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1
McCallum, Megan M., Premchendar Nandhikonda, Jonathan J. Temmer, Charles Eyermann, Anton Simeonov, Ajit Jadhav, Adam Yasgar, David Maloney, and Alexander (Leggy) Arnold. "High-Throughput Identification of Promiscuous Inhibitors from Screening Libraries with the Use of a Thiol-Containing Fluorescent Probe." Journal of Biomolecular Screening 18, no. 6 (February 27, 2013): 705–13. http://dx.doi.org/10.1177/1087057113476090.
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Testing small molecules for their ability to modify cysteine residues of proteins in the early stages of drug discovery is expected to accelerate our ability to develop more selective drugs with lesser side effects. In addition, this approach also enables the rapid evaluation of the mode of binding of new drug candidates with respect to thiol reactivity and metabolism by glutathione. Herein, we describe the development of a fluorescence-based high-throughput assay that allows the identification of thiol-reactive compounds. A thiol-containing fluorescent probe, MSTI, was synthesized and used to evaluate small molecules from the Library of Pharmacologically Active Compounds (LOPAC) collection of bioactive molecules. LOPAC compounds that are known to react with sulfur nucleophiles were identified with this assay, for example, irreversible protease inhibitors, nitric oxide–releasing compounds, and proton-pump inhibitors. The results confirm that both electrophilic and redox reactive compounds can be quickly identified in a high-throughput manner, enabling the assessment of screening libraries with respect to thiol-reactive compounds.
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Haus, Patricia, Michael Korbus, Michael Schröder, and Franz-Josef Meyer-Almes. "Identification of Selective Class II Histone Deacetylase Inhibitors Using a Novel Dual-Parameter Binding Assay Based on Fluorescence Anisotropy and Lifetime." Journal of Biomolecular Screening 16, no. 10 (October 25, 2011): 1206–16. http://dx.doi.org/10.1177/1087057111424605.
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Histone deacetylases (HDACs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. Consequently, HDACs have emerged as promising targets for cancer therapy. The drugability of HDACs has been shown by the discovery of several structural classes of inhibitors (HDACis), particularly by the recent approval of two HDACis, vorinostat (ZOLINZA) and romidepsin (Istodax), for the treatment of cutaneous T-cell lymphoma by the US Food and Drug Administration. The outstanding potential of HDACis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing high-throughput capable assays for screening campaigns. In this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the LOPAC (Sigma-Aldrich, St Louis, MO) library against the bacterial histone deacetylase homologue HDAH from Bordetella, which shares 35% identity with the second deacetylase domain of HDAC6. The binding assay proved to be highly suitable for high-throughput screening campaigns. Several LOPAC compounds have been identified to inhibit HDAH in the lower micromolar range. Most interestingly, some of the hit compounds turned out to be weak but selective inhibitors of human class IIa and IIb HDACs.
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Chen, Xian, Sudini Ranshaya Fernando, Yin-Lau Lee, William Shu-Biu Yeung, Ernest Hung-Yu Ng, Raymond Hang-Wun Li, and Kai-Fai Lee. "High-Throughput In Vitro Screening Identified Nemadipine as a Novel Suppressor of Embryo Implantation." International Journal of Molecular Sciences 23, no. 9 (May 3, 2022): 5073. http://dx.doi.org/10.3390/ijms23095073.
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Current contraceptive methods interfere with folliculogenesis, fertilization, and embryo implantation by physical or hormonal approaches. Although hormonal contraceptive pills are effective in regulating egg formation, they are less effective in preventing embryo implantation. To explore the use of non-hormonal compounds that suppress embryo implantation, we established a high-throughput spheroid-endometrial epithelial cell co-culture assay to screen the Library of Pharmacologically Active Compounds (LOPAC) for compounds that affect trophoblastic spheroid (blastocyst surrogate) attachment onto endometrial epithelial Ishikawa cells. We identified 174 out of 1280 LOPAC that significantly suppressed BeWo spheroid attachment onto endometrial Ishikawa cells. Among the top 20 compounds, we found the one with the lowest cytotoxicity in Ishikawa cells, P11B5, which was later identified as Nemadipine-A. Nemadipine-A at 10 µM also suppressed BeWo spheroid attachment onto endometrial epithelial RL95-2 cells and primary human endometrial epithelial cells (hEECs) isolated from LH +7/8-day endometrial biopsies. Mice at 1.5 days post coitum (dpc) treated with a transcervical injection of 100 µg/kg Nemadipine-A or 500 µg/kg PRI-724 (control, Wnt-inhibitor), but not 10 µg/kg Nemadipine-A, suppressed embryo implantation compared with controls. The transcript expressions of endometrial receptivity markers, integrin αV (ITGAV) and mucin 1 (MUC1), but not β-catenin (CTNNB1), were significantly decreased at 2.5 dpc in the uterus of treated mice compared with controls. The reduction of embryo implantation by Nemadipine-A was likely mediated through suppressing endometrial receptivity molecules ITGAV and MUC1. Nemadipine-A is a potential novel non-hormonal compound for contraception.
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Turek-Etienne, Tammy C., Eliza C. Small, Sharon C. Soh, Tianpei A. Xin, Priti V. Gaitonde, Ellen B. Barrabee, Richard F. Hart, and Robert W. Bryant. "Evaluation of Fluorescent Compound Interference in 4 Fluorescence Polarization Assays: 2 Kinases, 1 Protease, and 1 Phosphatase." Journal of Biomolecular Screening 8, no. 2 (April 2003): 176–84. http://dx.doi.org/10.1177/1087057103252304.
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With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC™, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential “hit” or false positives, depending on the assay format. Cy™ dyes (e.g., Cy3B™ and Cy5™) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC™ compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes. ( Journal of Biomolecular Screening 2003:176-184)
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Naghaei, R., and M. J. Monem. "Development of a Mathematical Model of Lopac Gates in Accordance with the ICSS Hydrodynamic Model." Journal of Irrigation and Drainage Engineering 142, no. 10 (October 2016): 04016043. http://dx.doi.org/10.1061/(asce)ir.1943-4774.0001066.
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Rabjohns, Jennifer L. A., Yoon-Dong Park, Jean Dehdashti, Wei Sun, Christina Henderson, Adrian Zelazny, Steven J. Metallo, Wei Zheng, and Peter R. Williamson. "A High-Throughput Screening Assay for Fungicidal Compounds against Cryptococcus neoformans." Journal of Biomolecular Screening 19, no. 2 (July 29, 2013): 270–77. http://dx.doi.org/10.1177/1087057113496847.
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Cryptococcus neoformans is a pathogenic fungus that causes meningitis worldwide, particularly in human immunodeficiency virus (HIV)–infected individuals. Although amphotericin B is the “gold standard” treatment for cryptococcal meningitis, the toxicity and inconvenience of intravenous injection emphasize a need for development of new anticryptocccal drugs. Recent data from humans and animal studies suggested that a nutrient-deprived host environment may exist in cryptococcal meningitis. Thus, a screening assay for identifying fungicidal compounds under nutrient-deprived conditions may provide an alternative strategy to develop new anticryptococcal drugs for this disease. A high-throughput fungicidal assay was developed using a profluorescent dye, alamarBlue, to detect residual metabolic activity of C. neoformans under nutrient-limiting conditions. Screening the Library of Pharmacologically Active Compounds (LOPAC) with this assay identified a potential chemical scaffold, 10058-F4, that exhibited fungicidal activity in the low micromolar range. These results thus demonstrate the feasibility of this alamarBlue-based assay for high-throughput screening of fungicidal compounds under nutrient-limiting conditions for new anticryptococcal drug development.
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Caporale, Andrea, Fabiola Mascanzoni, Biancamaria Farina, Mattia Sturlese, Gianluigi Di Sorbo, Roberto Fattorusso, Menotti Ruvo, and Nunzianna Doti. "FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay." Journal of Biomolecular Screening 21, no. 7 (July 10, 2016): 701–12. http://dx.doi.org/10.1177/1087057116650402.
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In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays.
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Ajit, Seena K., Mark H. Pausch, Jeffrey D. Kennedy, and Edward J. Kaftan. "Development of a FLIPR Assay for the Simultaneous Identification of MrgD Agonists and Antagonists from a Single Screen." Journal of Biomedicine and Biotechnology 2010 (2010): 1–8. http://dx.doi.org/10.1155/2010/326020.
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MrgD, a member of the Mas-related gene family, is expressed exclusively in small diameter neurons in the dorsal root ganglion. This unique expression pattern, the presence of a single copy of MrgD in rodents and humans, and the identification of a putative ligand, beta-alanine, make it an experimentally attractive therapeutic target for pain with limited likelihood of side effects. We have devised a high throughput calcium mobilization assay that enables identification of both agonists and antagonists from a single screen for MrgD. Screening of the Library of Pharmacologically Active Compounds (LOPAC) validated this assay approach, and we identified both agonists and antagonists active at micromolar concentrations in MrgD expressing but not in parental CHO-DUKX cell line. Further characterization was performed using a subset of these screening hits. Our results demonstrated that the dual agonist/antagonist assay format is feasible and likely can be extended to most GPCRs with known agonist.
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Camara, Ali, Alyssa George, Evan Hebner, Anika Mahmood, Jashun Paluru, and Seema Mattoo. "A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD." International Journal of Molecular Sciences 21, no. 19 (September 27, 2020): 7128. http://dx.doi.org/10.3390/ijms21197128.
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The covalent transfer of the AMP portion of ATP onto a target protein—termed adenylylation or AMPylation—by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore—Fl-ATP—we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE’s enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.
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Weizhen Wu, Jin Shang, Yue Feng, Chris M. Thompson, Sarah Horwitz, John R. Thompson, Euan D. Macintyre, et al. "Identification of Glucose-Dependent Insulin Secretion Targets in Pancreatic β Cells by Combining Defined-Mechanism Compound Library Screening and siRNA Gene Silencing." Journal of Biomolecular Screening 13, no. 2 (January 23, 2008): 128–34. http://dx.doi.org/10.1177/1087057107313763.
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Identification and validation of novel drug targets continues to be a major bottleneck in drug development, particularly for polygenic complex diseases such as type 2 diabetes. Here, the authors describe an approach that allows researchers to rapidly identify and validate potential drug targets by combining chemical tools and RNA interference technology. As a proof-of-concept study, the known mechanism Sigma LOPAC library was used to screen for glucose-dependent insulin secretion (GDIS) in INS-1 832/13 cells. In addition to several mechanisms that are known to regulate GDIS (such as cyclic adenosine monophosphate—specific phosphodiesterases, adrenoceptors, and Ca2+ channels), the authors find that several of the dopamine receptor ( DRD) antagonists significantly enhance GDIS, whereas DRD agonists profoundly inhibit GDIS. Subsequent siRNA studies in the same cell line indicate that knockdown of DRD2 enhanced GDIS. Furthermore, selective DRD2 antagonists and agonists also enhance or suppress, respectively, GDIS in isolated rat islets. The data support that the approach described here offers a rapid and effective way for target identification and validation. ( Journal of Biomolecular Screening 2008;128-134)
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Choi, Byoung-Soo, Jimin Lee, Sang-Hwan Kim, Seunghyuk Chang, JongHo Park, Sang-Jin Lee, and Jang-Kyoo Shin. "Analysis of Disparity Information for Depth Extraction Using CMOS Image Sensor with Offset Pixel Aperture Technique." Sensors 19, no. 3 (January 24, 2019): 472. http://dx.doi.org/10.3390/s19030472.
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A complementary metal oxide semiconductor (CMOS) image sensor (CIS), using offset pixel aperture (OPA) technique, was designed and fabricated using the 0.11-µm CIS process. In conventional cameras, an aperture is located on the camera lens. However, in a CIS camera using OPA technique, apertures are integrated as left-offset pixel apertures (LOPAs) and right-offset pixel apertures (ROPAs). A color pattern is built, comprising LOPA, blue, red, green, and ROPA pixels. The disparity information can be acquired from the LOPA and ROPA channels. Both disparity information and two-dimensional (2D) color information can be simultaneously acquired from the LOPA, blue, red, green, and ROPA channels. A geometric model of the OPA technique is constructed to estimate the disparity of the image, and the measurement results are compared with the estimated results. Depth extraction is thus achieved by a single CIS using the OPA technique, which can be easily adapted to commercial CIS cameras.
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Guo, Sheng, Zhong Liu, Lan-Feng Hui, Chuan-Ling Si, and Jin-Jiang Pang. "Application of polyoxometalate in hydrogen peroxide bleaching under acidic conditions." BioResources 6, no. 2 (February 28, 2011): 1251–61. http://dx.doi.org/10.15376/biores.6.2.1251-1261.
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The modified bleaching sequence OPAPPO from short-sequence bleaching OAP and OQP was studied in an effort to achieve higher quality straw pulp (with brightness 84.5% and acceptable viscosity 669 mL/g), which will be appropriate for more situations than straw pulp as presently produced. Though the OP and PO stages are recognized as the key processes used to increase the pulp’s brightness, addition of hydrogen peroxide in acid pretreatment with polyoxometalate (POM) as catalyst (AP stage) was mainly considered in this work. Phosphomolybdic acid was applied to improve straw pulp’s brightness, which was 4.8% ISO higher than the pulp treated without POM. The optimum conditions of the AP stage were: initial pH value 3, temperature 90 °C, H2O2 1.5%, and phosphomolybdic acid 1.0%. Comparison of the sequences OPQPO, OPAPO, and OPAPPO showed that the brightness of pulp bleached by OPAPPO was 2% and 4.7% higher than the same pulp subjected to OPAPO and OPQPO sequences, respectively. Four lignin samples (LOP, LOPA, LOPAP, LOPAPPO) were characterized by 31P NMR spectroscopy. The spectroscopic investigation showed that in LOPA and LOPAP, aliphatic hydroxyls, p-coumaryl units, and guaiacyl phenol moieties were degraded when compared with that in LOP. In LOPAPPO, all these aliphatic hydroxyls and guaiacyl phenols had been destroyed and carboxylic acid functionalities increased.
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Preuss, Janina, Michael Hedrick, Eduard Sergienko, Anthony Pinkerton, Arianna Mangravita-Novo, Layton Smith, Carolin Marx, et al. "High-Throughput Screening for Small-Molecule Inhibitors of Plasmodium falciparum Glucose-6-Phosphate Dehydrogenase 6-Phosphogluconolactonase." Journal of Biomolecular Screening 17, no. 6 (April 11, 2012): 738–51. http://dx.doi.org/10.1177/1087057112442382.
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Plasmodium falciparum causes severe malaria infections in millions of people every year. The parasite is developing resistance to the most common antimalarial drugs, which creates an urgent need for new therapeutics. A promising and attractive target for antimalarial drug design is the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) of P. falciparum, which catalyzes the key step in the parasites’ pentose phosphate pathway. In this study, we describe the development of a high-throughput screening assay to identify small-molecule inhibitors of recombinant PfGluPho. The optimized assay was used to screen three small-molecule compound libraries—namely, LOPAC (Sigma-Aldrich, 1280 compounds), Spectrum (MicroSource Discovery Systems, 1969 compounds), and DIVERSet (ChemBridge, 49 971 compounds). These pilot screens identified 899 compounds that inhibited PfGluPho activity by at least 50%. Selected compounds were further studied to determine IC50 values in an orthogonal assay, the type of inhibition and reversibility, and effects on P. falciparum growth. Screening results and follow-up studies for selected PfGluPho inhibitors are presented. Our high-throughput screening assay may provide the basis to identify novel and urgently needed antimalarial drugs.
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Shahane, Sampada A., Ruili Huang, David Gerhold, Ulrich Baxa, Christopher P. Austin, and Menghang Xia. "Detection of Phospholipidosis Induction." Journal of Biomolecular Screening 19, no. 1 (September 3, 2013): 66–76. http://dx.doi.org/10.1177/1087057113502851.
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Drug-induced phospholipidosis is characterized by the accumulation of intracellular phospholipids in cells exposed to cationic amphiphilic drugs. The appearance of unicentric or multicentric multilamellar bodies viewed under an electron microscope (EM) is the morphological hallmark of phospholipidosis. Although the EM method is the gold standard for detecting cellular phospholipidosis, this method has its drawbacks, including low throughput, high cost, and unsuitability for screening a large chemical library. In this study, a cell-based phospholipidosis assay has been developed using the LipidTOX Red reagent in HepG2 cells and miniaturized into a 1536-well plate format. To validate this assay for high-throughput screening (HTS), the LOPAC library of 1280 compounds was screened using a quantitative HTS platform. A group of known phospholipidosis inducers, such as amiodarone, propranolol, chlorpromazine, desipramine, promazine, clomipramine, and amitriptyline, was identified by the screen, consistent with previous reports. Several novel phospholipidosis inducers, including NAN-190, ebastine, GR127935, and cis-(Z)-flupentixol, were identified in this study and confirmed using the EM method. These results demonstrate that this assay can be used to evaluate and profile large numbers of chemicals for drug-induced phospholipidosis.
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Grant, Stephan K., Joseph G. Sklar, and Richard T. Cummings. "Development of Novel Assays for Proteolytic Enzymes Using Rhodamine-Based Fluorogenic Substrates." Journal of Biomolecular Screening 7, no. 6 (December 2002): 531–40. http://dx.doi.org/10.1177/1087057102238627.
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Components within synthetic chemical and natural product extract libraries often interfere with fluorescence-based assays. Fluorescence interference can result when the intrinsic spectral properties of colored compounds overlap with the fluorescent probes. Typically, fluorescence-based protease assays use peptide amidomethylcoumarin derivatives as substrates. However, because many organic compounds absorb in the ultraviolet region, they can interfere with coumarin-based fluorescence assays. Red-shifted fluorescent dyes such as peptidyl rhodamine derivatives are useful because there is generally less interference from organic compounds outside the ultraviolet wavelengths. In this report, rhodamine-based fluorogenic substrates, such as bis-(Leu)2-Rhod110 and bis-(Ala-Pro)2-Rhod110, were developed for leucine aminopeptidase and dipeptidyl aminopeptidase. Novel, tandem rhodamine substrates such as Ala-Pro-Rhod110-Leu were designed with 2 protease cleavage sites and used to assay 2 proteases in a multiplex format. General endpoint high-throughput screening (HTS) assays were also developed for leucine aminopeptidase, dipeptidyl aminopeptidase, and trypsin that incorporated both amidomethylcoumarin and rhodamine-based fluorogenic substrates into a single screening format. These dual-substrate assays allowed for the successful screening of the LOPAC™ collection and natural product extracts despite high levels of fluorescence interference.
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Tran, Elizabeth, and Ye Fang. "Duplexed Label-Free G Protein—Coupled Receptor Assays for High-Throughput Screening." Journal of Biomolecular Screening 13, no. 10 (November 21, 2008): 975–85. http://dx.doi.org/10.1177/1087057108326141.
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This article describes duplexed label-free optical biosensor cellular assays for simultaneously assaying 2 endogenous receptors, the Gq-coupled histamine receptor (H 1) and the Gs-coupled β2-adrenergic receptor (β2AR), in A431 cells. The biosensor cellular assays consist of 2 sequential steps—an initial agonist screening using Sigma LOPAC (Library of Pharmaceutically Active Compounds) and a subsequent antagonist screening using a solution mixture containing the H1 agonist histamine and the β2AR agonist epinephrine. Results showed that costimulating A431 cells with histamine and epinephrine led to an optical response additive to individual responses. The agonist screening not only identified all full agonists for both the H1 and β2 receptors, but also detected pathway-biased ligands for the β2AR. Furthermore, the succeeding antagonist screening documented all known antagonists in the library for either the H1 or β2 receptors. This is the 1st demonstration of a single cellular assay that is capable of screening ligands against 2 GPCRs coupled to distinct G proteins, and highlights the power of pathway-unbiased and label-free biosensor cellular assays for GPCR screens. ( Journal of Biomolecular Screening 2008:975-985)
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Fancher, Ashley T., Yun Hua, Daniel P. Camarco, David A. Close, Christopher J. Strock, and Paul A. Johnston. "Reconfiguring the AR-TIF2 Protein–Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen." ASSAY and Drug Development Technologies 14, no. 8 (October 2016): 453–77. http://dx.doi.org/10.1089/adt.2016.741.
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David, Alon Ben, Eran Diamant, Eyal Dor, Ada Barnea, Niva Natan, Lilach Levin, Shira Chapman, et al. "Identification of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries." Molecules 26, no. 11 (May 27, 2021): 3213. http://dx.doi.org/10.3390/molecules26113213.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.
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Shu, Chih-Wen, Charitha Madiraju, Dayong Zhai, Kate Welsh, Paul Diaz, Eduard Sergienko, Renata Sano, and John C. Reed. "High-Throughput Fluorescence Assay for Small-Molecule Inhibitors of Autophagins/Atg4." Journal of Biomolecular Screening 16, no. 2 (January 18, 2011): 174–82. http://dx.doi.org/10.1177/1087057110392996.
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Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA2) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z′ factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules ( n = 1280 for Lopac™ and 2000 for Spectrum™ library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA2 and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA2 reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.
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Preuss, Janina, Adam D. Richardson, Anthony Pinkerton, Michael Hedrick, Eduard Sergienko, Stefan Rahlfs, Katja Becker, and Lars Bode. "Identification and Characterization of Novel Human Glucose-6-Phosphate Dehydrogenase Inhibitors." Journal of Biomolecular Screening 18, no. 3 (September 27, 2012): 286–97. http://dx.doi.org/10.1177/1087057112462131.
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Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP+. Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC50 values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC50 values. The most potent hG6PD inhibitors presented IC50 values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC50 ~25 µM) more strongly than that of normal MCF10-A cells (IC50 >50 µM).
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Blevins, Melanie A., Jennifer Kouznetsova, Aaron B. Krueger, Rebecca King, Lesley Mathews Griner, Xin Hu, Noel Southall, et al. "Small Molecule, NSC95397, Inhibits the CtBP1-Protein Partner Interaction and CtBP1-Mediated Transcriptional Repression." Journal of Biomolecular Screening 20, no. 5 (December 4, 2014): 663–72. http://dx.doi.org/10.1177/1087057114561400.
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Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor that suppresses multiple proapoptotic and epithelial genes. CtBP is overexpressed in many human cancers, and its overexpression increases stem cell–like features, epithelial-mesenchymal transition, and cancer cell survival. Knockdown of CtBP also increases apoptosis independent of p53 in cell culture. Therefore, targeting CtBP with small molecules that disrupt its interaction with transcription factor partners may be an effective cancer therapy. To elicit its corepressing effect, CtBP binds to a conserved peptide motif in each transcription factor partner. We developed an AlphaScreen high-throughput screening assay to monitor the interaction between CtBP and E1A (which mimics the interaction between CtBP and its transcriptional partners). We screened the LOPAC library of 1280 bioactive compounds and identified NSC95397, which inhibits the CtBP-E1A interaction (IC50 = 2.9 µM). The inhibitory activity of NSC95397 was confirmed using two secondary assays and a counterscreen. NSC95397 also behaved as a weak substrate of CtBP dehydrogenase activity and did not inhibit another dehydrogenase, lactase dehydrogenase. Finally, NSC95397 was able to disrupt CtBP-mediated transcriptional repression of a target gene. These studies present a new possibility for the development of a therapeutic agent targeting tumors through disrupting the CtBP transcriptional complex.
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Rosenberg, Laura H., Marie Lafitte, Wayne Grant, Weimin Chen, John L. Cleveland, and Derek R. Duckett. "Development of an HTS-Compatible Assay for the Discovery of Ulk1 Inhibitors." Journal of Biomolecular Screening 20, no. 7 (April 7, 2015): 913–20. http://dx.doi.org/10.1177/1087057115579391.
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A rapidly accumulating body of work suggests the autophagy pathway is an attractive therapeutic target for neurodegenerative diseases and cancer. To validate autophagy as an anticancer strategy and to assess if systemic inhibition of the pathway will have deleterious effects on normal tissues and physiology, highly selective autophagy inhibitors are needed. While several inducers and inhibitors of autophagy are known, all are nonspecific and none target the enzymes that execute the pathway. A central upstream regulator of the autophagy pathway is the serine/threonine kinase Ulk1 (UNC-51-like kinase-1). Selective molecular probes that function as Ulk1-specific inhibitors are needed to improve our understanding of the autophagy pathway. To identify inhibitors of Ulk1 kinase activity, we developed an HTS-compatible, homogeneous biochemical assay using AlphaScreen technology. This novel assay design uses purified stress-activated Ulk1 and monitors phosphorylation of its full-length native substrate, Atg13. This assay was optimized and validated in a 384-well format by screening the Sigma LOPAC library. Here we report that the Ulk1 AlphaScreen assay is robust and reproducible, with a Z′ factor value of 0.83 ± 0.02 and a signal to background ratio of 20 ± 1.2. Thus, this assay can be used to screen large chemical libraries to discover novel inhibitors of Ulk1.
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Benjamin, Elfrida R., Farhana Pruthi, Shakira Olanrewaju, Victor I. Ilyin, Gregg Crumley, Elena Kutlina, Kenneth J. Valenzano, and Richard M. Woodward. "State-Dependent Compound Inhibition of Nav1.2 Sodium Channels Using the FLIPR Vm Dye: On-Target and Off-Target Effects of Diverse Pharmacological Agents." Journal of Biomolecular Screening 11, no. 1 (October 18, 2005): 29–39. http://dx.doi.org/10.1177/1087057105280918.
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Voltage-gated sodiumchannels (NaChs) are relevant targets for pain, epilepsy, and a variety of neurological and cardiac disorders. Traditionally, it has been difficult to develop structure-activity relationships for NaCh inhibitors due to rapid channel kinetics and state-dependent compound interactions. Membrane potential (V m)dyes in conjunctionwith a high-throughput fluorescence imaging plate reader (FLIPR) offer a satisfactory 1st-tier solution. Thus, the authors have developed a FLIPR V m assay of rat Na v1.2NaCh. Channels were opened by addition of veratridine, and Vm dye responses were measured. The IC50 values from various structural classes of compounds were compared to the resting state binding constant (K r)and inactivated state binding constant (K i)obtained using patch-clamp electrophysiology (EP). The FLIPR values correlated with Ki but not K r.FLIPRIC50 values fellwithin 0.1-to 1.5-fold of EPKi values, indicating that the assay generally reports use-dependent inhibition rather than resting state block. The Library of Pharmacologically Active Compounds (LOPAC, Sigma) was screened. Confirmed hits arose from diverse classes such as dopamine receptor antagonists, serotonin transport inhibitors, and kinase inhibitors. These data suggest that NaCh inhibition is inherent in a diverse set of biologically active molecules and may warrant counterscreening NaChs to avoid unwanted secondary pharmacology.
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Ullmann, Tatjana, Sonja Luckhardt, Markus Wolf, Michael J. Parnham, and Eduard Resch. "High-Throughput Screening for CEBPD-Modulating Compounds in THP-1-Derived Reporter Macrophages Identifies Anti-Inflammatory HDAC and BET Inhibitors." International Journal of Molecular Sciences 22, no. 6 (March 16, 2021): 3022. http://dx.doi.org/10.3390/ijms22063022.
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This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.
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Härmä, Harri, Anita Rozwandowicz-Jansen, Eija Martikkala, Heini Frang, Ilkka Hemmilä, Niko Sahlberg, Vidal Fey, Merja Perälä, and Pekka Hänninen. "A New Simple Cell-Based Homogeneous Time-Resolved Fluorescence QRET Technique for Receptor-Ligand Interaction Screening." Journal of Biomolecular Screening 14, no. 8 (August 14, 2009): 936–43. http://dx.doi.org/10.1177/1087057109341657.
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In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of β2-adrenoreceptor (β2AR) antagonists and agonists in intact human embryonic kidney HEK293i cells overexpressing human β2-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β2AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z′ values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the Ki values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β2AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening. ( Journal of Biomolecular Screening 2009:936-943)
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Wyhs, Nicolas, David Walker, Hugh Giovinazzo, Srinivasan Yegnasubramanian, and William G. Nelson. "Time-Resolved Fluorescence Resonance Energy Transfer Assay for Discovery of Small-Molecule Inhibitors of Methyl-CpG Binding Domain Protein 2." Journal of Biomolecular Screening 19, no. 7 (March 7, 2014): 1060–69. http://dx.doi.org/10.1177/1087057114526433.
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Methylated DNA binding proteins such as Methyl-CpG Binding Domain Protein 2 (MBD2) can transduce DNA methylation alterations into a repressive signal by recruiting transcriptional co-repressor complexes. Interfering with MBD2 could lead to reactivation of tumor suppressor genes and therefore represents an attractive strategy for epigenetic therapy. We developed and compared fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET)–based high-throughput screening (HTS) assays to identify small-molecule inhibitors of the interaction between the methyl binding domain of MBD2 (MBD2-MBD) and methylated DNA. Although both assays performed well in 96-well format, the TR-FRET assay (Z′ factor = 0.58) emerged as a superior screening strategy compared with FP (Z′ factor = 0.08) when evaluated in an HTS 384-well plate format. Using TR-FRET, we screened the Sigma LOPAC library for MBD2-MBD inhibitors and identified four compounds that also validated in a dose-response series. This included two known DNA intercalators (mitoxantrone and idarubicin) among two other inhibitory compounds (NF449 and aurintricarboxylic acid). All four compounds also inhibited the binding of SP-1, a transcription factor with a GC-rich binding sequence, to a methylated oligonucleotide, demonstrating that the activity was nonspecific. Our results provide proof of principle for using TR-FRET–based HTS to identify small-molecule inhibitors of MBD2 and other DNA-protein interactions.
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Teng, Christina T., Jui-Hua Hsieh, Jinghua Zhao, Ruili Huang, Menghang Xia, Negin Martin, Xiaohua Gao, et al. "Development of Novel Cell Lines for High-Throughput Screening to Detect Estrogen-Related Receptor Alpha Modulators." SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, no. 6 (January 31, 2017): 720–31. http://dx.doi.org/10.1177/2472555216689772.
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Estrogen-related receptor alpha (ERRα), the first orphan nuclear receptor discovered, is crucial for the control of cellular energy metabolism. ERRα and its coactivator, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), are required for rapid energy production in response to environmental challenges. They have been implicated in the etiology of metabolic disorders such as type 2 diabetes and metabolic syndrome. ERRα also plays a role in the pathogenesis of breast cancer. Identification of compounds that modulate ERRα signaling may elucidate environmental factors associated with these diseases. Therefore, we developed stable cell lines containing an intact ERRα signaling pathway, with and without the coactivator PGC-1α, to use as high-throughput screening tools to detect ERRα modulators. The lentiviral PGC-1α expression constructs and ERRα multiple hormone response element (MHRE) reporters were introduced into HEK293T cells that express endogenous ERRα. A cell line expressing the reporter alone was designated “ERR.” A second cell line expressing both reporter and PGC-1α was named “PGC/ERR.” Initial screenings of the Library of Pharmacologically Active Compounds (LOPAC) identified 33 ERR and 22 PGC/ERR agonists, and 54 ERR and 15 PGC/ERR antagonists. Several potent ERRα agonists were dietary plant compounds (e.g., genistein). In conclusion, these cell lines are suitable for high-throughput screens to identify environmental chemicals affecting metabolic pathways and breast cancer progression.
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Yan, Yu-Xin, Deborah M. Boldt-Houle, Bonnie P. Tillotson, Melissa A. Gee, Brian J. D'Eon, Xiao-Jia Chang, Corinne E. M. Olesen, and Michelle A. J. Palmer. "Cell-Based High-Throughput Screening Assay System for Monitoring G Protein-Coupled Receptor Activation Using β-Galactosidase Enzyme Complementation Technology." Journal of Biomolecular Screening 7, no. 5 (October 2002): 451–59. http://dx.doi.org/10.1177/108705702237677.
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A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between β-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX™ system, uses a pair of inactive β-galactosidase (β-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and β-arrestin-β-gal fusion proteins are generated. Following ligand stimulation, β-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the β-gal mutant fragments. GPCR activation is measured directly by quantitating restored β-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the β2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The β2-adrenergic receptor cell line was screened with the LOPAC™ compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.
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Lu, Zhuomei, Zhizhang Yin, Linda James, Rosalinda Syto, Jill M. Stafford, Sandra Koseoglu, Todd Mayhood, et al. "Development of a Fluorescence Polarization Bead-Based Coupled Assay to Target Different Activity/Conformation States of a Protein Kinase." Journal of Biomolecular Screening 9, no. 4 (June 2004): 309–21. http://dx.doi.org/10.1177/1087057104263506.
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Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP™)-based couple™d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z′ of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC™ and validated with the protein kinase C inhibitor staurosporine. The IC50 value generated was comparable to the value obtained by the radioactive 33P-γ-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.
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Joesch, Christoph, Emelie Guevarra, Serge P. Parel, Andreas Bergner, Peter Zbinden, Daniel Konrad, and Hugo Albrecht. "Use of FLIPR Membrane Potential Dyes for Validation of High-Throughput Screening with the FLIPR and µARCS Technologies: Identification of Ion Channel Modulators Acting on the GABAA Receptor." Journal of Biomolecular Screening 13, no. 3 (February 29, 2008): 218–28. http://dx.doi.org/10.1177/1087057108315036.
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Fluorometric imaging plate reader (FLIPR) membrane potential dyes (FMP-Red-Dye and FMP-Blue-Dye) were evaluated for the detection of compounds acting either as positive allosteric modulators or agonists on the GABAA receptor (GABAAR). A stable HEK293 cell line with constitutive expression of the rat GABA AR α1, β2, and γ2 genes was used to establish a functional high-throughput screening (HTS) assay based on measurement of the membrane potential change in living cells. The assay was validated with the FLIPR technology for identification of agonists and positive allosteric modulators using GABA and diazepam as model compounds. The FMP-Red-Dye showed better performance than the FMP-Blue-Dye, and the effects induced by GABA and diazepam were comparable to electrophysiology data. Subsequently, the assay was also validated with an ultra-HTS approach known as microarrayed compound screening (µARCS). The LOPAC library was used in a test screen for an initial assessment of the technology. Finally, the FLIPR and µARCS technologies were tested with a larger screening campaign. A focused library of 3520 putative positive modulators was tested with the FLIPR assay, and a diverse subset of 84,480 compounds was selected for screening with the µARCS technology. All hits were subjected to verification using the FLIPR technology, and confirmed hits were subsequently evaluated by EC50 determination. Finally, selected hits were further confirmed with electrophysiology testing. ( Journal of Biomolecular Screening 2008:218-228)
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Wu, Szu-Yu Szu, and Chi-Chang K. Shieh. "High-content screening assay for compounds that enhance or inhibit leukocyte superoxide production to identify drugs for treating immune-mediated diseases." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 237.21. http://dx.doi.org/10.4049/jimmunol.204.supp.237.21.
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Abstract Reactive oxygen species (ROS) are produced by cells to play important roles in the effector and regulatory functions of immune cells. Our previous studies revealed that ROS act as negative regulators in IL-1β-mediated inflammation. Enhanced severity of inflammation in immune-mediated arthritis was noted when leukocyte NADPH oxidase (NOX2), which mediates the production of ROS, was defective. We hence hypothesized that drugs that modulate the leukocyte ROS production may regulate the pathogenicity of immune-mediated arthritis. Here, we devised a high-content image assay using the dye H2DCF-DA for identifying LOPAC 1280 library compounds that may facilitate or suppress superoxide production in differentiated HL-60 (dHL-60) cells. ROS levels were measured after the cells were treated with phorbol 12-myristate 13-acetate (PMA) or menadione which may induce ROS production from NOX2 or mitochondria. From a screening of 640 compounds in a 3-point interpolate titration in cell-based imaging analysis, we identified 74 drugs that may enhance the ROS production and 72 compounds that may inhibit ROS production by PMA-stimulated-dHL-60 cells. The drugs that we identified included the agonist/antagonist of neurotransmitters and their receptors, ion channel blockers, serotonin antagonists, cell stress modulators and cell cycle inhibitors. To further clarified our hypothesis, we treated NOX2-deficient mice with menadione, which tended to increase the oxidant stress in the joint tissue. The severity of the immune-mediated arthritic joint was significantly suppressed after the treatment. This novel screening method hence may be used to identify potential drugs for treating redox-related inflammatory diseases.
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Lucumi, Edinson, Claire Darling, Hyunil Jo, Andrew D. Napper, Rajesh Chandramohanadas, Nicholas Fisher, Alison E. Shone, et al. "Discovery of Potent Small-Molecule Inhibitors of Multidrug-Resistant Plasmodium falciparum Using a Novel Miniaturized High-Throughput Luciferase-Based Assay." Antimicrobial Agents and Chemotherapy 54, no. 9 (June 14, 2010): 3597–604. http://dx.doi.org/10.1128/aac.00431-10.
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ABSTRACT Malaria is a global health problem that causes significant mortality and morbidity, with more than 1 million deaths per year caused by Plasmodium falciparum. Most antimalarial drugs face decreased efficacy due to the emergence of resistant parasites, which necessitates the discovery of new drugs. To identify new antimalarials, we developed an automated 384-well plate screening assay using P. falciparum parasites that stably express cytoplasmic firefly luciferase. After initial optimization, we tested two different types of compound libraries: known bioactive collections (Library of Pharmacologically Active Compounds [LOPAC] and the library from the National Institute of Neurological Disorders and Stroke [NINDS]) and a library of uncharacterized compounds (ChemBridge). A total of 12,320 compounds were screened at 5.5 μM. Selecting only compounds that reduced parasite growth by 85% resulted in 33 hits from the combined bioactive collection and 130 hits from the ChemBridge library. Fifteen novel drug-like compounds from the bioactive collection were found to be active against P. falciparum. Twelve new chemical scaffolds were found from the ChemBridge hits, the most potent of which was a series based on the 1,4-naphthoquinone scaffold, which is structurally similar to the FDA-approved antimalarial atovaquone. However, in contrast to atovaquone, which acts to inhibit the bc 1 complex and block the electron transport chain in parasite mitochondria, we have determined that our new 1,4-napthoquinones act in a novel, non-bc 1-dependent mechanism and remain potent against atovaquone- and chloroquine-resistant parasites. Ultimately, this study may provide new probes to understand the molecular details of the malaria life cycle and to identify new antimalarials.
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Dearmond, Patrick D., Graham M. West, Victor Anbalagan, Michael J. Campa, Edward F. Patz, and Michael C. Fitzgerald. "Discovery of Novel Cyclophilin A Ligands Using an H/D Exchange– and Mass Spectrometry–Based Strategy." Journal of Biomolecular Screening 15, no. 9 (September 20, 2010): 1051–62. http://dx.doi.org/10.1177/1087057110382775.
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Cyclophilin A (CypA) is an overexpressed protein in lung cancer tumors and as a result is a potential therapeutic and diagnostic target. Described here is use of an H/D exchange– and a matrix assisted laser desorption/ionization (MALDI) mass spectrometry–based assay, termed single-point SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange), to screen 2 chemical libraries, including the 1280-compound LOPAC library and the 9600-compound DIVERSet library, for binding to CypA. This work represents the first application of single-point SUPREX using a pooled ligand approach, which is demonstrated here to yield screening rates as fast as 6 s/ligand. The false-positive and false-negative rates determined in the current work using a set of control samples were 0% and 9%, respectively. A false-positive rate of 20% was found in screening the actual libraries. Eight novel ligands to CypA were discovered, including 2-(α-naphthoyl)ethyltrimethyl-ammonium iodide, (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile, 3-(N-benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one, cis-diammineplatinum (II) chloride, 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione, N-(3-chloro-1, 4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide, 1-[2-(3,4-dimethoxyphenyl)ethyl]-1H-pyrrole-2,5-dione, and 4-(2-methoxy-4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[5.2.1.0~2,6~]dec-8-ene-3,5-dione. These compounds, which had moderate binding affinities to CypA (i.e., Kd values in the low micromolar range), provide new molecular scaffolds that might be useful in the development of CypA-targeted diagnostic imaging or therapeutic agents for lung cancer.
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Hu, Zongyi, Keng-Hsin Lan, Shanshan He, Manju Swaroop, Xin Hu, Noel Southall, Wei Zheng, and T. Jake Liang. "Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds." Antimicrobial Agents and Chemotherapy 58, no. 2 (November 25, 2013): 995–1004. http://dx.doi.org/10.1128/aac.02094-13.
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ABSTRACTTherapy for hepatitis C virus (HCV) infection has advanced with the recent approval of direct-acting antivirals in combination with peginterferon and ribavirin. New antivirals with novel targets are still needed to further improve the treatment of hepatitis C. Previously reported screening methods for HCV inhibitors either are limited to a virus-specific function or apply a screening method at a single dose, which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screening (qHTS) assay platform with a cell-based HCV infection system. This highly sensitive assay can be miniaturized to a 1,536-well format for screening of large chemical libraries. All candidates are screened over a 7-concentration dose range to give EC50s (compound concentrations at 50% efficacy) and dose-response curves. Using this assay format, we screened a library of pharmacologically active compounds (LOPAC). Based on the profile of dose-dependent curves of HCV inhibition and cytotoxicity, 22 compounds with adequate curves and EC50s of <10 μM were selected for validation. In two additional independent assays, 17 of them demonstrated specific inhibition of HCV infection. Ten potential candidates with efficacies of >70% and CC50s (compound concentrations at 50% cytotoxicity) of <30 μM from these validated hits were characterized for their target stages in the HCV replication cycle. In this screen, we identified both known and novel hits with diverse structural and functional features targeting various stages of the HCV replication cycle. The pilot screen demonstrates that this assay system is highly robust and effective in identifying novel HCV inhibitors and that it can be readily applied to large-scale screening of small-molecule libraries.
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Kumar, Meera, Robert Lowery, and Vaishnav Kumar. "High-Throughput Screening Assays for Cancer Immunotherapy Targets: Ectonucleotidases CD39 and CD73." SLAS DISCOVERY: Advancing the Science of Drug Discovery 25, no. 3 (December 22, 2019): 320–26. http://dx.doi.org/10.1177/2472555219893632.
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Production of adenosine in the extracellular tumor microenvironment elicits strong immunosuppression and is associated with tumor progression. Thus, targeting adenosine-generating ectonucleotidases is a potential strategy to stimulate and prolong antitumor immunity. Because the reaction products of ectonucleotidases differ by a single phosphate group, selective detection in an assay format that is compatible with high-throughput screening (HTS) has been elusive. We report the development of biochemical assays capable of measuring the activity of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; also known as CD39) and ecto-5′-nucleotidase (CD73). Both assays leverage the Transcreener HTS Assay platform, which facilitates selective immunodetection of nucleotides with homogenous fluorescent readouts, fluorescence polarization or time-resolved fluorescence energy transfer. The Transcreener AMP2 Assay was used to measure CD39 activity, allowing detection of adenosine monophosphate (AMP) production (Z′ > 0.6) with subnanomolar amounts of CD39, allowing IC50 determination for tool compounds, consistent with previously reported values. To detect the production of adenosine by CD73, the Transcreener ADP2 Assay was coupled with adenosine kinase (AK); conversion of adenosine to AMP and adenosine diphosphate (ADP) by AK allows detection with ADP2 antibody. The Transcreener AMP2 Assay was used to screen a 1280 Library of Pharmacologically Active Compounds (LOPAC) library and a 1600-compound subset of a ChemBridge diversity library for CD39 inhibitors, allowing the identification of nine and eight candidate compounds from each library, respectively. The Transcreener ADP2 Assay was used to screen 1600 compounds from the ChemBridge diversity library for CD73 inhibitors and identified 14 potential candidates. HTS-compatible assays for ectonucleotidase activity may allow identification of purinergic signaling pathway inhibitors important for tumor-specific immune responses during tumor pathogenesis.
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Xiao, Jianpeng, Wei Shang, Zhiming Zhao, Jun Jiang, Jianping Chen, Hui Cai, Jinjin He, Zhihui Cai, and Zihan Zhao. "Pharmacodynamic Material Basis and Potential Mechanism Study of Spatholobi Caulis in Reversing Osteoporosis." Evidence-Based Complementary and Alternative Medicine 2023 (April 14, 2023): 1–19. http://dx.doi.org/10.1155/2023/3071147.
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Objective. To elucidate the mechanism of Spatholobi Caulis (SC) in treating osteoporosis (OP) integrated zebrafish model and bioinformatics. Methods. Skeleton staining coupled with image quantification was performed to evaluate the effects of SC on skeleton mineralization area (SSA) and total optical density (TOD). Zebrafish locomotor activity was monitored using the EthoVision XT. Bioactive compounds of SC and their corresponding protein targets were acquired from Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Potential therapeutic targets for OP were summarized through retrieving 5 databases, and then, the overlapping genes between SC and OP were acquired. The core genes were selected by CytoHubba. Subsequently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) functional analysis of the intersection target genes were carried out by R software. Finally, the molecular docking simulation was manipulated between the ingredients and the hub genes. Results. Compared with the model group, SC significantly increased the SSA and TOD at 10 mg/mL and improved the locomotor activity in a dose-dependent manner ( p < 0.001 ). 33 components of SC were associated with 72 OP-related genes including 10 core genes (MAPK1, VEGFA, MMP9, AKT1, AR, IL6, CALM3, TP53, EGFR, and CAT). Advanced Glycation End Product (AGE) Receptor for AGE (RAGE) signaling pathway was screened out as the principal pathway of SC in anti-OP. The bioactive components (Aloe-emodin, Emodin, Formononetin, Licochalcone A, Luteolin, and Lopac-I-3766) have excellent affinity to core genes (MAPK1, VEGFA, MMP9, AKT1, and IL6). Conclusion. SC had the hierarchical network characteristics of “multicomponents/multitargets/multifunctions/multipathways” in reversing OP, but AGE-RAGE signaling pathway may be the main regulatory mechanism.
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Murray, Clare, Anand Kornepati, Alvaro Padron, Yilun Deng, Myrna Garcia, Haiyan Bai, and Tyler Curiel. "421 Specific cephalosporin antibiotics deplete tumor PD-L1 to inhibit DNA damage sensing and sensitize to Chk1 inhibitors in vivo." Journal of Clinical and Translational Science 6, s1 (April 2022): 82. http://dx.doi.org/10.1017/cts.2022.245.
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OBJECTIVES/GOALS: Tumor PDL1 signals to immune cells for tumor immune evasion but has cell-intrinsic signals that promote tumor virulence. We identify novel tumor PDL1 depleting drugs (PDDs) to interrupt tumor-intrinsic PDL1 signals and sensitize tumors to targeted therapy in vitro and in vivo. METHODS/STUDY POPULATION: We screened the Prestwick and LOPAC libraries for FDA-approved drugs reducing B16 melanoma PDL1 > 2.6-fold. ?-lactam antibiotics were used at 80 ?M and Chk1 inhibitor rabusertib as indicated in T24 human bladder cancer, and murine ID8agg ovarian cancer, 4T1 breast cancer and B16. Genetic PDL1 KO was by CRISPR or shRNA and re-expression by lentivirus. Viability was by MTT and protein by immunoblot. We challenged 5 NSG mice/group with 2x106 T24 (SQ) cells and 5 BALB/c mice/group with 5x105 4T1 cells (mammary fat pad) and treated with cefepime (200 mg/kg), rabusertib (2.5 mg/kg), vehicle, or combo daily from day 3. RESULTS/ANTICIPATED RESULTS: Structurally-related ?-lactam antibiotics cefepime and ceftazidime are tumor PDDs. Cefepime or ceftazidime reduced tumor PD-L1 and thus its cell-intrinsic signals to deplete the DNA damage sensing Chk2 protein and promote rabusertib synthetic lethality in vitro and in vivo in a tumor PDL1-dependent manner independent of immunity. Structurally distinct ?-lactam antibiotics did not sensitize tumor cells to rabusertib, suggesting ?-lactam antimicrobial functions did not promote PDL1 depletion or rabusertib treatment effects in vivo. Although rabusertib effects were immune-independent, both PDDs induced immunogenic tumor STING signaling, suggesting they can improve tumor immunotherapy. DISCUSSION/SIGNIFICANCE: We show a rapidly translatable way to deplete detrimental tumor-intrinsic PDL1 signals, and sensitize tumors to rabusertib. We are testing PDD structure activity relationships to improve PDD effects and testing PDD effects on other treatments, e.g., PARP inhibitors, immunotherapy.
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Fontane, Ilaria Delle, Nuri Damayanti, Christopher Rupert, David E. Heppner, and Roberto Pili. "Abstract B020: A mechanistic study of the TFE3-splicing machinery gene fusions reveals a new druggable target for translocation renal cell carcinoma." Cancer Research 83, no. 16_Supplement (August 15, 2023): B020. http://dx.doi.org/10.1158/1538-7445.kidney23-b020.
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Abstract TFE3 is a member of basic helix-loop-helix leucine zipper MiT transcription factor family and its chimeric proteins are associated with translocation renal cell carcinoma (tRCC). Despite the variety of genes fusions, most of TFE3 fusions partner genes are linked to spliceosome machinery. Dissecting the function of TFE3 fused to spliceosome machinery factors (TFE3-SF) could direct the development of effective therapies for this lethal disease, which is refractory to standard treatments for kidney cancer. Here, by using a combination of in silico structure prediction, molecular cloning, next generation transcriptomics, FRET technology, mutagenesis, proteomics, and high-throughput high-content screening (HTHCS) we interrogated a series of oncogenic mechanisms of TFE3-SF-containing fusions, including spontaneous nuclear translocation, nuclear paraspeckle occupancy, enhanced in vitro proliferation and in vivo growth, transcriptome remodeling, alternative splicing reprogramming, and novel dimer partner recruitment. Molecular inhibition of TFE3-SF dimerization reverses its oncogenic activity and represents a potential new target for therapeutic intervention. Using HTHCS combined with FRET technology we screened FDA approved drugs library (LOPAC) and small molecule library (Microsource) and identified hits compounds which inhibit TFE3-SF dimerization. Hits compounds were validated in 2D and 3D models utilizing patient derived xenolines expressing TFE3-SF. Ouabain and terfenadine demonstrated decreased cell proliferation reduced spheroid growth and in vivo tumor growth. Overall, our results unmask synthetic vulnerabilities of TFE3-SF dimerization for novel therapeutic strategies in patients with this aggressive type of kidney cancer. Citation Format: Ilaria Delle Fontane, Nuri Damayanti, Christopher Rupert, David E. Heppner, Roberto Pili. A mechanistic study of the TFE3-splicing machinery gene fusions reveals a new druggable target for translocation renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B020.
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Damayanti, Nur, Sabrina Orsi, Ricardo Cordova, Christopher Rupert, Li Shen, William Marston Linehan, Kirk Staschke, Peter Hollenhorst, David Heppner, and Roberto Pili. "Abstract 4495: A mechanistic study of the TFE3-splicing machinery gene fusions reveals a new druggable target for translocation renal cell carcinoma." Cancer Research 83, no. 7_Supplement (April 4, 2023): 4495. http://dx.doi.org/10.1158/1538-7445.am2023-4495.
Full textAbstract:
Abstract TFE3 is a member of basic helix-loop-helix leucine zipper MiT transcription factor family and its chimeric proteins are associated with translocation renal cell carcinoma (tRCC). Despite the variety of genes fusions, most of TFE3 fusions partner genes are linked to spliceosome machinery. Dissecting the function of TFE3 fused to spliceosome machinery factors (TFE3-SF) could direct the development of effective therapies for this lethal disease, which is refractory to standard treatments for kidney cancer. Here, by using a combination of in silico structure prediction, molecular cloning, next generation transcriptomics, FRET technology, mutagenesis, proteomics, and high-throughput high-content screening (HTHCS) we interrogated a series of oncogenic mechanisms of TFE3-SF-containing fusions, including spontaneous nuclear translocation, nuclear paraspeckle occupancy, enhanced in vitro proliferation and in vivo growth, transcriptome remodeling, alternative splicing reprogramming, and novel dimer partner recruitment. Molecular inhibition of TFE3-SF dimerization reverses its oncogenic activity and represents a potential new target for therapeutic intervention. By using HTHCS combined with FRET technology we screened FDA approved drugs library (LOPAC) and small molecule library (Microsource) and identified hits compounds which inhibit TFE3-SF dimerization. Hits compounds were validated in 2D and 3D models utilizing patient derived xenolines expressing TFE3-SF. Ouabain and terfenadine demonstrated decreased cell proliferation, reduced spheroid growth and in vivo tumor growth. Overall, our results unmask synthetic vulnerabilities of TFE3-SF dimerization for novel therapeutic strategies in patients with this aggressive type of kidney cancer. Citation Format: Nur Damayanti, Sabrina Orsi, Ricardo Cordova, Christopher Rupert, Li Shen, William Marston Linehan, Kirk Staschke, Peter Hollenhorst, David Heppner, Roberto Pili. A mechanistic study of the TFE3-splicing machinery gene fusions reveals a new druggable target for translocation renal cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4495.
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Frank, Matthew I., Anant Agarwal, and Mary K. Vernon. "LoPC." ACM SIGPLAN Notices 32, no. 7 (July 1997): 276–87. http://dx.doi.org/10.1145/263767.263803.
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Arora, Reety, Masahiro Shuda, Anna Guastafierro, Tuna Toptan, Yanis Tolstov, Daniel Paul Normolle, Laura Vollmer, et al. "Rapid rational drug targeting of Merkel cell polyomavirus (MCV)-positive Merkel cell carcinoma (MCC) using the survivin inhibitor YM155." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): 8577. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.8577.
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8577 Background: MCC is an aggressive, chemoresistant skin cancer causing more deaths each year than chronic myelogenous leukemia. We discovered a new virus, Merkel cell polyomavirus (MCV), clonally integrated into ~80% of primary and metastatic MCC in 2008. To find therapeutic targets for this cancer, we examined cellular genes perturbed by MCV infection. Methods: Digital transcriptome subtraction was used to discover MCV and also to reveal survivin gene (BIRC5) upregulation in virus-positive tumors. MCV T antigen knockdown studies in seven MCC lines and large T (LT) transduction into BJ fibroblasts were used to confirm this. Drug screening was performed in vitro using Cell-Titer Glo assays in a two stage analysis. In vivo screening used an MKL-1 (MCV+) MCC NOD-SCIDg mouse xenograft model with a single three-week treatment round. Results: MCV large T oncoprotein induces survivin transcription through retinoblastoma protein sequestration by the LT LXCXE motif. MCV T antigen knockdown results in nonapoptotic MCC cell death and loss of survivin expression. YM155, a phase II survivin transcription inhibitor, causes MCV+ MCC cell necroptosis associated with autophagy at 1-12 nM EC50. Of 1359 other drugs from LOPAC and NCI Oncology Set II libraries, only bortezomib had in vitro potency comparable to YM155. In MKL-1 xenograft studies, mice were treated with saline, bortezomib or YM155 for three weeks using standard dosings. Bortezomib did not significantly improve mouse survival (33%) over saline (24%) during treatment. In contrast, all YM155-treated mice survived (100%, p<0.001) the 3 week treatment period. Tumors resumed growth once YM155 treatment was stopped suggesting that YM155 is cytostatic in vivo rather than cytotoxic. Conclusions: Survivin expression is induced by MCV LT and is critical to MCV+ MCC survival. A survivin inhibitor, YM155 was nontoxic to mice and cytostatic for MCV+ MCC xenografts. Using genomic technologies, in less than four years, the primary viral cause for most MCC was discovered, new diagnostic tests developed and a promising rational drug candidate identified. A cooperative group trial (E1611) for YM155 and bortezomib in MCC patients is currently planned.
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Ni, Liwen, Fanbin Zhao, Bolun Li, Tong Wei, Hang Guan, and Shixue Ren. "Antioxidant and Fluorescence Properties of Hydrogenolyzised Polymeric Proanthocyanidins Prepared Using SO42−/ZrO2 Solid Superacids Catalyst." Molecules 23, no. 10 (September 25, 2018): 2445. http://dx.doi.org/10.3390/molecules23102445.
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Larix bark oligomeric proanthocyanidins (LOPC) were prepared from larix bark polymeric proanthocyanidins (LPPC) by catalytic hydrogenolysis using SO42−/ZrO2 solid superacid as the catalyst. The catalyst to polymeric proanthocyanidins ratio was 0.2:1 (m/m). The LOPC, obtained after hydrogenolysis at 100 °C for 4 h under 3 MPa hydrogen pressure, retained the structural characteristics of proanthocyanidins. The average degree of polymerization was reduced from 9.50% to 4.76% and the depolymerization yield was 53.85%. LOPC has good antioxidant properties and, at the same concentration, the reducing ability of LOPC was much higher than that of LPPC. The IC50 values of LOPC for scavenging DPPH• and ABTS•+ radicals were 0.046 mg/mL and 0.051 mg/mL, respectively. LOPC is biocompatible and has fluorescent properties that are affected by external factors, such as solvent polarity, pH and the presence of different metal ions. These features indicate that LOPC could be developed as a new biological fluorescent marker. The depolymerization of low-value polymeric proanthocyanidins to provide high-value oligomeric proanthocyanidins and the development of new applications for proanthocyanidins represent significant advances.
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Yang, Zhenni, Meng Wang, Yanping Hou, Yucun Liu, Satheesh Chandran, Ravi Varma, Shengrong Lou, and Jun Chen. "Intercomparison of Ambient Nitrous Acid Measurements in a Shanghai Urban Site." Atmosphere 13, no. 2 (February 16, 2022): 329. http://dx.doi.org/10.3390/atmos13020329.
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Nitrous acid (HONO) is the major source of OH radicals in polluted regions and plays a key role in the nitrogen cycle of the atmosphere. Therefore, accurate measurements of HONO in the atmosphere is important. Long Path Absorption Photometer (LOPAP) is a common and highly sensitive method used for ambient HONO measurements. Incoherent Broadband Cavity Enhanced Absorption Spectroscopy (IBBCEAS) is a recent alternative for the detection of HONO with high temporal and spatial resolutions, which has shown a detection limit of 0.76 ppbv at a sampling average of 180 s. In this study, LOPAP and IBBCEAS-HONO instruments were deployed in a Shanghai Urban Site (Shanghai Academy of Environmental Sciences) and simultaneously recorded the data from both instruments for a quantitative intercomparison of the measured atmospheric HONO for four days from 30 December 2017–2 January 2018. The HONO concentration measured by IBBCEAS and LOPAP were well matched. The campaign average concentrations measured by IBBCEAS and LOPAP were 1.28 and 1.20 ppbv, respectively. The intercomparison results demonstrated that both the IBBCEAS-HONO instrument and LOPAP-HONO instrument are suitable for ambient monitoring of HONO in a polluted urban environment.
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Laksitaningtyas, Agatha Padma. "SISTEM IRIGASI DESA KASANG LOPAK ALAI." Jurnal Teknik Sipil 13, no. 4 (February 10, 2017): 314. http://dx.doi.org/10.24002/jts.v13i4.939.
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Desa Kasang Lopak Alai, merupakan salah satu desa yang terletak di KecamatanKumpeh Ulu, Kabupaten Muaro Jamb i, Provins i Jambi, yang menjadi lokasi dalam penelitian ini.Dalam Rencana Tata Ruang Wilayah Kab. Muaro Jambi (RTRW) untuk kawasan peta kawasanstrategis Desa Kasang Lopak Alai merupakan kawasan pertanian tanaman pangan holtikultura danperikanan, yang memiliki potensi yang sangat besar untuk dikembangkan menjadi desa yang lebihbesar lagi. Desa Kasang Lopak Alai merupakan desa yang mempunyai potensi daerah yang sangatbesar, salah satunya adalah irigasi yang sangat besar karena berada di pinggir Anak SungaiKumpeh, yang merupakan cabang dari Sungai Batang Hari yang merupakan sungai terbesar diProvinsi Jambi yang membelah Kota Jambi. Metode yang digunakan dalam penelitian ini adalahpenelitian kualitatif dengan penentuan informasi dengan menggunakan teknik sampling purposive.Metode pengumpulan data yang digunakan adalah observasi, wawancara, dan dokumentasi yanglangsung didapatkan di lapangan. Data yang diperoleh dalam penelitian ini dianalisis secaradeskriptif kualitatif. Hasil dari penelitian ini menemukan bahwa Desa Kasang Lopak Alaimerupakan wilayah desa yang mulai berkembang dalam berbagai segi salah satunya sistem irigasi,tetapi potensi tersebut belum d imanfaatkan secara maksimal. Desa Kasang Lopak Alai mempunyaibeberapa pola pertanian s istem irigasi, yaitu pola pertanian sistem irigasi sederhana dan sistemirigasi semi teknis.
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Gao, Han Chao, Zhi Jun Yin, Zi Qian Huang, Zhong Hui Li, and Zi Li Xie. "Investigation on p-In0.51Ga0.49As LOPC Mode by Raman Spectra." Advanced Materials Research 887-888 (February 2014): 442–45. http://dx.doi.org/10.4028/www.scientific.net/amr.887-888.442.
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Heavily P type In0.51Ga0.49As was grown with hole concentration from 3.3E19cm-3 to 4.6E19cm-3. Standard (001) surface Raman backscattering geometry was used to measure samples. Two mode behavior and LO phonon-plasmon-coupled mode (LOPC) were observed obviously. Raman peak of LOPC mode is insensitive to hole concentration. The full width at half maximum (FWHM) of LOPC and the intensity ratio of GaAs-like LO mode and LOPC mode depend on hole concentration. The coupling strength of GaAs-like LO mode and plasmons is weak when hole concentration is very high and Raman peak of LOPC mode is independence with increasing hole concentration.
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Freeman, Raymond “Randy.” "Quantifying LOPA uncertainty." Process Safety Progress 31, no. 3 (June 19, 2012): 240–47. http://dx.doi.org/10.1002/prs.11493.
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Romero Zarco, Carlos. "Bromus cincinnatus (Poaceae): perennial oat-grass or annual brome-grass?" Mediterranean Botany 44 (June 12, 2023): e84748. http://dx.doi.org/10.5209/mbot.84748.
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A former lectotype for Bromus cincinnatus Ten. (Poaceae) has been superseded, as it is in serious conflict with the protologue. Another element of the protologue has been designated as the effective lectotype. The new lectotype is identified as coespecific with Bromus intermedius Guss., a Mediterranean species of Bromus L. subg. Bromus. The new combination Helictochloa panormitana (Lojac.) Romero Zarco is proposed for the species originally described as Avena australis Parl., nom. illeg., which is currently known as Helictochloa cincinnata auct. The lectotypes of Avena panormitana Lojac., Avena opulenta Lojac., and Bromus intermedius Guss. are also designated.
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Cheson, B. D., D. M. Jasperse, R. Simon, and M. A. Friedman. "A critical appraisal of low-dose cytosine arabinoside in patients with acute non-lymphocytic leukemia and myelodysplastic syndromes." Journal of Clinical Oncology 4, no. 12 (December 1986): 1857–64. http://dx.doi.org/10.1200/jco.1986.4.12.1857.
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We reviewed 53 publications reporting 751 patients with hematologic malignancies treated with low doses (5 to 20 mg/m2/d) of cytosine arabinoside (LoDAC). Of 507 patients evaluable for response, complete remission (CR) rates varied from 32% for patients with primary acute non-lymphoblastic leukemia (1 degree ANLL) to 16% for patients with hematologic malignancies secondary to previous chemotherapy or following a myelodysplastic syndrome (MDS) (2 degrees ANLL), and 16% for MDS. Median duration of CR was 9.5 months for 1 degree ANLL, and 10.5 months for both 2 degrees ANLL and MDS. Based on limited available survival data, overall median survival for these groups was 9 months, 3 months, and 15 months, respectively. Only three CRs were reported of 31 evaluable patients treated for a variety of other hematologic malignancies. CR rates for patients with 1 degree ANLL greater than or equal to 50 years old was 56%, compared with 29% less than 50 years old (P = .10). While prior chemotherapy was more common in 1 degree ANLL patients less than 50 years of age (86% v 21%; P less than .001), it did not influence response rates in those greater than 50 years of age, suggesting other biases. Hematologic toxicity was mentioned in only 33 of 53 publications, affecting 254 of 289 patients (88%), with at least 15% treatment-related deaths. LoDAC hypothetically acts as a differentiating agent; however, correlative laboratory studies were rarely performed. Cytogenetic data were available for only 15%, and in vitro studies for 10% of all patients with marked discrepancies in the interpretation of results. LoDAC is clearly cytotoxic for both malignant and normal hematopoietic cells. While large numbers of patients have been reported, the lack of well-designed clinical trials prohibits definitive conclusions as to its role as a differentiating agent. Future LoDAC studies should determine optimal dose and schedule, with clinical laboratory correlates to assess differentiation. Trials in ANLL comparing LoDAC with conventional chemotherapy, and in MDS with supportive care alone, may help identify the role of LoDAC. Until appropriate indications can be identified, LoDAC should not be routinely used in clinical practice.
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Mendis, Shehara Ramyalini, Lara Rachel Lipton, Sumitra Ananda, Michael Michael, Sue-Anne McLachlan, Benjamin N. Thomson, Brett Knowles, et al. "Early-onset pancreatic cancer: Defining contemporary presentation, treatment, and outcomes in the under 50 age group using real-world data." Journal of Clinical Oncology 40, no. 4_suppl (February 1, 2022): 533. http://dx.doi.org/10.1200/jco.2022.40.4_suppl.533.
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533 Background: The incidence of pancreatic cancer is increasing in younger patients (pts). Early onset pancreatic cancer (EOPC) is reportedly diagnosed at a later stage, potentially compromising outcomes compared to later onset pts (LOPC). With recent gains in staging and neo/adjuvant regimens, we sought to elaborate on the characteristics of EOPC and LOPC in a contemporary real-world cohort. Methods: The PURPLE registry, a prospectively collected multi-site data set on consecutive pancreatic cancer pts was interrogated. Patient, tumor, treatment and outcome data were extracted for EOPC vs LOPC. EOPC were those diagnosed prior to age 50 and LOPC after age 50. Resectability status was per MDT consensus. Results: Of 1534 pts, 93 (6%) were EOPC (51% male) and 1442 (94%) LOPC (51% male). EOPC had better ECOG performance status (0-1: 95% vs 81%, Relative Risk [RR] 1.2, p < 0.001) and Charlson Comorbidity Index Score (0-2: 98% vs 28%, RR 3.5, p < 0.001). Primary tumor site (head/body/tail: 66%/11%/20% for EOPC and 68%/17%/14% for LOPC), and staging (resectable/borderline resectable/locally advanced/metastatic: 29%/16%/14%/41% for EOPC vs 28%/9%/21%/41% for LOPC) did not differ. 25 (93%) of EOPC and 320 (79%) LOPC resectable pts underwent resection (p = 0.13). 12 (80%) EOPC and 36 (26%) LOPC borderline resectable pts underwent resection (RR 3.0, p < 0.001). Resection margin status (R0 vs R1 vs R2) did not differ. Resected EOPC more frequently received neoadjuvant therapy (30% vs 9%, RR 3.2, p = 0.001). EOPC were more likely to receive palliative chemotherapy in the advanced/metastatic setting (77% vs 49%, RR 1.6, p < 0.001), and were more likely to receive first line (1L) FOLFIRINOX than gemcitabine-nab-paclitaxel (36% vs 18%, RR 2, p = 0.019). Median overall survival (OS) was superior for EOPC (24 vs 12 months, Hazard Ratio [HR] 0.55, p < 0.001). For resectable pts, relapse free survival (RFS) did not differ but OS was superior for EOPC (undefined vs 27.7 months, HR 0.26, p = 0.004). In borderline resectable pts, RFS was similar and OS only numerically superior for EOPC (31.2 vs 17.7 months, p = 0.20). For locally advanced disease, 1L progression free survival (PFS1) was similar and OS was superior for EOPC (27.8 vs 11 months, HR 0.40, p = 0.008). There was no difference in PFS1/OS for metastatic pts. Conclusions: EOPC are fitter, with similar stage at diagnosis as LOPC. EOPC are more likely to receive neoadjuvant chemotherapy and undergo resection when presenting with borderline resectable disease. EOPC receive more treatment and have superior OS, with RFS/PFS1 not statistically different to LOPC.
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Rimsa, Roberts, Artis Galvanovskis, Janis Plume, Felikss Rumnieks, Karlis Grindulis, Gunita Paidere, Sintija Erentraute, Gatis Mozolevskis, and Arturs Abols. "Lung on a Chip Development from Off-Stoichiometry Thiol–Ene Polymer." Micromachines 12, no. 5 (May 11, 2021): 546. http://dx.doi.org/10.3390/mi12050546.
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Current in vitro models have significant limitations for new respiratory disease research and rapid drug repurposing. Lung on a chip (LOAC) technology offers a potential solution to these problems. However, these devices typically are fabricated from polydimethylsiloxane (PDMS), which has small hydrophobic molecule absorption, which hinders the application of this technology in drug repurposing for respiratory diseases. Off-stoichiometry thiol–ene (OSTE) is a promising alternative material class to PDMS. Therefore, this study aimed to test OSTE as an alternative material for LOAC prototype development and compare it to PDMS. We tested OSTE material for light transmission, small molecule absorption, inhibition of enzymatic reactions, membrane particle, and fluorescent dye absorption. Next, we microfabricated LOAC devices from PDMS and OSTE, functionalized with human umbilical vein endothelial cell (HUVEC) and A549 cell lines, and analyzed them with immunofluorescence. We demonstrated that compared to PDMS, OSTE has similar absorption of membrane particles and effect on enzymatic reactions, significantly lower small molecule absorption, and lower light transmission. Consequently, the immunofluorescence of OSTE LOAC was significantly impaired by OSTE optical properties. In conclusion, OSTE is a promising material for LOAC, but optical issues should be addressed in future LOAC prototypes to benefit from the material properties.