to molecular targeted tyrosine kinase inhibitors.

    PubMed

    Wei, Yuming; Poon, Daniel C; Fei, Rong; Lam, Amy S M; Au-Yeung, Steve C F; To, Kenneth K W

    2016-05-06

    Three molecular targeted tyrosine kinase inhibitors (TKI) were conjugated to classical platinum-based drugs with an aim to circumvent TKI resistance, predominately mediated by the emergence of secondary mutations on oncogenic kinases. The hybrids were found to maintain specificity towards the same oncogenic kinases as the original TKI. Importantly, they are remarkably less affected by TKI resistance, presumably due to their unique structure and the observed dual mechanism of anticancer activity (kinase inhibition and DNA damage). The study is also the first to report the application of a hybrid drug approach to switch TKIs from being efflux transporter substrates into non-substrates. TKIs cannot penetrate into the brain for treating metastases because of efflux transporters at the blood brain barrier. The hybrids were found to escape drug efflux and they accumulate more than the original TKI in the brain in BALB/c mice. Further development of the hybrid compounds is warranted.

  3. Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target

    PubMed Central

    Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M. Michael; Taguchi, Y-h.; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A. Mary; Velmurugan, D.; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

    2015-01-01

    A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective. PMID:26607293

  4. A platinum-based hybrid drug design approach to circumvent acquired resistance to molecular targeted tyrosine kinase inhibitors

    PubMed Central

    Wei, Yuming; Poon, Daniel C.; Fei, Rong; Lam, Amy S. M.; Au-Yeung, Steve C. F.; To, Kenneth K. W.

    2016-01-01

    Three molecular targeted tyrosine kinase inhibitors (TKI) were conjugated to classical platinum-based drugs with an aim to circumvent TKI resistance, predominately mediated by the emergence of secondary mutations on oncogenic kinases. The hybrids were found to maintain specificity towards the same oncogenic kinases as the original TKI. Importantly, they are remarkably less affected by TKI resistance, presumably due to their unique structure and the observed dual mechanism of anticancer activity (kinase inhibition and DNA damage). The study is also the first to report the application of a hybrid drug approach to switch TKIs from being efflux transporter substrates into non-substrates. TKIs cannot penetrate into the brain for treating metastases because of efflux transporters at the blood brain barrier. The hybrids were found to escape drug efflux and they accumulate more than the original TKI in the brain in BALB/c mice. Further development of the hybrid compounds is warranted. PMID:27150583

  5. Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

    PubMed

    Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M Michael; Taguchi, Y-H; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A Mary; Velmurugan, D; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

    2015-11-26

    A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

  6. Control of the flow properties of DNA by topoisomerase II and its targeting inhibitor.

    PubMed

    Kundukad, Binu; van der Maarel, Johan R C

    2010-09-22

    The flow properties of DNA are important for understanding cell division and, indirectly, cancer therapy. DNA topology controlling enzymes such as topoisomerase II are thought to play an essential role. We report experiments showing how double-strand passage facilitated by topoisomerase II controls DNA rheology. For this purpose, we have measured the elastic storage and viscous loss moduli of a model system comprising bacteriophage λ-DNA and human topoisomerase IIα using video tracking of the Brownian motion of colloidal probe particles. We found that the rheology is critically dependent on the formation of temporal entanglements among the DNA molecules with a relaxation time of ∼1 s. We observed that topoisomerase II effectively removes these entanglements and transforms the solution from an elastic physical gel to a viscous fluid depending on the consumption of ATP. A second aspect of this study is the effect of the generic topoisomerase II inhibitor adenylyl-imidodiphosphate (AMP-PNP). In mixtures of AMP-PNP and ATP, the double-strand passage reaction gets blocked and progressively fewer entanglements are relaxed. A total replacement of ATP by AMP-PNP results in a temporal increase in elasticity at higher frequencies, but no transition to an elastic gel with fixed cross-links. PMID:20858436

  7. ETV6-NTRK3 as a therapeutic target of small molecule inhibitor PKC412

    SciTech Connect

    Chi, Hoang Thanh; Ly, Bui Thi Kim; Kano, Yasuhiko; Tojo, Arinobu; Sato, Yuko

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer ETV6-NTRK3 is an oncogene with transformation activity in multiple cell lineages. Black-Right-Pointing-Pointer PKC412 could block ETV6-NTRK3 activation. Black-Right-Pointing-Pointer Loss of ETV6-NTRK3 phosphorylation leads to inactivation of its downstream signaling pathway. Black-Right-Pointing-Pointer Inhibition of ETV6-NTRK3 activation by PKC412 could be a novel strategy for the treatment. -- Abstract: The ETV6-NTRK3 (EN) fusion gene which encodes a chimeric tyrosine kinase was first identified by cloning of the t(12;15)(p13;q25) translocation in congenital fibrosarcoma (CFS). Since then, EN has been also found in congenital mesoblastic nephroma (CMN), secretory breast carcinoma (SBC) and acute myelogenous leukemia (AML). Using IMS-M2 and M0-91 cell lines harboring the EN fusion gene, and Ba/F3 cells stably transfected with EN, we demonstrated that PKC412, also known as midostaurin, is an inhibitor of EN. Inhibition of EN activity by PKC412 suppressed the activity of it downstream molecules leading to inhibition of cell proliferation and induction of apoptosis. Our data for the first time suggested that PKC412 could serve as therapeutic drug for treatment of patients with this fusion.

  8. A novel inhibitor of dengue virus replication that targets the capsid protein.

    PubMed

    Byrd, Chelsea M; Dai, Dongcheng; Grosenbach, Douglas W; Berhanu, Aklile; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Wineinger, Kristin A; Page, Jessica M; Harver, Chris; Stavale, Eric; Tyavanagimatt, Shanthakumar; Stone, Melialani A; Bartenschlager, Ralf; Scaturro, Pietro; Hruby, Dennis E; Jordan, Robert

    2013-01-01

    Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.

  9. The Rac1 inhibitor NSC23766 suppresses CREB signaling by targeting NMDA receptor function.

    PubMed

    Hou, Hailong; Chávez, Andrés E; Wang, Chih-Chieh; Yang, Hongtian; Gu, Hua; Siddoway, Benjamin A; Hall, Benjamin J; Castillo, Pablo E; Xia, Houhui

    2014-10-15

    NMDA receptor signaling plays a complex role in CREB activation and CREB-mediated gene transcription, depending on the subcellular location of NMDA receptors, as well as how strongly they are activated. However, it is not known whether Rac1, the prototype of Rac GTPase, plays a role in neuronal CREB activation induced by NMDA receptor signaling. Here, we report that NSC23766, a widely used specific Rac1 inhibitor, inhibits basal CREB phosphorylation at S133 (pCREB) and antagonizes changes in pCREB levels induced by NMDA bath application in rat cortical neurons. Unexpectedly, we found that NSC23766 affects the levels of neuronal pCREB in a Rac1-independent manner. Instead, our results indicate that NSC23766 can directly regulate NMDA receptors as indicated by their strong effects on both exogenous and synaptically evoked NMDA receptor-mediated currents in mouse and rat neurons, respectively. Our findings strongly suggest that Rac1 does not affect pCREB signaling in cortical neurons and reveal that NSC23766 could be a novel NMDA receptor antagonist.

  10. MicroRNAs as targets for dietary and pharmacological inhibitors of mutagenesis and carcinogenesis

    PubMed Central

    Izzotti, Alberto; Cartiglia, Cristina; Steele, Vernon E.; De Flora, Silvio

    2012-01-01

    MicroRNAs (miRNAs) have been implicated in many biological processes, cancer, and other diseases. In addition, miRNAs are dysregulated following exposure to toxic and genotoxic agents. Here we review studies evaluating modulation of miRNAs by dietary and pharmacological agents, which could potentially be exploited for inhibition of mutagenesis and carcinogenesis. This review covers natural agents, including vitamins, oligoelements, polyphenols, isoflavones, indoles, isothiocyanates, phospholipids, saponins, anthraquinones and polyunsaturated fatty acids, and synthetic agents, including thiols, nuclear receptor agonists, histone deacetylase inhibitors, antiinflammatory drugs, and selective estrogen receptor modulators. As many as 145 miRNAs, involved in the control of a variety of carcinogenesis mechanisms, were modulated by these agents, either individually or in combination. Most studies used cancer cells in vitro with the goal of modifying their phenotype by changing miRNA expression profiles. In vivo studies evaluated regulation of miRNAs by chemopreventive agents in organs of mice and rats, either untreated or exposed to carcinogens, with the objective of evaluating their safety and efficacy. The tissue specificity of miRNAs could be exploited for the chemoprevention of site-specific cancers, and the study of polymorphic miRNAs is expected to predict the individual response to chemopreventive agents as a tool for developing new prevention strategies. PMID:22683846

  11. Preclinical Activity of ARQ 087, a Novel Inhibitor Targeting FGFR Dysregulation

    PubMed Central

    Hall, Terence G.; Yu, Yi; Eathiraj, Sudharshan; Wang, Yunxia; Savage, Ronald E.; Lapierre, Jean-Marc; Schwartz, Brian; Abbadessa, Giovanni

    2016-01-01

    Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers (e.g. liver, gastric, ovarian, endometrial, and bladder). ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent in vitro and in vivo activity against FGFR addicted cell lines and tumors. Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway (FRS2α, AKT, ERK) was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920). PMID:27627808

  12. Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bisubstrate Adenylation Inhibitors

    PubMed Central

    Petrelli, Riccardo; De la Mora-Rey, Teresa; Tiwari, Divya; Liu, Feng; Dawadi, Surrendra; Nandakumar, Madhumitha; Rhee, Kyu Y.; Schnappinger, Dirk; Finzel, Barry C.; Aldrich, Courtney C.

    2015-01-01

    Mycobacterium tuberculosis (Mtb) responsible for both latent and symptomatic tuberculosis (TB) remains the second leading cause of mortality among infectious diseases worldwide. Mycobacterial biotin protein ligase (MtBPL) is an essential enzyme in Mtb and regulates lipid metabolism through the post-translational biotinylation of acyl coenzyme A carboxylases. We report the synthesis and evaluation of a systematic series of potent nucleoside-based inhibitors of MtBPL that contain modifications to the ribofuranosyl ring of the nucleoside. All compounds were characterized by isothermal titration calorimetry (ITC) and shown to bind potently with KD's below 2 nM. Additionally, we obtained high-resolution co-crystal structures for a majority of the compounds. Despite fairly uniform biochemical potency, the whole-cell Mtb activity varied greatly with minimum inhibitory concentrations (MIC) ranging from 0.78 to >100 μM. Cellular accumulation studies showed a nearly 10-fold enhanced accumulation of a C-2′-α analog over the corresponding C-2′-β analog, consistent with their differential whole-cell activity. PMID:26299766

  13. A novel inhibitor of dengue virus replication that targets the capsid protein.

    PubMed

    Byrd, Chelsea M; Dai, Dongcheng; Grosenbach, Douglas W; Berhanu, Aklile; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Wineinger, Kristin A; Page, Jessica M; Harver, Chris; Stavale, Eric; Tyavanagimatt, Shanthakumar; Stone, Melialani A; Bartenschlager, Ralf; Scaturro, Pietro; Hruby, Dennis E; Jordan, Robert

    2013-01-01

    Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle. PMID:23070172

  14. Identification and analysis of inhibitors targeting the hepatitis C virus NS3 helicase.

    PubMed

    Hanson, Alicia M; Hernandez, John J; Shadrick, William R; Frick, David N

    2012-01-01

    This chapter describes two types of FRET-based fluorescence assays that can be used to identify and analyze compounds that inhibit the helicase encoded by the hepatitis C virus (HCV). Both assays use a fluorescently labeled DNA or RNA oligonucleotide to monitor helicase-catalyzed strand separation, and they differ from other real-time helicase assays in that they do not require the presence of other nucleic acids to trap the reaction products. The first assay is a molecular beacon-based helicase assay (MBHA) that monitors helicase-catalyzed displacement of a hairpin-forming oligonucleotide with a fluorescent moiety on one end and a quencher on the other. DNA-based MBHAs have been used extensively for high-throughput screening (HTS), but RNA-based MBHAs are typically less useful because of poor signal to background ratios. In the second assay discussed, the fluorophore and quencher are split between two hairpin-forming oligonucleotides annealed in tandem to a third oligonucleotide. This split beacon helicase assay can be used for HTS with either DNA or RNA oligonucleotides. These assays should be useful to the many labs searching for HCV helicase inhibitors in order to develop new HCV therapies that are still desperately needed.

  15. Preclinical Activity of ARQ 087, a Novel Inhibitor Targeting FGFR Dysregulation.

    PubMed

    Hall, Terence G; Yu, Yi; Eathiraj, Sudharshan; Wang, Yunxia; Savage, Ronald E; Lapierre, Jean-Marc; Schwartz, Brian; Abbadessa, Giovanni

    2016-01-01

    Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers (e.g. liver, gastric, ovarian, endometrial, and bladder). ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent in vitro and in vivo activity against FGFR addicted cell lines and tumors. Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway (FRS2α, AKT, ERK) was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920). PMID:27627808

  16. Novel benzoxazole inhibitor of dengue virus replication that targets the NS3 helicase.

    PubMed

    Byrd, Chelsea M; Grosenbach, Douglas W; Berhanu, Aklile; Dai, Dongcheng; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Yang, Guang; Tyavanagimatt, Shanthakumar; Harver, Chris; Wineinger, Kristin A; Page, Jessica; Stavale, Eric; Stone, Melialani A; Fuller, Kathleen P; Lovejoy, Candace; Leeds, Janet M; Hruby, Dennis E; Jordan, Robert

    2013-04-01

    Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

  17. Role of inhibitors of mammalian target of rapamycin in the treatment of luminal breast cancer.

    PubMed

    Ciruelos, Eva; Cortes-Funes, Hernán; Ghanem, Ismael; Manso, Luis; Arteaga, Carlos

    2013-09-01

    Approximately 75% of patients with breast cancer present hormone receptor-positive tumors. This subtype of breast cancer initially shows a high overall response rate to hormonal treatments. However, resistance eventually develops, resulting in tumor progression. The PI3K/Akt/mTOR pathway regulates several cellular functions in cancer such as cell growth, survival, and proliferation. In addition, a high activation level of the PI3K/Akt/mTOR pathway is related to resistance to conventional chemotherapy and hormone therapy. The mTOR inhibitor everolimus, in combination with hormonal treatments, has led to excellent results in progression-free survival in patients with metastatic breast cancer resistant to hormone therapies. Therefore, everolimus has entered the National Comprehensive Cancer Network (NCCN) guidelines 2012 and its combination with exemestane was approved recently by the US Food and Drug Administration and the European Medicines Agency. This is the first time that a drug will have been approved for the restoration of hormone sensitivity in breast cancer.

  18. Design, Synthesis, and Biological Evaluation of Novel Conformationally Constrained Inhibitors Targeting EGFR.

    PubMed

    Wu, Jianwei; Chen, Wenteng; Xia, Guangxin; Zhang, Jing; Shao, Jiaan; Tan, Biqin; Zhang, Chunchun; Yu, Wanwan; Weng, Qinjie; Liu, Haiyan; Hu, Miao; Deng, Hailin; Hao, Yu; Shen, Jingkang; Yu, Yongping

    2013-10-10

    This letter describes the construction of conformationally constrained quinazoline analogues. Structure-activity relationship studies led to the identification of the lead compound 9n . Compound 9n exhibits effective in vitro activity against A431(WT,overexpression) and H1975([L858R/T790M]) cancer cell lines but is significantly less effective against EGFR negative cancer cell lines (SW620, A549, and K562). Compound 9n was also assessed for potency in enzymatic assays and in vivo antitumor studies. The results indicated that 9n is a potent kinase inhibitor against both wild-type and T790M mutant EGFR kinase. Meanwhile, an oral administration of 9n at a dose of 200 mg/kg produced a considerable antitumor effect in a A431 xenograft model, as compared to gefitinib. A preliminary pharmacokinetic study of 9n also indicates it has good pharmacokinetic properties, and therefore, it is a good starting point for further development. PMID:24900594

  19. Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells

    PubMed Central

    Lai, I-Lu; Chou, Chih-Chien; Lai, Po-Ting; Fang, Chun-Sheng; Shirley, Lawrence A.; Yan, Ribai; Mo, Xiaokui; Bloomston, Mark; Kulp, Samuel K.; Bekaii-Saab, Tanios; Chen, Ching-Shih

    2014-01-01

    Gemcitabine resistance remains a significant clinical challenge. Here, we used a novel glucose transporter (Glut) inhibitor, CG-5, as a proof-of-concept compound to investigate the therapeutic utility of targeting the Warburg effect to overcome gemcitabine resistance in pancreatic cancer. The effects of gemcitabine and/or CG-5 on viability, survival, glucose uptake and DNA damage were evaluated in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cell lines. Mechanistic studies were conducted to determine the molecular basis of gemcitabine resistance and the mechanism of CG-5-induced sensitization to gemcitabine. The effects of CG-5 on gemcitabine sensitivity were investigated in a xenograft tumor model of gemcitabine-resistant pancreatic cancer. In contrast to gemcitabine-sensitive pancreatic cancer cells, the resistant Panc-1 and Panc-1GemR cells responded to gemcitabine by increasing the expression of ribonucleotide reductase M2 catalytic subunit (RRM2) through E2F1-mediated transcriptional activation. Acting as a pan-Glut inhibitor, CG-5 abrogated this gemcitabine-induced upregulation of RRM2 through decreased E2F1 expression, thereby enhancing gemcitabine-induced DNA damage and inhibition of cell survival. This CG-5-induced inhibition of E2F1 expression was mediated by the induction of a previously unreported E2F1-targeted microRNA, miR-520f. The addition of oral CG-5 to gemcitabine therapy caused greater suppression of Panc-1GemR xenograft tumor growth in vivo than either drug alone. Glut inhibition may be an effective strategy to enhance gemcitabine activity for the treatment of pancreatic cancer. PMID:24879635

  20. Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells.

    PubMed

    Lai, I-Lu; Chou, Chih-Chien; Lai, Po-Ting; Fang, Chun-Sheng; Shirley, Lawrence A; Yan, Ribai; Mo, Xiaokui; Bloomston, Mark; Kulp, Samuel K; Bekaii-Saab, Tanios; Chen, Ching-Shih

    2014-10-01

    Gemcitabine resistance remains a significant clinical challenge. Here, we used a novel glucose transporter (Glut) inhibitor, CG-5, as a proof-of-concept compound to investigate the therapeutic utility of targeting the Warburg effect to overcome gemcitabine resistance in pancreatic cancer. The effects of gemcitabine and/or CG-5 on viability, survival, glucose uptake and DNA damage were evaluated in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cell lines. Mechanistic studies were conducted to determine the molecular basis of gemcitabine resistance and the mechanism of CG-5-induced sensitization to gemcitabine. The effects of CG-5 on gemcitabine sensitivity were investigated in a xenograft tumor model of gemcitabine-resistant pancreatic cancer. In contrast to gemcitabine-sensitive pancreatic cancer cells, the resistant Panc-1 and Panc-1(GemR) cells responded to gemcitabine by increasing the expression of ribonucleotide reductase M2 catalytic subunit (RRM2) through E2F1-mediated transcriptional activation. Acting as a pan-Glut inhibitor, CG-5 abrogated this gemcitabine-induced upregulation of RRM2 through decreased E2F1 expression, thereby enhancing gemcitabine-induced DNA damage and inhibition of cell survival. This CG-5-induced inhibition of E2F1 expression was mediated by the induction of a previously unreported E2F1-targeted microRNA, miR-520f. The addition of oral CG-5 to gemcitabine therapy caused greater suppression of Panc-1(GemR) xenograft tumor growth in vivo than either drug alone. Glut inhibition may be an effective strategy to enhance gemcitabine activity for the treatment of pancreatic cancer.

  1. MicroRNA-31 Is a Transcriptional Target of Histone Deacetylase Inhibitors and a Regulator of Cellular Senescence*

    PubMed Central

    Cho, Joon-Ho; Dimri, Manjari; Dimri, Goberdhan P.

    2015-01-01

    MicroRNAs (miRNAs) have emerged as important regulators of tumorigenesis. Several miRNAs, which can function either as oncomiRs or tumor suppressive miRs are deregulated in cancer cells. The microRNA-31 (miR-31) has been shown to be overexpressed in metastatic breast cancer. It promotes multiple oncogenic phenotypes, including proliferation, motility, and invasion of cancer cells. Using a breast cancer-related miRNA array analysis, we identified miR-31 as a novel target of histone deacetylase inhibitors (HDACi) in breast cancer cells. Specifically, we show that sodium butyrate (NaB) and panobinostat (LBH589), two broad-spectrum HDAC inhibitors up-regulate hsa-miR-31 (miR-31). The up-regulation of miR-31 was accompanied by repression of the polycomb group (PcG) protein BMI1 and induction of cellular senescence. We further show that inhibition of miR-31 overcomes the senescence-inducing effect of HDACi, and restores expression of the PcG protein BMI1. Interestingly, BMI1 also acts as a repressor of miR-31 transcription, suggesting a cross-negative feedback loop between the expression of miR-31 and BMI1. Our data suggest that miR-31 is an important physiological target of HDACi, and that it is an important regulator of senescence relevant to cancer. These studies further suggest that manipulation of miR-31 expression can be used to modulate senescence-related pathological conditions such as cancer, and the aging process. PMID:25737447

  2. Use of mammalian target of rapamycin inhibitors after failure of tyrosine kinase inhibitors in patients with metastatic renal cell carcinoma undergoing hemodialysis: A single-center experience with four cases.

    PubMed

    Omae, Kenji; Kondo, Tsunenori; Takagi, Toshio; Iizuka, Junpei; Kobayashi, Hirohito; Hashimoto, Yasunobu; Tanabe, Kazunari

    2016-07-01

    We retrospectively identified patients with end-stage renal disease undergoing hemodialysis treated with the mammalian target of rapamycin inhibitors as a second- and/or third-line targeted therapy after treatment failure with the tyrosine kinase inhibitors for metastatic renal cell carcinoma. Patient medical records were reviewed to evaluate the response to therapies and treatment-related toxicities. Four patients were identified. All patients had undergone nephrectomy, and one had received immunotherapy before targeted therapy. Two patients had clear cell histology, and the other two had papillary histology. All patients were classified into the intermediate risk group according to the Memorial Sloan-Kettering Cancer Center risk model. All patients were treated with everolimus as a second- or third-line therapy, and two patients were treated with temsirolimus as a second- or third-line therapy after treatment failure with sorafenib or sunitinib. The median duration of everolimus therapy was 6.7 months, whereas that of temsirolimus was 9.5 months. All patients had stable disease as the best response during each period of therapy. There were no severe adverse events. The use of mammalian target of rapamycin inhibitors in patients who previously failed to respond to tyrosine kinase inhibitors appears to be feasible in patients with end-stage renal disease requiring hemodialysis.

  3. Identification of Novel Ezrin Inhibitors Targeting Metastatic Osteosarcoma by Screening Open Access Malaria Box.

    PubMed

    Çelik, Haydar; Hong, Sung-Hyeok; Colón-López, Daisy D; Han, Jenny; Kont, Yasemin Saygideger; Minas, Tsion Z; Swift, Matthew; Paige, Mikell; Glasgow, Eric; Toretsky, Jeffrey A; Bosch, Jürgen; Üren, Aykut

    2015-11-01

    Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins and functions as a linker between the plasma membrane and the actin cytoskeleton. Ezrin is a key driver of tumor progression and metastatic spread of osteosarcoma. We discovered a quinoline-based small molecule, NSC305787, that directly binds to ezrin and inhibits its functions in promoting invasive phenotype. NSC305787 possesses a very close structural similarity to commonly used quinoline-containing antimalarial drugs. On the basis of this similarity and of recent findings that ezrin has a likely role in the pathogenesis of malaria infection, we screened antimalarial compounds in an attempt to identify novel ezrin inhibitors with better efficacy and drug properties. Screening of Medicines for Malaria Venture (MMV) Malaria Box compounds for their ability to bind to recombinant ezrin protein yielded 12 primary hits with high selective binding activity. The specificity of the hits on ezrin function was confirmed by inhibition of the ezrin-mediated cell motility of osteosarcoma cells. Compounds were further tested for phenocopying the morphologic defects associated with ezrin suppression in zebrafish embryos as well as for inhibiting the lung metastasis of high ezrin-expressing osteosarcoma cells. The compound MMV667492 exhibited potent anti-ezrin activity in all biologic assays and had better physicochemical properties for drug-likeness than NSC305787. The drug-like compounds MMV020549 and MMV666069 also showed promising activities in functional assays. Thus, our study suggests further evaluation of antimalarial compounds as a novel class of antimetastatic agents for the treatment of metastatic osteosarcoma. PMID:26358752

  4. Identification of Novel Ezrin Inhibitors Targeting Metastatic Osteosarcoma by Screening Open Access Malaria Box.

    PubMed

    Çelik, Haydar; Hong, Sung-Hyeok; Colón-López, Daisy D; Han, Jenny; Kont, Yasemin Saygideger; Minas, Tsion Z; Swift, Matthew; Paige, Mikell; Glasgow, Eric; Toretsky, Jeffrey A; Bosch, Jürgen; Üren, Aykut

    2015-11-01

    Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins and functions as a linker between the plasma membrane and the actin cytoskeleton. Ezrin is a key driver of tumor progression and metastatic spread of osteosarcoma. We discovered a quinoline-based small molecule, NSC305787, that directly binds to ezrin and inhibits its functions in promoting invasive phenotype. NSC305787 possesses a very close structural similarity to commonly used quinoline-containing antimalarial drugs. On the basis of this similarity and of recent findings that ezrin has a likely role in the pathogenesis of malaria infection, we screened antimalarial compounds in an attempt to identify novel ezrin inhibitors with better efficacy and drug properties. Screening of Medicines for Malaria Venture (MMV) Malaria Box compounds for their ability to bind to recombinant ezrin protein yielded 12 primary hits with high selective binding activity. The specificity of the hits on ezrin function was confirmed by inhibition of the ezrin-mediated cell motility of osteosarcoma cells. Compounds were further tested for phenocopying the morphologic defects associated with ezrin suppression in zebrafish embryos as well as for inhibiting the lung metastasis of high ezrin-expressing osteosarcoma cells. The compound MMV667492 exhibited potent anti-ezrin activity in all biologic assays and had better physicochemical properties for drug-likeness than NSC305787. The drug-like compounds MMV020549 and MMV666069 also showed promising activities in functional assays. Thus, our study suggests further evaluation of antimalarial compounds as a novel class of antimetastatic agents for the treatment of metastatic osteosarcoma.

  5. Ebsulfur Is a Benzisothiazolone Cytocidal Inhibitor Targeting the Trypanothione Reductase of Trypanosoma brucei *

    PubMed Central

    Lu, Jun; Vodnala, Suman K.; Gustavsson, Anna-Lena; Gustafsson, Tomas N.; Sjöberg, Birger; Johansson, Henrik A.; Kumar, Sangit; Tjernberg, Agneta; Engman, Lars; Rottenberg, Martin E.; Holmgren, Arne

    2013-01-01

    Trypanosoma brucei is the causing agent of African trypanosomiasis. These parasites possess a unique thiol redox system required for DNA synthesis and defense against oxidative stress. It includes trypanothione and trypanothione reductase (TryR) instead of the thioredoxin and glutaredoxin systems of mammalian hosts. Here, we show that the benzisothiazolone compound ebsulfur (EbS), a sulfur analogue of ebselen, is a potent inhibitor of T. brucei growth with a favorable selectivity index over mammalian cells. EbS inhibited the TryR activity and decreased non-protein thiol levels in cultured parasites. The inhibition of TryR by EbS was irreversible and NADPH-dependent. EbS formed a complex with TryR and caused oxidation and inactivation of the enzyme. EbS was more toxic for T. brucei than for Trypanosoma cruzi, probably due to lower levels of TryR and trypanothione in T. brucei. Furthermore, inhibition of TryR produced high intracellular reactive oxygen species. Hydrogen peroxide, known to be constitutively high in T. brucei, enhanced the EbS inhibition of TryR. The elevation of reactive oxygen species production in parasites caused by EbS induced a programmed cell death. Soluble EbS analogues were synthesized and cured T. brucei brucei infection in mice when used together with nifurtimox. Altogether, EbS and EbS analogues disrupt the trypanothione system, hampering the defense against oxidative stress. Thus, EbS is a promising lead for development of drugs against African trypanosomiasis. PMID:23900839

  6. Targeted Metabolomics Approach To Detect the Misuse of Steroidal Aromatase Inhibitors in Equine Sports by Biomarker Profiling.

    PubMed

    Chan, George Ho Man; Ho, Emmie Ngai Man; Leung, David Kwan Kon; Wong, Kin Sing; Wan, Terence See Ming

    2016-01-01

    The use of anabolic androgenic steroids (AAS) is prohibited in both human and equine sports. The conventional approach in doping control testing for AAS (as well as other prohibited substances) is accomplished by the direct detection of target AAS or their characteristic metabolites in biological samples using hyphenated techniques such as gas chromatography or liquid chromatography coupled with mass spectrometry. Such an approach, however, falls short when dealing with unknown designer steroids where reference materials and their pharmacokinetics are not available. In addition, AASs with fast elimination times render the direct detection approach ineffective as the detection window is short. A targeted metabolomics approach is a plausible alternative to the conventional direct detection approach for controlling the misuse of AAS in sports. Because the administration of AAS of the same class may trigger similar physiological responses or effects in the body, it may be possible to detect such administrations by monitoring changes in the endogenous steroidal expression profile. This study attempts to evaluate the viability of using the targeted metabolomics approach to detect the administration of steroidal aromatase inhibitors, namely androst-4-ene-3,6,17-trione (6-OXO) and androsta-1,4,6-triene-3,17-dione (ATD), in horses. Total (free and conjugated) urinary concentrations of 31 endogenous steroids were determined by gas chromatography-tandem mass spectrometry for a group of 2 resting and 2 in-training thoroughbred geldings treated with either 6-OXO or ATD. Similar data were also obtained from a control (untreated) group of in-training thoroughbred geldings (n = 28). Statistical processing and chemometric procedures using principle component analysis and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) have highlighted 7 potential biomarkers that could be used to differentiate urine samples obtained from the control and the treated groups

  7. Chemical genomics in Escherichia coli identifies an inhibitor of bacterial lipoprotein targeting.

    PubMed

    Pathania, Ranjana; Zlitni, Soumaya; Barker, Courtney; Das, Rahul; Gerritsma, David A; Lebert, Julie; Awuah, Emilia; Melacini, Giuseppe; Capretta, Fred A; Brown, Eric D

    2009-11-01

    One of the most significant hurdles to developing new chemical probes of biological systems and new drugs to treat disease is that of understanding the mechanism of action of small molecules discovered with cell-based small-molecule screening. Here we have assembled an ordered, high-expression clone set of all of the essential genes from Escherichia coli and used it to systematically screen for suppressors of growth inhibitory compounds. Using this chemical genomic approach, we demonstrate that the targets of well-known antibiotics can be identified as high copy suppressors of chemical lethality. This approach led to the discovery of MAC13243, a molecule that belongs to a new chemical class and that has a unique mechanism and promising activity against multidrug-resistant Pseudomonas aeruginosa. We show that MAC13243 inhibits the function of the LolA protein and represents a new chemical probe of lipoprotein targeting in bacteria with promise as an antibacterial lead with Gram-negative selectivity. PMID:19783991

  8. Cargo-Delivery Platforms for Targeted Delivery of Inhibitor Cargos Against Botulism

    PubMed Central

    Wilson, Brenda A.; Ho, Mengfei

    2015-01-01

    Delivering therapeutic cargos to specific cell types in vivo poses many technical challenges. There is currently a plethora of drug leads and therapies against numerous diseases, ranging from small molecule compounds to nucleic acids to peptides to proteins with varying binding or enzymatic functions. Many of these candidate therapies have documented potential for mitigating or reversing disease symptoms, if only a means for gaining access to the intracellular target were available. Recent advances in our understanding of the biology of cellular uptake and transport processes and the mode of action of bacterial protein toxins have accelerated the development of toxin-based cargo-delivery vehicle platforms. This review provides an updated survey of the status of available platforms for targeted delivery of therapeutic cargos, outlining various strategies that have been used to deliver different types of cargo into cells. Particular emphasis is placed on the application of toxin-based approaches, examining critical issues that have hampered realization of post-intoxication antitoxins against botulism. PMID:25335885

  9. The biofilm inhibitor Carolacton inhibits planktonic growth of virulent pneumococci via a conserved target

    PubMed Central

    Donner, Jannik; Reck, Michael; Bergmann, Simone; Kirschning, Andreas; Müller, Rolf; Wagner-Döbler, Irene

    2016-01-01

    New antibacterial compounds, preferentially exploiting novel cellular targets, are urgently needed to fight the increasing resistance of pathogens against conventional antibiotics. Here we demonstrate that Carolacton, a myxobacterial secondary metabolite previously shown to damage Streptococcus mutans biofilms, inhibits planktonic growth of Streptococcus pneumoniae TIGR4 and multidrug-resistant clinical isolates of serotype 19A at nanomolar concentrations. A Carolacton diastereomer is inactive in both streptococci, indicating a highly specific interaction with a conserved cellular target. S. mutans requires the eukaryotic-like serine/threonine protein kinase PknB and the cysteine metabolism regulator CysR for susceptibility to Carolacton, whereas their homologues are not needed in S. pneumoniae, suggesting a specific function for S. mutans biofilms only. A bactericidal effect of Carolacton was observed for S. pneumoniae TIGR4, with a reduction of cell numbers by 3 log units. The clinical pneumonia isolate Sp49 showed immediate growth arrest and cell lysis, suggesting a bacteriolytic effect of Carolacton. Carolacton treatment caused a reduction in membrane potential, but not membrane integrity, and transcriptome analysis revealed compensatory reactions of the cell. Our data show that Carolacton might have potential for treating pneumococcal infections. PMID:27404808

  10. Whole Cell Target Engagement Identifies Novel Inhibitors of Mycobacterium tuberculosis Decaprenylphosphoryl-β-d-ribose Oxidase.

    PubMed

    Batt, Sarah M; Cacho Izquierdo, Monica; Castro Pichel, Julia; Stubbs, Christopher J; Vela-Glez Del Peral, Laura; Pérez-Herrán, Esther; Dhar, Neeraj; Mouzon, Bernadette; Rees, Mike; Hutchinson, Jonathan P; Young, Robert J; McKinney, John D; Barros Aguirre, David; Ballell, Lluis; Besra, Gurdyal S; Argyrou, Argyrides

    2015-12-11

    We have targeted the Mycobacterium tuberculosis decaprenylphosphoryl-β-d-ribose oxidase (Mt-DprE1) for potential chemotherapeutic intervention of tuberculosis. A multicopy suppression strategy that overexpressed Mt-DprE1 in M. bovis BCG was used to profile the publically available GlaxoSmithKline antimycobacterial compound set, and one compound (GSK710) was identified that showed an 8-fold higher minimum inhibitory concentration relative to the control strain. Analogues of GSK710 show a clear relationship between whole cell potency and in vitro activity using an enzymatic assay employing recombinant Mt-DprE1, with binding affinity measured by fluorescence quenching of the flavin cofactor of the enzyme. M. bovis BCG spontaneous resistant mutants to GSK710 and a closely related analogue were isolated and sequencing of ten such mutants revealed a single point mutation at two sites, E221Q or G248S within DprE1, providing further evidence that DprE1 is the main target of these compounds. Finally, time-lapse microscopy experiments showed that exposure of M. tuberculosis to a compound of this series arrests bacterial growth rapidly followed by a slower cytolysis phase. PMID:27623058

  11. Cargo-delivery platforms for targeted delivery of inhibitor cargos against botulism.

    PubMed

    Wilson, Brenda A; Ho, Mengfei

    2014-01-01

    Delivering therapeutic cargos to specific cell types in vivo poses many technical challenges. There is currently a plethora of drug leads and therapies against numerous diseases, ranging from small molecule compounds to nucleic acids to peptides to proteins with varying binding or enzymatic functions. Many of these candidate therapies have documented potential for mitigating or reversing disease symptoms, if only a means for gaining access to the intracellular target were available. Recent advances in our understanding of the biology of cellular uptake and transport processes and the mode of action of bacterial protein toxins have accelerated the development of toxin-based cargo-delivery vehicle platforms. This review provides an updated survey of the status of available platforms for targeted delivery of therapeutic cargos, outlining various strategies that have been used to deliver different types of cargo into cells. Particular emphasis is placed on the application of toxin-based approaches, examining critical issues that have hampered realization of post-intoxication antitoxins against botulism.

  12. The biofilm inhibitor Carolacton inhibits planktonic growth of virulent pneumococci via a conserved target.

    PubMed

    Donner, Jannik; Reck, Michael; Bergmann, Simone; Kirschning, Andreas; Müller, Rolf; Wagner-Döbler, Irene

    2016-01-01

    New antibacterial compounds, preferentially exploiting novel cellular targets, are urgently needed to fight the increasing resistance of pathogens against conventional antibiotics. Here we demonstrate that Carolacton, a myxobacterial secondary metabolite previously shown to damage Streptococcus mutans biofilms, inhibits planktonic growth of Streptococcus pneumoniae TIGR4 and multidrug-resistant clinical isolates of serotype 19A at nanomolar concentrations. A Carolacton diastereomer is inactive in both streptococci, indicating a highly specific interaction with a conserved cellular target. S. mutans requires the eukaryotic-like serine/threonine protein kinase PknB and the cysteine metabolism regulator CysR for susceptibility to Carolacton, whereas their homologues are not needed in S. pneumoniae, suggesting a specific function for S. mutans biofilms only. A bactericidal effect of Carolacton was observed for S. pneumoniae TIGR4, with a reduction of cell numbers by 3 log units. The clinical pneumonia isolate Sp49 showed immediate growth arrest and cell lysis, suggesting a bacteriolytic effect of Carolacton. Carolacton treatment caused a reduction in membrane potential, but not membrane integrity, and transcriptome analysis revealed compensatory reactions of the cell. Our data show that Carolacton might have potential for treating pneumococcal infections. PMID:27404808

  13. Build an Emergency Preparedness Kit

    MedlinePlus

    ... tire traction -Red or brightly- colored cloth -NOAA weather radio For more information on building emergency kits, ... and a flashlight with extra batteries. A NOAA weather radio warns the public of severe weather and ...

  14. Energy Management Curriculum Starter Kit

    SciTech Connect

    Turner, W.C.

    1987-02-01

    The Energy Management Curriculum Starter Kit was designed to help engineering educators develop and teach energy management courses. Montana State University and Oklahoma State University courses are embodied in the model curriculum given. The curricula offered at many other universities throughout the United States are also presented. The kit was designed specifically to train engineering students to be good energy managers. Courses at both the undergraduate and postgraduate level are presented.

  15. Phenotypic Screening of Small-Molecule Inhibitors: Implications for Therapeutic Discovery and Drug Target Development in Traumatic Brain Injury.

    PubMed

    Al-Ali, Hassan; Lemmon, Vance P; Bixby, John L

    2016-01-01

    The inability of central nervous system (CNS) neurons to regenerate damaged axons and dendrites following traumatic brain injury (TBI) creates a substantial obstacle for functional recovery. Apoptotic cell death, deposition of scar tissue, and growth-repressive molecules produced by glia further complicate the problem and make it challenging for re-growing axons to extend across injury sites. To date, there are no approved drugs for the treatment of TBI, accentuating the need for relevant leads. Cell-based and organotypic bioassays can better mimic outcomes within the native CNS microenvironment than target-based screening methods and thus should speed the discovery of therapeutic agents that induce axon or dendrite regeneration. Additionally, when used to screen focused chemical libraries such as small-molecule protein kinase inhibitors, these assays can help elucidate molecular mechanisms involved in neurite outgrowth and regeneration as well as identify novel drug targets. Here, we describe a phenotypic cellular (high content) screening assay that utilizes brain-derived primary neurons for screening small-molecule chemical libraries. PMID:27604745

  16. Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence.

    PubMed

    Kortlever, Roderik M; Higgins, Paul J; Bernards, René

    2006-08-01

    p53 limits the proliferation of primary diploid fibroblasts by inducing a state of growth arrest named replicative senescence - a process which protects against oncogenic transformation and requires integrity of the p53 tumour suppressor pathway. However, little is known about the downstream target genes of p53 in this growth-limiting response. Here, we report that suppression of the p53 target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference (RNAi) leads to escape from replicative senescence both in primary mouse embryo fibroblasts and primary human BJ fibroblasts. PAI-1 knockdown results in sustained activation of the PI(3)K-PKB-GSK3beta pathway and nuclear retention of cyclin D1, consistent with a role for PAI-1 in regulating growth factor signalling. In agreement with this, we find that the PI(3)K-PKB-GSK3beta-cyclin D1 pathway is also causally involved in cellular senescence. Conversely, ectopic expression of PAI-1 in proliferating p53-deficient murine or human fibroblasts induces a phenotype displaying all the hallmarks of replicative senescence. Our data indicate that PAI-1 is not merely a marker of senescence, but is both necessary and sufficient for the induction of replicative senescence downstream of p53.

  17. Traffic jam at the bacterial sec translocase: targeting the SecA nanomotor by small-molecule inhibitors.

    PubMed

    Segers, Kenneth; Anné, Jozef

    2011-06-24

    The rapid rise of drug-resistant bacteria is one of the most serious unmet medical needs facing the world. Despite this increasing problem of antibiotic resistance, the number of different antibiotics available for the treatment of serious infections is dwindling. Therefore, there is an urgent need for new antibacterial drugs, preferably with novel modes of action to potentially avoid cross-resistance with existing antibacterial agents. In recent years, increasing attention has been paid to bacterial protein secretion as a potential antibacterial target. Among the different protein secretion pathways that are present in bacterial pathogens, the general protein secretory (Sec) pathway is widely considered as an attractive target for antibacterial therapy. One of the key components of the Sec pathway is the peripheral membrane ATPase SecA, which provides the energy for the translocation of preproteins across the bacterial cytoplasmic membrane. In this review, we will provide an overview of research efforts on the discovery and development of small-molecule SecA inhibitors. Furthermore, recent advances on the structure and function of SecA and their potential impact on antibacterial drug discovery will be discussed.

  18. Multi-scaled explorations of binding-induced folding of intrinsically disordered protein inhibitor IA3 to its target enzyme.

    PubMed

    Wang, Jin; Wang, Yong; Chu, Xiakun; Hagen, Stephen J; Han, Wei; Wang, Erkang

    2011-04-01

    Biomolecular function is realized by recognition, and increasing evidence shows that recognition is determined not only by structure but also by flexibility and dynamics. We explored a biomolecular recognition process that involves a major conformational change - protein folding. In particular, we explore the binding-induced folding of IA3, an intrinsically disordered protein that blocks the active site cleft of the yeast aspartic proteinase saccharopepsin (YPrA) by folding its own N-terminal residues into an amphipathic alpha helix. We developed a multi-scaled approach that explores the underlying mechanism by combining structure-based molecular dynamics simulations at the residue level with a stochastic path method at the atomic level. Both the free energy profile and the associated kinetic paths reveal a common scheme whereby IA3 binds to its target enzyme prior to folding itself into a helix. This theoretical result is consistent with recent time-resolved experiments. Furthermore, exploration of the detailed trajectories reveals the important roles of non-native interactions in the initial binding that occurs prior to IA3 folding. In contrast to the common view that non-native interactions contribute only to the roughness of landscapes and impede binding, the non-native interactions here facilitate binding by reducing significantly the entropic search space in the landscape. The information gained from multi-scaled simulations of the folding of this intrinsically disordered protein in the presence of its binding target may prove useful in the design of novel inhibitors of aspartic proteinases.

  19. Angiogenic inhibitors delivered by the type III secretion system of tumor-targeting Salmonella typhimurium safely shrink tumors in mice.

    PubMed

    Shi, Lei; Yu, Bin; Cai, Chun-Hui; Huang, Jian-Dong

    2016-12-01

    Despite of a growing number of bacterial species that apparently exhibit intrinsic tumor-targeting properties, no bacterium is able to inhibit tumor growth completely in the immunocompetent hosts, due to its poor dissemination inside the tumors. Oxygen and inflammatory reaction form two barriers and restrain the spread of the bacteria inside the tumors. Here, we engineered a Salmonella typhimurium strain named ST8 which is safe and has limited ability to spread beyond the anaerobic regions of tumors. When injected systemically to tumor-bearing immunocompetent mice, ST8 accumulated in tumors at levels at least 100-fold greater than parental obligate anaerobic strain ST4. ST8/pSEndo harboring therapeutic plasmids encoding Endostatin fused with a secreted protein SopA could target vasculature at the tumor periphery, can stably maintain and safely deliver a therapeutic vector, release angiogenic inhibitors through a type III secretion system (T3SS) to interfere with the pro-angiogenic action of growth factors in tumors. Mice with murine CT26 colon cancer that had been injected with ST8/pSEndo showed efficient tumor suppression by inducing more severe necrosis and inhibiting blooding vessel density within tumors. Our findings provide a therapeutic platform for indirectly acting therapeutic strategies such as anti-angiogenesis and immune therapy. PMID:27558018

  20. Acquisition of a single EZH2 D1 domain mutation confers acquired resistance to EZH2-targeted inhibitors

    PubMed Central

    Baker, Theresa; Nerle, Sujata; Pritchard, Justin; Zhao, Boyang; Rivera, Victor M.

    2015-01-01

    Although targeted therapies have revolutionized cancer treatment, overcoming acquired resistance remains a major clinical challenge. EZH2 inhibitors (EZH2i), EPZ-6438 and GSK126, are currently in the early stages of clinical evaluation and the first encouraging signs of efficacy have recently emerged in the clinic. To anticipate mechanisms of resistance to EZH2i, we used a forward genetic platform combining a mutagenesis screen with next generation sequencing technology and identified a hotspot of secondary mutations in the EZH2 D1 domain (Y111 and I109). Y111D mutation within the WT or A677G EZH2 allele conferred robust resistance to both EPZ-6438 and GSK126, but it only drove a partial resistance within the Y641F allele. EZH2 mutants required histone methyltransferase (HMT) catalytic activity and the polycomb repressive complex 2 (PRC2) components, SUZ12 and EED, to drive drug resistance. Furthermore, D1 domain mutations not only blocked the ability of EZH2i to bind to WT and A677G mutant, but also abrogated drug binding to the Y641F mutant. These data provide the first cellular validation of the mechanistic model underpinning the oncogenic function of WT and mutant EZH2. Importantly, our findings suggest that acquired-resistance to EZH2i may arise in WT and mutant EZH2 patients through a single mutation that remains targetable by second generation EZH2i. PMID:26360609

  1. Small-Molecule Inhibitors That Target Protein–Protein Interactions in the RAD51 Family of Recombinases

    PubMed Central

    Scott, Duncan E; Coyne, Anthony G; Venkitaraman, Ashok; Blundell, Tom L; Abell, Chris; Hyvönen, Marko

    2015-01-01

    The development of small molecules that inhibit protein–protein interactions continues to be a challenge in chemical biology and drug discovery. Herein we report the development of indole-based fragments that bind in a shallow surface pocket of a humanised surrogate of RAD51. RAD51 is an ATP-dependent recombinase that plays a key role in the repair of double-strand DNA breaks. It both self-associates, forming filament structures with DNA, and interacts with the BRCA2 protein through a common “FxxA” tetrapeptide motif. We elaborated previously identified fragment hits that target the FxxA motif site and developed small-molecule inhibitors that are approximately 500-fold more potent than the initial fragments. The lead compounds were shown to compete with the BRCA2-derived Ac-FHTA-NH2 peptide and the self-association peptide of RAD51, but they had no effect on ATP binding. This study is the first reported elaboration of small-molecular-weight fragments against this challenging target. PMID:25470112

  2. Some phosphonic acid analogs as inhibitors of pyrophosphate-dependent phosphofructokinase, a novel target in Toxoplasma gondii.

    PubMed

    Peng, Z Y; Mansour, J M; Araujo, F; Ju, J Y; McKenna, C E; Mansour, T E

    1995-01-01

    Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was identified previously in Toxoplasma gondii as the only kinase that phosphorylates fructose-6-P to fructose-1,6-bisP. Since such an enzyme is not present in mammals, it was considered to be a good target for prospective selective inhibitors of the parasite. We have examined the effects of several phosphonic acid derivatives, analogs of pyrophosphate, on PPi-PFK activity, as well as on the replication of T. gondii in human foreskin fibroblast (HFF) cells. The most active compound in inhibiting PPi-PFK was tetrasodium carbonyldiphosphonate. Several bisphosphonates and related arylhydrazones showed inhibition of the enzyme, but with higher IC50 values. Although several phosphonoacetic acid derivatives also inhibited PPi-PFK, as a group they were less potent than the bisphosphonate derivatives. Comparison among the structures of various inhibitors and their effects against PPi-PFK indicates that a carbonyl (C=O) or amino (C=N) group between two phosphoryl moieties is associated with more potent enzyme inhibiton. Tetrasodium carbonyldiphosphonate did not show a significant effect against replication of T. gondii cells, probably because, as a charged molecule, it could not cross the cell membrane to reach the intracellular parasite. Tetraisopropyl carbonyldiphosphonate 2,4-dinitrophenylhydrazone showed some selective inhibitory effect against replication of the parasite in the HFF cells and protected the mammalian cells from damage by T. gondii. The results indicate that carbonyldiphosphonic acid is a good prototype compound that is amenable to chemical manipulation, which, in turn, may optimize selective inhibition of T. gondii PPi-PFK and increase accessibility to the intracellular parasite.

  3. A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding

    PubMed Central

    Lin, Pin-Fang; Blair, Wade; Wang, Tao; Spicer, Timothy; Guo, Qi; Zhou, Nannan; Gong, Yi-Fei; Wang, H.-G. Heidi; Rose, Ronald; Yamanaka, Gregory; Robinson, Brett; Li, Chang-Ben; Fridell, Robert; Deminie, Carol; Demers, Gwendeline; Yang, Zheng; Zadjura, Lisa; Meanwell, Nicholas; Colonno, Richard

    2003-01-01

    BMS-378806 is a recently discovered small molecule HIV-1 inhibitor that blocks viral entrance to cells. The compound exhibits potent inhibitory activity against a panel of R5-(virus using the CCR5 coreceptor), X4-(virus using the CXCR4 coreceptor), and R5/X4 HIV-1 laboratory and clinical isolates of the B subtype (median EC50 of 0.04 μM) in culture assays. BMS-378806 is selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses, and exhibits no significant cytotoxicity in the 14 cell types tested (concentration for 50% reduction of cell growth, >225 μM). Mechanism of action studies demonstrated that BMS-378806 binds to gp120 and inhibits the interactions of the HIV-1 envelope protein to cellular CD4 receptors. Further confirmation that BMS-378806 targets the envelope in infected cells was obtained through the isolation of resistant variants and the mapping of resistance substitutions to the HIV-1 envelope. In particular, two substitutions, M426L and M475I, are situated in the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to compound inhibition. BMS-378806 displays many favorable pharmacological traits, such as low protein binding, minimal human serum effect on anti-HIV-1 potency, good oral bioavailability in animal species, and a clean safety profile in initial animal toxicology studies. Together, the data show that BMS-378806 is a representative of a new class of HIV inhibitors that has the potential to become a valued addition to our current armamentarium of antiretroviral drugs. PMID:12930892

  4. Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation

    PubMed Central

    Polireddy, Kishore; Dong, Ruochen; McDonald, Peter R.; Wang, Tao; Luke, Brendan; Chen, Ping; Broward, Melinda; Roy, Anuradha; Chen, Qi

    2016-01-01

    Background Pancreatic cancer has an enrichment of stem-like cancer cells (CSCs) that contribute to chemoresistant tumors prone to metastasis and recurrence. Drug screening assays based on cytotoxicity cannot identify specific CSC inhibitors, because CSCs comprise only a small portion of cancer cell population, and it is difficult to propagate stable CSC populations in vitro for high-throughput screening (HTS) assays. Based on the important role of cancer cell epithelial-to-mesenchymal transition (EMT) in promoting CSCs, we hypothesized that inhibition of EMT can be a useful strategy for inhibiting CSCs, and therefore a feasible approach for HTS can be built for identification of CSC inhibitors, based on assays detecting EMT inhibition. Methods An immunofluorescent assay was established and optimized for HTS to identify compounds that enhance E-cadherin expression, as a hallmark of inhibition of EMT. Four chemical libraries containing 41,472 compounds were screened in PANC-1 pancreatic cancer cell line. Positive hits were validated for EMT and CSC inhibition in vitro using sphere formation assay, western blotting, immune fluorescence, and scratch assay. Results Initial hits were refined to 73 compounds with a secondary screening, among which 17 exhibited concentration dependent induction of E-cadherin expression. Six compounds were selected for further study which belonged to 2 different chemical structural clusters. A novel compound 1-(benzylsulfonyl) indoline (BSI, Compound #38) significantly inhibited pancreatic cancer cell migration and invasion. BSI inhibited histone deacetylase, increased histone 4 acetylation preferably, resulting in E-cadherin up-regulation. BSI effectively inhibited tumor spheres formation. Six more analogues of BSI were tested for anti-migration and anti-CSC activities. Conclusion This study demonstrated a feasible approach for discovery of agents targeting EMT and CSCs using HTS, and identified a class of novel chemicals that could be

  5. Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

    PubMed Central

    Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; Bensen, Daniel C.; Chen, Zhiyong; Lam, Thanh; Zhang, Junhu; Lee, Suk Joong; Hough, Grayson; Phillipson, Doug; Akers-Rodriguez, Suzanne; Cunningham, Mark L.; Kwan, Bryan P.; Nelson, Kirk J.; Castellano, Amanda; Locke, Jeff B.; Brown-Driver, Vickie; Murphy, Timothy M.; Ong, Voon S.; Pillar, Chris M.; Shinabarger, Dean L.; Nix, Jay; Lightstone, Felice C.; Wong, Sergio E.; Nguyen, Toan B.; Shaw, Karen J.; Finn, John

    2013-01-01

    Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models. PMID:24386374

  6. Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

    DOE PAGES

    Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; Bensen, Daniel C.; Chen, Zhiyong; Lam, Thanh; Zhang, Junhu; Lee, Suk Joong; Hough, Grayson; Phillipson, Doug; et al

    2013-12-26

    Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highlymore » conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.« less

  7. Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

    SciTech Connect

    Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; Bensen, Daniel C.; Chen, Zhiyong; Lam, Thanh; Zhang, Junhu; Lee, Suk Joong; Hough, Grayson; Phillipson, Doug; Akers-Rodriguez, Suzanne; Cunningham, Mark L.; Kwan, Bryan P.; Nelson, Kirk J.; Castellano, Amanda; Locke, Jeff B.; Brown-Driver, Vickie; Murphy, Timothy M.; Ong, Voon S.; Pillar, Chris M.; Shinabarger, Dean L.; Nix, Jay; Lightstone, Felice C.; Wong, Sergio E.; Nguyen, Toan B.; Shaw, Karen J.; Finn, John

    2013-12-26

    Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.

  8. Design, synthesis and characterization of dual inhibitors against new targets FabG4 and HtdX of Mycobacterium tuberculosis.

    PubMed

    Banerjee, Deb Ranjan; Biswas, Rupam; Das, Amit K; Basak, Amit

    2015-07-15

    Herein, we present dual inhibitors of new targets FabG4 and HtdX for the first time. In this work, eight compounds have been designed, synthesized, characterized and evaluated for bio-activities. Amongst them, six compounds have shown inhibitory activities. Three of them (12-14) demonstrate dual inhibition of both FabG4 and HtdX at low micromolar concentration. In addition, the dual inhibitors show good anti-mycobacterial properties against both planktonic growth and biofilm culture of Mycobacterium species. This study is an important addition to tuberculosis drug discovery because it explores two new enzymes as drug targets and presents their dual inhibitors as good candidates for pre-clinical trials. PMID:26092447

  9. 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

  10. 4,5-Diarylisoxazol-3-carboxylic acids: A new class of leukotriene biosynthesis inhibitors potentially targeting 5-lipoxygenase-activating protein (FLAP).

    PubMed

    Banoglu, Erden; Çelikoğlu, Erşan; Völker, Susanna; Olgaç, Abdurrahman; Gerstmeier, Jana; Garscha, Ulrike; Çalışkan, Burcu; Schubert, Ulrich S; Carotti, Andrea; Macchiarulo, Antonio; Werz, Oliver

    2016-05-01

    In this article, we report novel leukotriene (LT) biosynthesis inhibitors that may target 5-lipoxygenase-activating protein (FLAP) based on the previously identified isoxazole derivative (8). The design and synthesis was directed towards a subset of 4,5-diaryl-isoxazole-3-carboxylic acid derivatives as LT biosynthesis inhibitors. Biological evaluation disclosed a new skeleton of potential anti-inflammatory agents, exemplified by 39 and 40, which potently inhibit cellular 5-LO product synthesis (IC50 = 0.24 μM, each) seemingly by targeting FLAP with weak inhibition on 5-LO (IC50 ≥ 8 μM). Docking studies and molecular dynamic simulations with 5-LO and FLAP provide valuable insights into potential binding modes of the inhibitors. Together, these diaryl-isoxazol-3-carboxylic acids may possess potential as leads for development of effective anti-inflammatory drugs through inhibition of LT biosynthesis. PMID:26922224

  11. A high-affinity [18F]-labeled phosphoramidate peptidomimetic PSMA-targeted inhibitor for PET imaging of prostate cancer

    PubMed Central

    Ganguly, Tanushree; Dannoon, Shorouk; Hopkins, Mark R.; Murphy, Stephanie; Cahaya, Hendry; Blecha, Joseph E.; Jivan, Salma; Drake, Christopher R.; Barinka, Cyril; Jones, Ella F.; VanBrocklin, Henry F.; Berkman, Clifford E.

    2015-01-01

    Introduction In this study, a structurally modified phosphoramidate scaffold, with improved prostate-specific membrane antigen (PSMA) avidity, stability and in vivo characteristics, as a PET imaging agent for prostate cancer (PCa), was prepared and evaluated. Methods p-Fluorobenzoyl-aminohexanoate and 2-(3-hydroxypropyl)glycine were introduced into the PSMA-targeting scaffold yielding phosphoramidate 5. X-ray crystallography was performed on the PSMA/5 complex. [18F]5 was synthesized, and cell uptake and internalization studies were conducted in PSMA(+) LNCaP and CWR22Rv1 cells and PSMA(−) PC-3 cells. In vivo PET imaging and biodistribution studies were performed at 1 and 4 h post injection in mice bearing CWR22Rv1 tumor, with or without blocking agent. Results The crystallographic data showed interaction of the p-fluorobenzoyl group with an arene-binding cleft on the PSMA surface. In vitro studies revealed elevated uptake of [18F]5 in PSMA(+) cells (2.2% in CWR22Rv1 and 12.1% in LNCaP) compared to PSMA(−) cells (0.08%) at 4 h. In vivo tumor uptake of 2.33% ID/g and tumor-to-blood ratio of 265:1 was observed at 4 h. Conclusions We have successfully synthesized, radiolabeled and evaluated a new PSMA-targeted PET agent. The crystal structure of the PSMA/5 complex highlighted the interactions within the arene-binding cleft contributing to the overall complex stability. The high target uptake and rapid non-target clearance exhibited by [18F]5 in PSMA(+) xenografts substantiates its potential use for PET imaging of PCa. Advances in Knowledge The only FDA-approved imaging agent for PCa, Prostascint®, targets PSMA but suffers from inherent shortcomings. The data acquired in this manuscript confirmed that our new generation of [18F]-labeled PSMA inhibitor exhibited promising in vivo performance as a PET imaging agent for PCa and is well-positioned for subsequent clinical trials. Implications for Patient Care Our preliminary data demonstrate that this tracer possesses

  12. Narrow-spectrum inhibitors targeting an alternative menaquinone biosynthetic pathway of Helicobacter pylori.

    PubMed

    Yamamoto, Tsuyoshi; Matsui, Hidenori; Yamaji, Kenzaburo; Takahashi, Tetsufumi; Øverby, Anders; Nakamura, Masahiko; Matsumoto, Atsuko; Nonaka, Kenichi; Sunazuka, Toshiaki; Ōmura, Satoshi; Nakano, Hirofumi

    2016-09-01

    We aimed to identify narrow-spectrum natural compounds that specifically inhibit an alternative menaquinone (MK; vitamin K2) biosynthetic pathway (the futalosine pathway) of Helicobacter pylori. Culture broth samples of 6183 microbes were examined using the paper disc method with different combinations of 2 of the following 3 indicator microorganisms: Bacillus halodurans C-125 and Kitasatospora setae KM-6054(T), which have only the futalosine pathway of MK biosynthesis, and Bacillus subtilis H17, which has only the canonical MK biosynthetic pathway. Most of the active compounds isolated from culture broth samples were from the families of polyunsaturated fatty acids (PUFAs). Only one compound isolated from the culture broth of Streptomyces sp. K12-1112, siamycin I (a 21-residue lasso peptide antibiotic), targeted the futalosine pathway. The inhibitory activities of representative PUFAs and siamycin I against the growth of B. halodurans or K. setae were abrogated by supplementation with MK. Thereafter, the growth of H. pylori strains SS1 and TN2GF4 in broth cultures was dose-dependently suppressed by eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or siamycin I, and these inhibitory effects were reduced by supplementation with MK. Daily administration of EPA (100 μM), DHA (100 μM), or siamycin I (2.5 μM) in drinking water reduced the H. pylori SS1 colonization in the gastric mucosa of C57BL/6 mice by 96%, 78%, and 68%, respectively. These data suggest that EPA, DHA, and siamycin I prevented H. pylori infection by inhibiting the futalosine pathway of MK biosynthesis. PMID:27346378

  13. Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor.

    PubMed

    Ferreira de Freitas, Renato; Eram, Mohammad S; Szewczyk, Magdalena M; Steuber, Holger; Smil, David; Wu, Hong; Li, Fengling; Senisterra, Guillermo; Dong, Aiping; Brown, Peter J; Hitchcock, Marion; Moosmayer, Dieter; Stegmann, Christian M; Egner, Ursula; Arrowsmith, Cheryl; Barsyte-Lovejoy, Dalia; Vedadi, Masoud; Schapira, Matthieu

    2016-02-11

    Protein methyltransferases (PMTs) are a promising target class in oncology and other disease areas. They are composed of SET domain methyltransferases and structurally unrelated Rossman-fold enzymes that include protein arginine methyltransferases (PRMTs). In the absence of a well-defined medicinal chemistry tool-kit focused on PMTs, most current inhibitors were identified by screening large and diverse libraries of leadlike molecules. So far, no successful fragment-based approach was reported against this target class. Here, by deconstructing potent PRMT inhibitors, we find that chemical moieties occupying the substrate arginine-binding site can act as efficient fragment inhibitors. Screening a fragment library against PRMT6 produced numerous hits, including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased global histone 3 arginine 2 methylation in cells, and can serve as a warhead for the development of PRMT chemical probes.

  14. A recommended practical approach to the management of target therapy and angiogenesis inhibitors cardiotoxicity: an opinion paper of the working group on drug cardiotoxicity and cardioprotection, Italian Society of Cardiology

    PubMed Central

    Maurea, Nicola; Spallarossa, Paolo; Cadeddu, Christian; Madonna, Rosalinda; Mele, Donato; Monte, Ines; Novo, Giuseppina; Pagliaro, Pasquale; Pepe, Alessia; Tocchetti, Carlo G.; Zito, Concetta; Mercuro, Giuseppe

    2016-01-01

    The US National Cancer Institute estimates that cardiotoxicity (CTX) from target therapy refers mostly to four groups of drugs: epidermal growth factor receptor 2 inhibitors, angiogenic inhibitors, directed Abelson murine leukemia viral oncogene homolog inhibitors, and proteasome inhibitors. The main cardiotoxic side-effects related to antiepidermal growth factor receptor 2 therapy are left ventricular systolic dysfunction and heart failure. Angiogenesis inhibitors are associated with hypertension, left ventricular dysfunction/heart failure, myocardial ischemia, QT prolongation, and thrombosis. Moreover, other agents may be related to CTX induced by treatment. In this study, we review the guidelines for a practical approach for the management of CTX in patients under anticancer target therapy. PMID:27183530

  15. Nucleic acid detection kits

    DOEpatents

    Hall, Jeff G.; Lyamichev, Victor I.; Mast, Andrea L.; Brow, Mary Ann; Kwiatkowski, Robert W.; Vavra, Stephanie H.

    2005-03-29

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of nucleic acid from various viruses in a sample.

  16. Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ.

    PubMed

    Sonamoto, Rie; Kii, Isao; Koike, Yuka; Sumida, Yuto; Kato-Sumida, Tomoe; Okuno, Yukiko; Hosoya, Takamitsu; Hagiwara, Masatoshi

    2015-01-01

    The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer's disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases. PMID:26234946

  17. Targeted Metabolomics Approach To Detect the Misuse of Steroidal Aromatase Inhibitors in Equine Sports by Biomarker Profiling.

    PubMed

    Chan, George Ho Man; Ho, Emmie Ngai Man; Leung, David Kwan Kon; Wong, Kin Sing; Wan, Terence See Ming

    2016-01-01

    The use of anabolic androgenic steroids (AAS) is prohibited in both human and equine sports. The conventional approach in doping control testing for AAS (as well as other prohibited substances) is accomplished by the direct detection of target AAS or their characteristic metabolites in biological samples using hyphenated techniques such as gas chromatography or liquid chromatography coupled with mass spectrometry. Such an approach, however, falls short when dealing with unknown designer steroids where reference materials and their pharmacokinetics are not available. In addition, AASs with fast elimination times render the direct detection approach ineffective as the detection window is short. A targeted metabolomics approach is a plausible alternative to the conventional direct detection approach for controlling the misuse of AAS in sports. Because the administration of AAS of the same class may trigger similar physiological responses or effects in the body, it may be possible to detect such administrations by monitoring changes in the endogenous steroidal expression profile. This study attempts to evaluate the viability of using the targeted metabolomics approach to detect the administration of steroidal aromatase inhibitors, namely androst-4-ene-3,6,17-trione (6-OXO) and androsta-1,4,6-triene-3,17-dione (ATD), in horses. Total (free and conjugated) urinary concentrations of 31 endogenous steroids were determined by gas chromatography-tandem mass spectrometry for a group of 2 resting and 2 in-training thoroughbred geldings treated with either 6-OXO or ATD. Similar data were also obtained from a control (untreated) group of in-training thoroughbred geldings (n = 28). Statistical processing and chemometric procedures using principle component analysis and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) have highlighted 7 potential biomarkers that could be used to differentiate urine samples obtained from the control and the treated groups

  18. Modelling of human leucyl aminopeptidases for in silico off target binding analysis of potential Plasmodium falciparum leucine aminopeptidase (PfA-M17) specific inhibitors.

    PubMed

    Sahi, Shakti; Raj, Utkarsh; Chaudhary, Meenakshi; Nain, Vikrant

    2014-01-01

    Malaria is one of the most widespread infectious diseases in the world. Emergence of multi-drug resistant Plasmodium strains makes it crucial to identify new classes of compounds for anti-malarial therapy. Novel anti-malarial compounds from natural sources (Gomphostema niveum) as well as synthetic chemicals (5-aminolevulinic acid) have been reported in recent patents. Plasmodium falciparum leucyl aminopeptidase (PfA-M17) is a validated target for antimalarial drug development. However, known aminopeptidase inhibitors beset with the problem of non-specificity. Therefore, 3D structural models of PfA-M17 human homologs, Leucine aminopeptidase3 (hLAP3) and probable leucine aminopeptidase (hNPEPL1) were predicted for molecular docking based screening of potential inhibitors for their off target activity. Comparison of IC50 and docking scores of highly active hLAP3 inhibitors shows good correlation (r(2)≈ 0.8). Further, docking analysis with potential PfA-M17 inhibitor Compound-X (identified through virtual screening) shows much higher binding affinity towards PfA-M17 (docking score -11.44) than hLAP3 (docking score -4.26) and hNPEPL1 (docking score -5.08). This lead compound, Compound-X can act as a scaffold for further increasing PfA-M17 binding affinity and hLAP3 and hNPEPL1 3D structure models will be useful for screening of PfA-M17 specific inhibitors.

  19. Targeting Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MAPKAPK2, MK2): Medicinal Chemistry Efforts To Lead Small Molecule Inhibitors to Clinical Trials.

    PubMed

    Fiore, Mario; Forli, Stefano; Manetti, Fabrizio

    2016-04-28

    The p38/MAPK-activated kinase 2 (MK2) pathway is involved in a series of pathological conditions (inflammation diseases and metastasis) and in the resistance mechanism to antitumor agents. None of the p38 inhibitors entered advanced clinical trials because of their unwanted systemic side effects. For this reason, MK2 was identified as an alternative target to block the pathway but avoiding the side effects of p38 inhibition. However, ATP-competitive MK2 inhibitors suffered from low solubility, poor cell permeability, and scarce kinase selectivity. Fortunately, non-ATP-competitive inhibitors of MK2 have been already discovered that allowed circumventing the selectivity issue. These compounds showed the additional advantage to be effective at lower concentrations in comparison to the ATP-competitive inhibitors. Therefore, although the significant difficulties encountered during the development of these inhibitors, MK2 is still considered as an attractive target to treat inflammation and related diseases to prevent tumor metastasis and to increase tumor sensitivity to chemotherapeutics.

  20. Compensatory activation of Akt in response to mTOR and Raf inhibitors - a rationale for dual-targeted therapy approaches in neuroendocrine tumor disease.

    PubMed

    Zitzmann, Kathrin; Rüden, Janina von; Brand, Stephan; Göke, Burkhard; Lichtl, Jennifer; Spöttl, Gerald; Auernhammer, Christoph J

    2010-09-01

    Several studies have established a link between aberrant PI(3)K-Akt-mTOR- and Ras-Raf-MEK-Erk1/2 signaling and neuroendocrine tumor disease. In this study, we comparatively investigate the antitumor potential of novel small-molecule inhibitors targeting mTOR (RAD001), mTOR/PI(3)K (NVP-BEZ235) and Raf (Raf265) on human NET cell lines of heterogeneous origin. All inhibitors induced potent antitumor effects which involved the induction of apoptosis and G0/G1 arrest. However, the dual mTOR/PI(3)K inhibitor NVP-BEZ235 was more efficient compared to the single mTOR inhibitor RAD001. Consistently, NVP-BEZ235 prevented the negative feedback activation of Akt as observed after treatment with RAD001. Raf265 inhibited Erk1/2 phosphorylation but strongly induced Akt phosphorylation and VEGF secretion, suggesting the existence of a compensatory feedback loop on PI3K-Akt signaling. Finally, combined treatment with RAD001 or NVP-BEZ235 and Raf265 was more efficient than single treatment with either kinase inhibitor. Together, our data provide a rationale for dual targeting of PI(3)K-Akt-mTOR- and Ras-Raf-MEK-Erk1/2 signaling in NET disease.

  1. miR-17 targets tissue inhibitor of metalloproteinase 1 and 2 to modulate cardiac matrix remodeling.

    PubMed

    Li, Shu-Hong; Guo, Jian; Wu, Jun; Sun, Zhuo; Han, Mihan; Shan, Sze Wan; Deng, Zhaoqun; Yang, Burton B; Weisel, Richard D; Li, Ren-Ke

    2013-10-01

    We aimed to investigate the role of miR-17 in cardiac matrix remodeling following myocardial infarction (MI). Using real-time PCR, we quantified endogenous miR-17 in infarcted mouse hearts. Compared with related microRNAs, miR-17 was up-regulated most dramatically: 3.7-fold and 2.4-fold in the infarct region 3 and 7 d post-MI, respectively, and 2.4-fold in the border zone at d 3 compared to sham control (P<0.01). Chimeric luciferase reporter constructs were cloned for miR-17 target validation. miR-17 targeted the 3'-UTR of TIMP2 and the protein coding region of TIMP1. The miR-17 mimic decreased TIMP2 (P<0.01) and TIMP1 (P<0.05) protein expression compared with the scrambled control. Inhibition of endogenous miR-17 by in vivo antagomir delivery enhanced TIMP2 (P<0.01) and TIMP1 (P<0.05) protein expression compared to the mismatch group, decreased MMP9 activity (P<0.05), reduced infarct size as early as 7 d post-MI (P<0.05), and improved cardiac function (fractional shortening and fractional area contraction, P<0.05) at d 21 and 28 post-MI. Transgenic mice overexpressing miR-17 in the heart confirmed the deleterious role of miR-17 in matrix modulation. Our study suggests that miR-17 participates in the regulation of cardiac matrix remodeling and provides a novel therapeutic approach using miR-17 inhibitors to prevent remodeling and heart failure after MI.