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https://www.readbyqxmd.com/read/28165734/phantom-pains-problems-with-the-utility-of-alerts-for-pan-assay-interference-compounds
#1
Stephen J Capuzzi, Eugene N Muratov, Alexander Tropsha
The use of substructural alerts to identify Pan-Assay INterference compoundS (PAINS) has become a common component of the triage process in biological screening campaigns. These alerts, however, were originally derived from a proprietary library tested in just six assays measuring protein-protein interaction (PPI) inhibition using the AlphaScreen detection technology only; moreover, 68% (328 out of the 480 alerts) were derived from four or fewer compounds. In an effort to assess the reliability of these alerts as indicators of pan-assay interference, we performed a large-scale analysis of the impact of PAINS alerts on compound promiscuity in bioassays using publicly available data in PubChem...
February 25, 2017: Journal of Chemical Information and Modeling
https://www.readbyqxmd.com/read/28153734/high-throughput-screening-for-survivin-and-borealin-interaction-inhibitors-in-hepatocellular-carcinoma
#2
Liyun Yue, Lu Li, Dan Li, Zhuo Yang, Shuai Han, Ming Chen, Shujue Lan, Xiaojun Xu, Lijian Hui
Survivin, a key member of the chromatin passenger complex (CPC), is often highly expressed in human cancers, making it a promising target for cancer treatment. Out of the numerous reported Survivin inhibitors, YM155 is only one entering clinical trial, but was recently failed in the Phase II trial. It is important to develop Survivin inhibitors with new strategies. We recently reported that both Survivin and its binding protein Borealin in the CPC complex are essential for the development of hepatocellular carcinoma, suggesting that disrupting the interaction between Survivin and Borealin would be a promising strategy...
March 11, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28125678/inhibition-of-stat3-by-anticancer-drug-bendamustine
#3
Kazunori Iwamoto, Yutaka Uehara, Yukie Inoue, Kyoko Taguchi, Daisuke Muraoka, Naohisa Ogo, Kenji Matsuno, Akira Asai
Bendamustine (BENDA), which bears the bis(2-chloroethyl)amino moiety, is an alkylating agent that stops the growth of cancer cells by binding to DNA and interfering with its replication. However, the mechanism of action underlying its excellent clinical efficacy remains unclear. In this work, we report that BENDA inhibits signal transducer and activator of transcription 3 (STAT3). In an AlphaScreen-based biochemical assay using recombinant human STAT3, binding of STAT3-Src homology 2 (SH2) to the phosphotyrosine (pTyr, pY) peptide was inhibited by BENDA but not by the inactive metabolite dihydroxy bendamustine (HP2)...
2017: PloS One
https://www.readbyqxmd.com/read/28092042/alpha-based-multiplexed-assay-for-identifying-sh2-domain-antagonists
#4
Akira Asai, Kazuyuki Takakuma
Constitutive activation of STAT3/5b frequently occurs in various human malignancies. STAT3/5b activation involves dimerization via intermolecular pTyr-SH2 binding; therefore, antagonizing this interaction is a feasible approach to inhibit STAT3/5b activation for cancer therapy. We have developed a multiplexed assay to assess STAT3- and STAT5b-SH2 binding in a single well by combining AlphaLISA and AlphaScreen beads. In this chapter, we describe application of the method for the purpose of identifying new STAT3 and STAT5b antagonists...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27909234/a-high-throughput-screening-strategy-for-development-of-rnf8-ubc13-protein-protein-interaction-inhibitors
#5
Elisabeth Weber, Ina Rothenaigner, Stefanie Brandner, Kamyar Hadian, Kenji Schorpp
The ubiquitin-proteasome system plays an essential role in a broad range of cellular signaling pathways. Ubiquitination is a posttranslational protein modification that involves the action of an enzymatic cascade (E1, E2, and E3 enzymes) for the covalent attachment of ubiquitin to target proteins. The emerging knowledge of the molecular mechanisms and correlation of deregulation of the ubiquitin system in human diseases is uncovering new opportunities for therapeutics development. The E3 ligase RNF8 acts in cooperation with the heterodimeric E2 enzyme Ubc13/Uev1a to generate ubiquitin conjugates at the sides of DNA double-strand breaks, and recent findings suggest RNF8 as a potential therapeutic target for the treatment of breast cancer...
March 2017: SLAS Discov
https://www.readbyqxmd.com/read/27827304/development-of-an-hts-compatible-assay-for-discovery-of-melanoma-related-microphthalmia-transcription-factor-disruptors-using-alphascreen-technology
#6
Jing Wang, Pengfei Fang, Peter Chase, Sagi Tshori, Ehud Razin, Timothy P Spicer, Louis Scampavia, Peter Hodder, Min Guo
Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro...
November 8, 2016: Journal of Biomolecular Screening
https://www.readbyqxmd.com/read/27628689/a-cell-based-high-throughput-assay-for-gap-junction-communication-suitable-for-assessing-connexin-43-ezrin-interaction-disruptors-using-incucyte-zoom
#7
Aleksandra R Dukic, David W McClymont, Kjetil Taskén
Connexin 43 (Cx43), the predominant gap junction (GJ) protein, directly interacts with the A-kinase-anchoring protein (AKAP) Ezrin in human cytotrophoblasts and a rat liver epithelial cells (IAR20). The Cx43-Ezrin-protein kinase (PKA) complex facilitates Cx43 phosphorylation by PKA, which triggers GJ opening in cytotrophoblasts and IAR20 cells and may be a general mechanism regulating GJ intercellular communication (GJIC). Considering the importance of Cx43 GJs in health and disease, they are considered potential pharmaceutical targets...
September 14, 2016: Journal of Biomolecular Screening
https://www.readbyqxmd.com/read/27613056/using-alphascreen-%C3%A2-to-identify-small-molecule-inhibitors-targeting-a-conserved-host-pathogen-interaction
#8
Sisley Austin, Saïd Taouji, Eric Chevet, Harald Wodrich, Fabienne Rayne
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...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27494158/evaluation-of-lipopeptides-as-toll-like-receptor-2-ligands
#9
Waleed M Hussein, Phil Choi, Cheng Zhang, Mei Su, Emma Sierecki, Wayne Johnston, Vincent Fagan, Kirill Alexandrov, Mariusz Skwarczynski, Yann Gambin, Istvan Toth, P Simerska
Peptide-based vaccines are considered to be the next generation of modern immunizations, as they are safe, easy to produce and well-defined. However, due to their weak immunogenic effect, it is important to first develop an appropriate adjuvant for peptide-based vaccines. In this work, a series of four adjuvanting moieties was synthesized as alkyne derivatives, incorporating dipalmitoyl serine (DPS), 1,3-diglyceride (DG), two hexadecane lipoamino acids (diLAA), and 2,3-dipalmitoyl-S-glycerylcysteine (Pam2Cys)...
August 4, 2016: Current Drug Delivery
https://www.readbyqxmd.com/read/27379031/h11-hspb8-restricts-hiv-2-vpx-to-restore-the-anti-viral-activity-of-samhd1
#10
Ayumi Kudoh, Kei Miyakawa, Satoko Matsunaga, Yuki Matsushima, Isao Kosugi, Hirokazu Kimura, Satoshi Hayakawa, Tatsuya Sawasaki, Akihide Ryo
Virus-host interactions play vital roles in viral replication and virus-induced pathogenesis. Viruses rely entirely upon host cells to reproduce progeny viruses; however, host factors positively or negatively regulate virus replication by interacting with viral proteins. The elucidation of virus-host protein interaction not only provides a better understanding of the molecular mechanisms by which host cells combat viral infections, but also facilitates the development of new anti-viral therapeutics. Identification of relevant host factors requires techniques that enable comprehensive characterization of virus-host protein interactions...
2016: Frontiers in Microbiology
https://www.readbyqxmd.com/read/27374490/discovery-and-structural-optimization-of-4-4-benzyloxy-phenyl-3-4-dihydropyrimidin-2-1h-ones-as-rorc-inverse-agonists
#11
Xi-Shan Wu, Rui Wang, Yan-Li Xing, Xiao-Qian Xue, Yan Zhang, Yong-Zhi Lu, Yu Song, Xiao-Yu Luo, Chun Wu, Yu-Lai Zhou, Jian-Qin Jiang, Yong Xu
AIM: Retinoic acid receptor-related orphan nuclear receptors (RORs) are orphan nuclear receptors that show constitutive activity in the absence of ligands. Among 3 subtypes of RORs, RORc is a promising therapeutic target for the treatment of Th17-mediated autoimmune diseases. Here, we report novel RORc inverse agonists discovered through structure-based drug design. METHODS: Based on the structure of compound 8, a previously described agonist of RORa, a series of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives were designed and synthesized...
November 2016: Acta Pharmacologica Sinica
https://www.readbyqxmd.com/read/27316989/alphascreen-based-assays-ultra-high-throughput-screening-for-small-molecule-inhibitors-of-challenging-enzymes-and-protein-protein-interactions
#12
Adam Yasgar, Ajit Jadhav, Anton Simeonov, Nathan P Coussens
AlphaScreen technology has been routinely utilized in high-throughput screening assays to quantify analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range, and sensitivity associated with AlphaScreens as well as the homogenous assay format and reagent stability make the technology particularly well suited for high-throughput screening applications. Here, we describe the development of AlphaScreen assays to identify small-molecule inhibitors of enzymes and protein-protein interactions using the highly miniaturized 1536-well format...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27249653/establishment-of-a-wheat-cell-free-synthesized-protein-array-containing-250-human-and-mouse-e3-ubiquitin-ligases-to-identify-novel-interaction-between-e3-ligases-and-substrate-proteins
#13
Hirotaka Takahashi, Atsushi Uematsu, Satoshi Yamanaka, Mei Imamura, Tatsuro Nakajima, Kousuke Doi, Saki Yasuoka, Chikako Takahashi, Hiroyuki Takeda, Tatsuya Sawasaki
Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3). Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell...
2016: PloS One
https://www.readbyqxmd.com/read/27245893/a-functional-assay-for-gpr55-envision-protocol
#14
Sharon Anavi-Goffer, Ruth A Ross
AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27243554/signal-transduction-pathways-activated-by-insulin-like-peptide-5-insl5-at-the-relaxin-family-peptide-receptor-4-rxfp4
#15
Sheng Y Ang, Dana S Hutchinson, Nitin Patil, Bronwyn A Evans, Ross A D Bathgate, Michelle L Halls, Mohammed A Hossain, Roger J Summers, Martina Kocan
BACKGROUND AND PURPOSE: Insulin-like peptide 5 (INSL5) is a two-chain, three-disulphide bonded peptide of the insulin/relaxin superfamily, uniquely expressed in enteroendocrine L-cells of the colon. It is the cognate ligand of relaxin family peptide receptor 4 (RXFP4) that is mainly expressed in the colorectum and enteric nervous system. This study identifies new signalling pathways activated by INSL5 acting on RXFP4. EXPERIMENTAL APPROACH: INSL5/RXFP4 signalling was investigated using AlphaScreen® proximity assays...
May 31, 2016: British Journal of Pharmacology
https://www.readbyqxmd.com/read/27110299/the-bromodomain-inhibitor-n-methyl-pyrrolidone-reduced-fat-accumulation-in-an-ovariectomized-rat-model
#16
Bebeka Gjoksi, Chafik Ghayor, Indranil Bhattacharya, Marcy Zenobi-Wong, Franz E Weber
BACKGROUND: Weight gain is one of the consequences of estrogen deficiency and constitutes a major health problem. The present study highlights the effects of N-methyl pyrrolidone (NMP) on adipogenesis in osteoporosis induced by estrogen deficiency in an ovariectomized rat model. RESULTS: Ovariectomy resulted in body weight gain, increased femoral marrow adipocytes, and hypertrophic adipocytes in white adipose tissue, distorted serum leptin, and TNF-α and PPARγ levels...
2016: Clinical Epigenetics
https://www.readbyqxmd.com/read/27108602/generation-and-characterization-of-a-human-nanobody-against-vegfr-2
#17
Lin Ma, Kai Gu, Cheng-Hai Zhang, Xue-Tao Chen, Yi Jiang, Karsten Melcher, Juan Zhang, Min Wang, H Eric Xu
AIM: Nanobody is an antibody fragment consisting of a single monomeric variable antibody domain, which can be used for a variety of biotechnological and therapeutic purposes. The aim of this work was to isolate and characterize a human signal domain antibody against VEGFR-2 domain3 (VEGFR D3) from a phage display library. METHODS: To produce antigen-specific recombinant nanobodies with high affinity to VEGFR2 D3, a liquid phase panning strategy was used for all rounds of panning...
June 2016: Acta Pharmacologica Sinica
https://www.readbyqxmd.com/read/27089417/modulation-of-l-%C3%AE-lysophosphatidylinositol-gpr55-map-kinase-signalling-by-cb2-receptor-agonists-identifying-novel-gpr55-inhibitors
#18
Sharon Anavi-Goffer, Andrew J Irving, Ruth A Ross
BACKGROUND: GPR55 is a lipid-sensing G protein-coupled receptor that is activated by the endogenous lipid l-α-lysophosphatidylinositol (LPI) and can be modulated by certain cannabinoid ligands. METHODS: In this study we investigated the GPR55 activity of four synthetic CB2 receptor agonists using the AlphaScreen® SureFire® assay. RESULTS: Here we show that the CB2 receptor-selective agonists HU-308, HU-433 and HU-910 do not promote GPR55-mediated ERK1/2 phosphorylation up to a concentration of 3 μM...
May 1, 2016: Journal of Basic and Clinical Physiology and Pharmacology
https://www.readbyqxmd.com/read/27044684/identification-of-small-molecule-frequent-hitters-of-glutathione-s-transferase-glutathione-interaction
#19
Jara K Brenke, Elena S Salmina, Larissa Ringelstetter, Scarlett Dornauer, Maria Kuzikov, Ina Rothenaigner, Kenji Schorpp, Fabian Giehler, Jay Gopalakrishnan, Arnd Kieser, Sheraz Gul, Igor V Tetko, Kamyar Hadian
In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction...
July 2016: Journal of Biomolecular Screening
https://www.readbyqxmd.com/read/27043267/identification-of-n-phenyl-2-n-phenylphenylsulfonamido-acetamides-as-new-ror%C3%AE-inverse-agonists-virtual-screening-structure-based-optimization-and-biological-evaluation
#20
Yu Song, Xiaoqian Xue, Xishan Wu, Rui Wang, Yanli Xing, Weiqun Yan, Yulai Zhou, Chao-Nan Qian, Yan Zhang, Yong Xu
Retinoic acid receptor-related orphan receptors (RORs) are ligand-dependent transcriptional factors and members of the nuclear receptor superfamily. RORs regulate inflammation, metabolic disorders and circadian rhythm. RORγ is a promising therapeutic drug target for treating Th17-mediated autoimmune diseases. In our study, we performed structure-based virtual screening and ligand-based virtual screening targeting the RORγ ligand-binding domain and successfully identified N-phenyl-2-(N-phenylphenylsulfonamido) acetamides as a type of RORγ inverse agonist...
June 30, 2016: European Journal of Medicinal Chemistry
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