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Sample records for high-throughput screening assays

  1. Design and implementation of high throughput screening assays.

    PubMed

    Macarrón, Ricardo; Hertzberg, Robert P

    2011-03-01

    High throughput screening (HTS) is at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful analysis of many variables, starting with the choice of assay target and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to insure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces. PMID:20865348

  2. Mining Chemical Activity Status from High-Throughput Screening Assays

    PubMed Central

    Soufan, Othman; Ba-alawi, Wail; Afeef, Moataz; Essack, Magbubah; Rodionov, Valentin; Kalnis, Panos; Bajic, Vladimir B.

    2015-01-01

    High-throughput screening (HTS) experiments provide a valuable resource that reports biological activity of numerous chemical compounds relative to their molecular targets. Building computational models that accurately predict such activity status (active vs. inactive) in specific assays is a challenging task given the large volume of data and frequently small proportion of active compounds relative to the inactive ones. We developed a method, DRAMOTE, to predict activity status of chemical compounds in HTP activity assays. For a class of HTP assays, our method achieves considerably better results than the current state-of-the-art-solutions. We achieved this by modification of a minority oversampling technique. To demonstrate that DRAMOTE is performing better than the other methods, we performed a comprehensive comparison analysis with several other methods and evaluated them on data from 11 PubChem assays through 1,350 experiments that involved approximately 500,000 interactions between chemicals and their target proteins. As an example of potential use, we applied DRAMOTE to develop robust models for predicting FDA approved drugs that have high probability to interact with the thyroid stimulating hormone receptor (TSHR) in humans. Our findings are further partially and indirectly supported by 3D docking results and literature information. The results based on approximately 500,000 interactions suggest that DRAMOTE has performed the best and that it can be used for developing robust virtual screening models. The datasets and implementation of all solutions are available as a MATLAB toolbox online at www.cbrc.kaust.edu.sa/dramote and can be found on Figshare. PMID:26658480

  3. A Colloidal Stability Assay Suitable for High-Throughput Screening.

    PubMed

    Abarca, Carla; Ali, M Monsur; Yang, Songtao; Dong, Xiaofei; Pelton, Robert H

    2016-03-01

    A library of 32 polystyrene copolymer latexes, with diameters ranging between 53 and 387 nm, was used to develop and demonstrate a high-throughput assay using a 96-well microplate platform to measure critical coagulation concentrations, a measure of colloidal stability. The most robust assay involved an automated centrifugation-decantation step to remove latex aggregates before absorbance measurements, eliminating aggregate interference with optical measurements made through the base of the multiwell plates. For smaller nanoparticles (diameter <150 nm), the centrifugation-decantation step was not required as the interference was less than with larger particles. Parallel measurements with a ChemiDoc MP plate scanner gave indications of aggregation; however, the results were less sensitive than the absorbance measurements. PMID:26857643

  4. High-throughput screening assays for antibacterial and antifungal activities of Lactobacillus species.

    PubMed

    Inglin, Raffael C; Stevens, Marc J A; Meile, Lukas; Lacroix, Christophe; Meile, Leo

    2015-07-01

    We describe high-throughput screening techniques to rapidly detect either antimicrobial activity, using an agar-well diffusion assay in microtiter plates, or antifungal activity using an agar-spot assay in 24-well plates. 504 Lactobacillus isolates were screened with minimal laboratory equipment and screening rates of 2000-5000 individual antimicrobial interactions. PMID:25937247

  5. Design and Implementation of High-Throughput Screening Assays.

    PubMed

    Powell, David J; Hertzberg, Robert P; Macarrόn, Ricardo

    2016-01-01

    HTS remains at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful consideration of many options and variables, starting with the choice of screening strategy and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to ensure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces. PMID:27316985

  6. Design and implementation of high throughput screening assays.

    PubMed

    Macarrón, Ricardo; Hertzberg, Robert P

    2002-01-01

    HTS is at the core of the drug-discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful analysis of many variables, starting with the choice of assay target and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines can be established to ensure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces. PMID:12029816

  7. Design and implementation of high-throughput screening assays.

    PubMed

    Macarrón, Ricardo; Hertzberg, Robert P

    2009-01-01

    HTS is at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful analysis of many variables, starting with the choice of assay target and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to ensure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces. PMID:19551355

  8. ToxCast Workflow: High-throughput screening assay data processing, analysis and management (SOT)

    EPA Science Inventory

    US EPA’s ToxCast program is generating data in high-throughput screening (HTS) and high-content screening (HCS) assays for thousands of environmental chemicals, for use in developing predictive toxicity models. Currently the ToxCast screening program includes over 1800 unique c...

  9. Development of a thyroperoxidase inhibition assay for high-throughput screening

    EPA Science Inventory

    High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluores...

  10. A click chemistry-based microRNA maturation assay optimized for high-throughput screening.

    PubMed

    Lorenz, Daniel A; Garner, Amanda L

    2016-07-01

    Catalytic enzyme-linked click-chemistry assays (cat-ELCCA) are an emerging class of biochemical assay. Herein we report on expanding the toolkit of cat-ELCCA to include the kinetically superior inverse-electron demand Diels-Alder (IEDDA) reaction. The result is a technology with improved sensitivity and reproducibility, enabling automated high-throughput screening. PMID:27284591

  11. A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening.

    PubMed

    Krammer, B; Rumbold, K; Tschemmernegg, M; Pöchlauer, P; Schwab, H

    2007-03-30

    Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities. PMID:17157404

  12. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    SciTech Connect

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-02-20

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  13. An Enzymatic Assay for High-Throughput Screening of Cytidine-Producing Microbial Strains

    PubMed Central

    Dong, Huina; Liu, Yongfei; Zu, Xin; Li, Ning; Li, Feiran; Zhang, Dawei

    2015-01-01

    Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH3, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058 - 10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more). PMID:25816248

  14. Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

    PubMed

    El-Mowafi, S A; Sineva, E; Alumasa, J N; Nicoloff, H; Tomsho, J W; Ades, S E; Keiler, K C

    2015-01-01

    Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway. PMID:25331704

  15. A High Throughput Assay for Screening Host Restriction Factors and Antivirals Targeting Influenza A Virus

    PubMed Central

    Wang, Lingyan; Li, Wenjun; Li, Shitao

    2016-01-01

    Influenza A virus (IAV) is a human respiratory pathogen that causes seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. Currently approved treatments against influenza are losing effectiveness, as new viral strains are often refractory to conventional treatments. Thus, there is an urgent need to find new therapeutic targets with which to develop novel antiviral drugs. The common strategy to discover new drug targets and antivirals is high throughput screening. However, most current screenings for IAV rely on the engineered virus carrying a reporter, which prevents the application to newly emerging wild type flu viruses, such as 2009 pandemic H1N1 flu. Here we developed a simple and sensitive screening assay for wild type IAV by quantitatively analyzing viral protein levels using a Dot Blot Assay in combination with the LI-COR Imaging System (DBALIS). We first validated DBALIS in overexpression and RNAi assays, which are suitable methods for screening host factors regulating viral infection. More importantly, we also validated and initiated drug screening using DBALIS. A pilot compound screening identified a small molecule that inhibited IAV infection. Taken together, our method represents a reliable and convenient high throughput assay for screening novel host factors and antiviral compounds. PMID:27375580

  16. A High Throughput Assay for Screening Host Restriction Factors and Antivirals Targeting Influenza A Virus.

    PubMed

    Wang, Lingyan; Li, Wenjun; Li, Shitao

    2016-01-01

    Influenza A virus (IAV) is a human respiratory pathogen that causes seasonal epidemics and occasional global pandemics with devastating levels of morbidity and mortality. Currently approved treatments against influenza are losing effectiveness, as new viral strains are often refractory to conventional treatments. Thus, there is an urgent need to find new therapeutic targets with which to develop novel antiviral drugs. The common strategy to discover new drug targets and antivirals is high throughput screening. However, most current screenings for IAV rely on the engineered virus carrying a reporter, which prevents the application to newly emerging wild type flu viruses, such as 2009 pandemic H1N1 flu. Here we developed a simple and sensitive screening assay for wild type IAV by quantitatively analyzing viral protein levels using a Dot Blot Assay in combination with the LI-COR Imaging System (DBALIS). We first validated DBALIS in overexpression and RNAi assays, which are suitable methods for screening host factors regulating viral infection. More importantly, we also validated and initiated drug screening using DBALIS. A pilot compound screening identified a small molecule that inhibited IAV infection. Taken together, our method represents a reliable and convenient high throughput assay for screening novel host factors and antiviral compounds. PMID:27375580

  17. Fluorometric High-Throughput Screening Assay for Secreted Phospholipases A2 Using Phospholipid Vesicles.

    PubMed

    Ewing, Heather; Fernández-Vega, Virneliz; Spicer, Timothy P; Chase, Peter; Brown, Steven; Scampavia, Louis; Roush, William R; Riley, Sean; Rosen, Hugh; Hodder, Peter; Lambeau, Gerard; Gelb, Michael H

    2016-08-01

    There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate. PMID:27146384

  18. Development of fluorescence-based high-throughput screening assays: choice of appropriate instrumentation

    NASA Astrophysics Data System (ADS)

    Burns, David J.; Alder, Elisabeth; Fan, Yi-Hong; McKeegan, Evelyn; Warrior, Usha; Beutel, Bruce

    1998-04-01

    Fluorescence-based assays have become increasingly popular in high throughput screening for a variety of reasons (e.g. sensitivity). However, new screening technologies are pushing the limits of conventional fluorescence plate readers. For example, instruments that have optical sensitivities beyond most of the commercially available plate readers are required to reproducibly measure the fluorescence generated by the green fluorescent protein (GFP)--a novel reporter gene. Also, miniaturization of screening formats (with densities higher than the conventional 96-well plate) requires high resolution instrumentation to measure fluorescence. Several assays based on optical fluorescence measurements have been developed and screened in our Biological Screening group. These assays include various fluorescence-based protease assays (standard end-point and kinetic modes) and a functional cell-based screen using the green fluorescent protein as a reporter gene. The choice of instrumentation was the critical factor in the performance and success of each of these arrays. Data will be presented for the cell- based reporter assay including the type of instrumentation (fluorescence plate readers; fluorescence imaging systems) used for detection of GFP fluorescence.

  19. Development of a thyroperoxidase inhibition assay for high-throughput screening.

    PubMed

    Paul, Katie B; Hedge, Joan M; Rotroff, Daniel M; Hornung, Michael W; Crofton, Kevin M; Simmons, Steven O

    2014-03-17

    High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including Z', dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z' score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2',4,4'-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach. PMID:24383450

  20. Optimization and validation of two miniaturized glucocerebrosidase enzyme assays for high throughput screening.

    PubMed

    Urban, Daniel J; Zheng, Wei; Goker-Alpan, Ozlem; Jadhav, Ajit; Lamarca, Mary E; Inglese, James; Sidransky, Ellen; Austin, Christopher P

    2008-12-01

    Glucocerebrosidase (GC) catalyzes the hydrolysis of beta-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene (GBA) result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein. Some inhibitors of GC have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. High throughput screening (HTS) of small molecule libraries for such compounds with potential for chaperone therapy requires an accurate, reproducible and sensitive assay method. We have adapted and optimized two fluorogenic GC enzyme assays and miniaturized them into the 1536-well plate format for HTS. The two substrates, 4-methylumbelliferyl beta-D-glucopyranoside and resorufin beta-D-glucopyranoside, have K(m) values of 768 microM and 33 microM, respectively, and different emission spectra. Paired screening with the two assays helps to eliminate false inference of activity due to autofluorescence or fluorescence quenching by the screened compounds. Test screens with the LOPAC library indicated that both assays were robust for HTS, and gave comparable results for GC inhibitor activities. These two assays can be used to identify both GC activators and inhibitors with potential therapeutic value. PMID:19075603

  1. Optimization and Validation of Two Miniaturized Glucocerebrosidase Enzyme Assays for High-Throughput Screening

    PubMed Central

    Urban, Daniel J.; Zheng, Wei; Goker-Alpan, Ozlem; Jadhav, Ajit; LaMarca, Mary E.; Inglese, James; Sidransky, Ellen; Austin, Christopher P.

    2009-01-01

    Glucocerebrosidase (GC) catalyzes the hydrolysis of β-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene (GBA) result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein. Some inhibitors of GC have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. High-throughput screening (HTS) of small molecule libraries for such compounds with potential for chaperone therapy requires an accurate, reproducible and sensitive assay method. We have adapted and optimized two fluorogenic GC enzyme assays and miniaturized them into the 1536-well plate format for HTS. The two substrates, 4-methylumbelliferyl β-D-glucopyranoside and resorufin β-D-glucopyranoside, have Km values of 768 μM and 33 μM, respectively, and different emission spectra. Paired screening with the two assays helps to eliminate false inference of activity due to autofluorescence or fluorescence quenching by the screened compounds. Test screens with the LOPAC library indicated that both assays were robust for HTS, and gave comparable results for GC inhibitor activities. These two assays can be used to identify both GC activators and inhibitors with potential therapeutic value. PMID:19075603

  2. Validating a Firefly Luciferase-Based High-Throughput Screening Assay for Antimalarial Drug Discovery

    PubMed Central

    Che, Pulin; Cui, Long; Kutsch, Olaf; Cui, Liwang

    2012-01-01

    Abstract The emergence and spread of multidrug-resistant Plasmodium falciparum and recent detection of potential artemisinin-resistant strains in Southeast Asia highlight the importance of developing novel antimalarial therapies. Using a previously generated stable transgenic P. falciparum line with high-level firefly luciferase expression, we report the adaptation, miniaturization, optimization, and validation of a high-throughput screening assay in 384-well plates. Assay conditions, including the percentage of parasitemia and hematocrit, were optimized. Parameters of assay robustness, including Z′-value, coefficient variation (CV), and signal-to-background (S/B) ratio, were determined. The LOPAC1280 small-compound library was used to validate this assay. Our results demonstrated that this assay is robust and reliable, with an average Z′-value of >0.7 and CV of <10%. Moreover, this assay showed a very low background, with the S/B ratio up to 71. Further, identified hits were selected and confirmed using a SYBR Green I-based confirmatory assay. It is evident that this assay is suitable for large-scale screening of chemical libraries for antimalarial drug discovery. PMID:22050430

  3. High-throughput functional screening using a homemade dual-glow luciferase assay.

    PubMed

    Baker, Jessica M; Boyce, Frederick M

    2014-01-01

    We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest. PMID:24962249

  4. High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

    PubMed Central

    Baker, Jessica M.; Boyce, Frederick M.

    2014-01-01

    We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson's disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest. PMID:24962249

  5. Assay development and high throughput antiviral drug screening against Bluetongue virus

    PubMed Central

    Li, Qianjun; Maddox, Clinton; Rasmussen, Lynn; Hobrath, Judith V.; White, Lucile E.

    2009-01-01

    Bluetongue virus (BTV) infection is one of the most important diseases of domestic livestock. There are no antivirals available against BTV disease. In this paper, we present the development, optimization and validation of an in vitro cell-based high-throughput screening (HTS) assay using the luminescent-based CellTiter-Glo reagent to identify novel antivirals against BTV. Conditions of the cytopathic effect (CPE)-based assay were optimized at cell density of 5 000 cells/well in medium containing 1% FBS and a multiplicity of infection at 0.01 in 384-well plate, with Z'-values ≥ 0.70, Coefficient of Variations ≥ 5.68 and signal-to-background ratio ≥ 7.10. This assay was further validated using a 9 532 compound library. The fully validated assay was then used to screen the 194 950 compound collection, which identified 693 compounds with > 30% CPE inhibition. The ten-concentration dose response assay identified 185 structures with IC50 ≤ 100 μM, out of which 42 compounds were grouped into six analog series corresponding to six scaffolds enriched within the active set compared to their distribution in the library. The CPE-based assay development demonstrated its robustness and reliability, and its application in the HTS campaign will make significant contribution to the antiviral drug discovery against BTV disease. PMID:19559054

  6. A Novel High-Throughput Screening Assay for Discovery of Molecules That Increase Cellular Tetrahydrobiopterin

    PubMed Central

    LI, LI; DU, YUHONG; CHEN, WEI; FU, HAIAN; HARRISON, DAVID G.

    2015-01-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH4 has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH4. The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH4 levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH4 levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z′ factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein–protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH4 levels. PMID:21693765

  7. A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin.

    PubMed

    Li, Li; Du, Yuhong; Chen, Wei; Fu, Haian; Harrison, David G

    2011-09-01

    Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels. PMID:21693765

  8. Fluorescence polarization assays in high-throughput screening and drug discovery: a review

    NASA Astrophysics Data System (ADS)

    Hall, Matthew D.; Yasgar, Adam; Peryea, Tyler; Braisted, John C.; Jadhav, Ajit; Simeonov, Anton; Coussens, Nathan P.

    2016-06-01

    The sensitivity of fluorescence polarization (FP) and fluorescence anisotropy (FA) to molecular weight changes has enabled the interrogation of diverse biological mechanisms, ranging from molecular interactions to enzymatic activity. Assays based on FP/FA technology have been widely utilized in high-throughput screening (HTS) and drug discovery due to the homogenous format, robust performance and relative insensitivity to some types of interferences, such as inner filter effects. Advancements in assay design, fluorescent probes, and technology have enabled the application of FP assays to increasingly complex biological processes. Herein we discuss different types of FP/FA assays developed for HTS, with examples to emphasize the diversity of applicable targets. Furthermore, trends in target and fluorophore selection, as well as assay type and format, are examined using annotated HTS assays within the PubChem database. Finally, practical considerations for the successful development and implementation of FP/FA assays for HTS are provided based on experience at our center and examples from the literature, including strategies for flagging interference compounds among a list of hits.

  9. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays

    PubMed Central

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R.

    2015-01-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise–filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC50 (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds. PMID:25904095

  10. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays.

    PubMed

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R

    2015-08-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds. PMID:25904095

  11. Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors.

    PubMed

    Mukherjee, Abhisek; Syeb, Kathleen; Concannon, John; Callegari, Keri; Soto, Claudio; Glicksman, Marcie A

    2015-01-01

    Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer's and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies. PMID:26176772

  12. Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors

    PubMed Central

    Mukherjee, Abhisek; Syeb, Kathleen; Concannon, John; Callegari, Keri; Soto, Claudio; Glicksman, Marcie A.

    2015-01-01

    Currently there is no effective treatment available for major neurodegenerative disorders associated to protein misfolding, including Alzheimer’s and Parkinson's disease. One of most promising therapeutic approaches under development focuses on inhibiting the misfolding and aggregation pathway. However, it is likely that by the time clinical symptoms appear, there is a large accumulation of misfolded aggregates and a very substantial damage to the brain. Thus, it seems that at the clinical stage of the disease it is necessary also to develop strategies aiming to prevent the neuronal damage produced by already formed misfolded aggregates. Chronic activation of calcineurin (CaN), a type IIB phosphatase, has been implicated as a pivotal molecule connecting synaptic loss and neuronal damage to protein misfolding. The fact that the crystal structure of CaN is also well established makes it an ideal target for drug discovery. CaN activity assays for High Throughput Screening (HTS) reported so far are based on absorbance. In this article we report the development of a fluorescent quenching based CaN activity assay suitable for robotic screening of large chemical libraries to find novel inhibitors. The assay yielded a Z score of 0.84 with coefficient of variance ≤ 15%. Our results also show that this assay can be used to identify CaN inhibitors with a wide range of potencies. PMID:26176772

  13. Robust ridge regression estimators for nonlinear models with applications to high throughput screening assay data.

    PubMed

    Lim, Changwon

    2015-03-30

    Nonlinear regression is often used to evaluate the toxicity of a chemical or a drug by fitting data from a dose-response study. Toxicologists and pharmacologists may draw a conclusion about whether a chemical is toxic by testing the significance of the estimated parameters. However, sometimes the null hypothesis cannot be rejected even though the fit is quite good. One possible reason for such cases is that the estimated standard errors of the parameter estimates are extremely large. In this paper, we propose robust ridge regression estimation procedures for nonlinear models to solve this problem. The asymptotic properties of the proposed estimators are investigated; in particular, their mean squared errors are derived. The performances of the proposed estimators are compared with several standard estimators using simulation studies. The proposed methodology is also illustrated using high throughput screening assay data obtained from the National Toxicology Program. PMID:25490981

  14. A high-throughput screening assay to identify bacterial antagonists against Fusarium verticillioides.

    PubMed

    Figueroa-López, Alejandro Miguel; Cordero-Ramírez, Jesús Damián; Quiroz-Figueroa, Francisco Roberto; Maldonado-Mendoza, Ignacio Eduardo

    2014-07-01

    A high-throughput antagonistic assay was developed to screen for bacterial isolates capable of controlling the maize fungal phytopathogen Fusarium verticillioides. This assay combines a straightforward methodology, in which the fungus is challenged with bacterial isolates in liquid medium, with a novel approach that uses the plant lectin wheat germ agglutinin (WGA) coupled to a fluorophore (Alexa-Fluor® 488) under the commercial name of WGA, Alexa Fluor® 488 conjugate. The assay is performed in a 96-well plate format, which reduces the required laboratory space and streamlines quantitation and automation of the process, making it fast and accurate. The basis of our assay is that fungal biomass can be assessed by WGA, Alexa Fluor® 488 conjugate staining, which recognizes the chitin in the fungal cell wall and thus permits the identification of potential antagonistic bacteria that inhibit fungal growth. This principle was validated by chitin-competition binding assays against WGA, Alexa Fluor® 488 conjugate; confocal laser microscopy confirmed that the fluorescent WGA, Alexa Fluor® 488 conjugate binds to the chitin of the fungal cell wall. The majority of bacterial isolates did not bind to the WGA, Alexa Fluor® 488 conjugate. Furthermore, including washing steps significantly reduced any bacterial staining to background levels, even in the rare cases where bacterial isolates were capable of binding to WGA. Confirmatory conventional agar plate antagonistic assays were also conducted to validate our technique. We are now successfully employing this large-scale antagonistic assay as a pre-screening step for potential fungal antagonists in extensive bacteria collections (on the order of thousands of isolates). PMID:23787812

  15. Quantitative microtiter fibronectin fibrillogenesis assay: use in high throughput screening for identification of inhibitor compounds

    PubMed Central

    Tomasini-Johansson, Bianca R.; Johnson, Ian A.; Hoffmann, F. Michael; Mosher, Deane F.

    2012-01-01

    Fibronectin (FN) is a plasma glycoprotein that circulates in the near micromolar concentration range and is deposited along with locally produced FN in the extracellular matrices of many tissues. Control of FN deposition is tightly controlled by cells. Agents that modulate FN assembly may be useful therapeutically in conditions characterized by excessive FN deposition, such as fibrosis, inflammatory diseases, and malignancies. To identify such agents by high throughput screening (HTS), we developed a microtiter assay of FN deposition by human fibroblasts. The assay provides a robust read-out of FN assembly. Alexa 488-FN (A488-FN) was added to cell monolayers, and the total fluorescence intensity of deposited A488-FN was quantified. The fluorescence intensity of deposited A488-FN correlated with the presence of FN fibrils visualized by fluorescence microscopy. The assay Z’ values were 0.67 or 0.54, respectively, when using background values of fluorescence either with no added A488-FN or with A488-FN added together with a known inhibitor of FN deposition. The assay was used to screen libraries comprising 4160 known bioactive compounds. Nine compounds were identified as non- or low-cytotoxic inhibitors of FN assembly. Four (ML-9, HA-100, tyrphostin and imatinib mesylate) are kinase inhibitors, a category of compounds known to inhibit FN assembly; two (piperlongumine and cantharidin) are promoters of cancer cell apoptosis; and three (maprotiline, CGS12066B, and aposcopolamine) are modulators of biogenic amine signaling. The latter six compounds have not been recognized heretofore as affecting FN assembly. The assay is straight-forward, adapts to 96- and 384-well formats, and should be useful for routine measurement of FN deposition and HTS. Screening of more diverse chemical libraries and identification of specific and efficient modulators of FN fibrillogenesis may result in therapeutics to control excessive connective tissue deposition. PMID:22986508

  16. Evaluating the Impact of Uncertainties in Clearance and Exposure When Prioritizing Chemicals Screened in High-Throughput Assays

    EPA Science Inventory

    The toxicity-testing paradigm has evolved to include high-throughput (HT) methods for addressing the increasing need to screen hundreds to thousands of chemicals rapidly. Approaches that involve in vitro screening assays, in silico predictions of exposure concentrations, and phar...

  17. Development of a Novel Phosphorylated AMPK Protection Assay for High-Throughput Screening Using TR-FRET Assay.

    PubMed

    Xu, Yazhou; Wang, Yunjie; Xu, Yuan; Li, Jia; Liao, Hong; Zhang, Luyong; Pang, Tao

    2015-08-01

    AMP-activated protein kinase (AMPK), a conserved heterotrimeric kinase, serves as an energy sensor maintaining energy balance at both cellular and whole-body levels and plays multiple beneficial roles in carbohydrate and lipid metabolism, which makes AMPK an attractive target for diabetes and other metabolic disorders. To date, establishment of the physiologically relevant biochemical assay for AMPK has not been reported. Here we developed a phosphorylated AMPK protection assay based on a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, using the protein phosphatase 2A (PP2A) to dephosphorylate AMPK. The partially dephosphorylated AMPK by PP2A had lower activity than phosphorylated AMPK. This specific TR-FRET assay for AMPK was optimized in the 384-well format and produced similar EC(50) values for AMPK activators AMP and A769662 and a similar IC(50) value for AMPK inhibitor compound C, as previously reported. Under the optimized conditions, the assay Z' factor calculated over 160 data points has an optimal value greater than 0.5, which is suitable for high-throughput screening. In conclusion, this phosphorylated AMPK protection assay we developed is very robust, sensitive, and simple to perform and may be useful as a high-throughput assay for identifying AMPK activators with the ability of preventing activated AMPK against dephosphorylation by phosphatase in the physiological conditions. PMID:25956678

  18. Hematin Polymerization Assay as a High-Throughput Screen for Identification of New Antimalarial Pharmacophores

    PubMed Central

    Kurosawa, Yae; Dorn, Arnulf; Kitsuji-Shirane, Michiko; Shimada, Hisao; Satoh, Tomoko; Matile, Hugues; Hofheinz, Werner; Masciadri, Raffaello; Kansy, Manfred; Ridley, Robert G.

    2000-01-01

    Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of 14C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100,000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the “Catalyst” program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified “hit” compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program. PMID:10991837

  19. Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores.

    PubMed

    Kurosawa, Y; Dorn, A; Kitsuji-Shirane, M; Shimada, H; Satoh, T; Matile, H; Hofheinz, W; Masciadri, R; Kansy, M; Ridley, R G

    2000-10-01

    Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program. PMID:10991837

  20. A High-Throughput Screening Assay to Identify Kidney Toxic Compounds.

    PubMed

    Ramm, Susanne; Adler, Melanie; Vaidya, Vishal S

    2016-01-01

    Kidney toxicity due to drugs and chemicals poses a significant health burden for patients and a financial risk for pharmaceutical companies. However, currently no sensitive and high-throughput in vitro method exists for predictive nephrotoxicity assessment. Primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells, making them a desirable model to use in in vitro screening systems. Additionally, heme oxygenase 1 (HO-1) protein expression is upregulated as a protective mechanism during kidney toxicant-induced oxidative stress or inflammation in HPTECs and can therefore be used as a biomarker for nephrotoxicity. In this article, we describe two different methods to screen for HO-1 increase: A homogeneous time resolved fluorescence (HTRF) assay and an immunofluorescence assay. The latter provides lower throughput but higher sensitivity due to the combination of two readouts, HO-1 intensity and cell number. The methods described in the protocol are amendable for other cell types as well. © 2016 by John Wiley & Sons, Inc. PMID:27479365

  1. BioAssay Ontology (BAO): a semantic description of bioassays and high-throughput screening results

    PubMed Central

    2011-01-01

    Background High-throughput screening (HTS) is one of the main strategies to identify novel entry points for the development of small molecule chemical probes and drugs and is now commonly accessible to public sector research. Large amounts of data generated in HTS campaigns are submitted to public repositories such as PubChem, which is growing at an exponential rate. The diversity and quantity of available HTS assays and screening results pose enormous challenges to organizing, standardizing, integrating, and analyzing the datasets and thus to maximize the scientific and ultimately the public health impact of the huge investments made to implement public sector HTS capabilities. Novel approaches to organize, standardize and access HTS data are required to address these challenges. Results We developed the first ontology to describe HTS experiments and screening results using expressive description logic. The BioAssay Ontology (BAO) serves as a foundation for the standardization of HTS assays and data and as a semantic knowledge model. In this paper we show important examples of formalizing HTS domain knowledge and we point out the advantages of this approach. The ontology is available online at the NCBO bioportal http://bioportal.bioontology.org/ontologies/44531. Conclusions After a large manual curation effort, we loaded BAO-mapped data triples into a RDF database store and used a reasoner in several case studies to demonstrate the benefits of formalized domain knowledge representation in BAO. The examples illustrate semantic querying capabilities where BAO enables the retrieval of inferred search results that are relevant to a given query, but are not explicitly defined. BAO thus opens new functionality for annotating, querying, and analyzing HTS datasets and the potential for discovering new knowledge by means of inference. PMID:21702939

  2. High-Throughput/High-Content Screening Assays with Engineered Nanomaterials in ToxCast

    EPA Science Inventory

    High-throughput and high-content screens are attractive approaches for prioritizing nanomaterial hazards and informing targeted testing due to the impracticality of using traditional toxicological testing on the large numbers and varieties of nanomaterials. The ToxCast program a...

  3. Adapting High-Throughput Screening Methods and Assays for Biocontainment Laboratories

    PubMed Central

    Tigabu, Bersabeh; White, E. Lucile; Bostwick, Robert; Tower, Nichole; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W.; Noah, James W.

    2015-01-01

    Abstract High-throughput screening (HTS) has been integrated into the drug discovery process, and multiple assay formats have been widely used in many different disease areas but with limited focus on infectious agents. In recent years, there has been an increase in the number of HTS campaigns using infectious wild-type pathogens rather than surrogates or biochemical pathogen-derived targets. Concurrently, enhanced emerging pathogen surveillance and increased human mobility have resulted in an increase in the emergence and dissemination of infectious human pathogens with serious public health, economic, and social implications at global levels. Adapting the HTS drug discovery process to biocontainment laboratories to develop new drugs for these previously uncharacterized and highly pathogenic agents is now feasible, but HTS at higher biosafety levels (BSL) presents a number of unique challenges. HTS has been conducted with multiple bacterial and viral pathogens at both BSL-2 and BSL-3, and pilot screens have recently been extended to BSL-4 environments for both Nipah and Ebola viruses. These recent successful efforts demonstrate that HTS can be safely conducted at the highest levels of biological containment. This review outlines the specific issues that must be considered in the execution of an HTS drug discovery program for high-containment pathogens. We present an overview of the requirements for HTS in high-level biocontainment laboratories. PMID:25710545

  4. A high-throughput in vivo micronucleus assay for genome instability screening in mice

    PubMed Central

    Balmus, Gabriel; Karp, Natasha A; Ng, Bee Ling; Jackson, Stephen P; Adams, David J; McIntyre, Rebecca E

    2016-01-01

    We describe a sensitive, robust, high-throughput method for quantifying the formation of micronuclei, markers of genome instability, in mouse erythrocytes. Micronuclei are whole chromosomes or chromosome segments that have been separated from the nucleus. Other methods of detection rely on labour-intensive, microscopy-based techniques. Here, we describe a 2-d, 96-well plate-based flow cytometric method of micronucleus scoring that is simple enough for a research technician experienced in flow cytometry to perform. The assay detects low levels of genome instability that cannot be readily identified by classic phenotyping, using 25 μl of blood. By using this assay, we have screened >10,000 blood samples and discovered novel genes that contribute to vertebrate genome maintenance, as well as novel disease models and mechanisms of genome instability disorders. We discuss experimental design considerations, including statistical power calculation, we provide troubleshooting tips, and we discuss factors that contribute to a false-positive increase in the number of micronucleated red blood cells and to experimental variability. PMID:25551665

  5. Using adverse outcome pathway analysis to guide development of high-throughput screening assays for thyroid-disruptors

    EPA Science Inventory

    Using Adverse Outcome Pathway Analysis to Guide Development of High-Throughput Screening Assays for Thyroid-Disruptors Katie B. Paul1,2, Joan M. Hedge2, Daniel M. Rotroff4, Kevin M. Crofton4, Michael W. Hornung3, Steven O. Simmons2 1Oak Ridge Institute for Science Education Post...

  6. A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of gsp

    PubMed Central

    Bhattacharyya, Nisan; Hu, Xin; Chen, Catherine Z.; Mathews Griner, Lesley A.; Zheng, Wei; Inglese, James; Austin, Christopher P.; Marugan, Juan J.; Southall, Noel; Neumann, Susanne; Northup, John K.; Ferrer, Marc; Collins, Michael T.

    2014-01-01

    Mis-sense mutations in the α-subunit of the G-protein, Gsα, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gsα and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant Gsα protein, the so-called gsp oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant Gsα proteins (R201C and R201H). Stable cell lines with equivalent transfected Gsα protein expression that had relatively lower (WT) or higher (R201C and R201H) cAMP levels were generated. These cell lines were used to develop a fluorescence resonance energy transfer (FRET)–based cAMP assay in 1536-well microplate format for high throughput screening of small molecule libraries. A small molecule library of 343,768 compounds was screened to identify modulators of gsp activity. A total of 1,356 compounds with inhibitory activity were initially identified and reconfirmed when tested in concentration dose responses. Six hundred eighty-six molecules were selected for further analysis after removing cytotoxic compounds and those that were active in forskolin-induced WT cells. These molecules were grouped by potency, efficacy, and structural similarities to yield 22 clusters with more than 5 of structurally similar members and 144 singleton molecules. Seven chemotypes of the major clusters were identified for further testing and analyses. PMID:24667240

  7. Development and Optimization of a Novel 384-Well Anti-Malarial Imaging Assay Validated for High-Throughput Screening

    PubMed Central

    Duffy, Sandra; Avery, Vicky M.

    2012-01-01

    With the increasing occurrence of drug resistance in the malaria parasite, Plasmodium falciparum, there is a great need for new and novel anti-malarial drugs. We have developed a 384-well, high-throughput imaging assay for the detection of new anti-malarial compounds, which was initially validated by screening a marine natural product library, and subsequently used to screen more than 3 million data points from a variety of compound sources. Founded on another fluorescence-based P. falciparum growth inhibition assay, the DNA-intercalating dye 4′,6-diamidino-2-phenylindole, was used to monitor changes in parasite number. Fluorescent images were acquired on the PerkinElmer Opera High Throughput confocal imaging system and analyzed with a spot detection algorithm using the Acapella data processing software. Further optimization of this assay sought to increase throughput, assay stability, and compatibility with our high-throughput screening equipment platforms. The assay typically yielded Z'-factor values of 0.5–0.6, with signal-to-noise ratios of 12. PMID:22232455

  8. Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

    PubMed

    Ngo, Tony; Coleman, James L J; Smith, Nicola J

    2015-01-01

    Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system. PMID:25563179

  9. Large-scale drug screening against Babesia divergens parasite using a fluorescence-based high-throughput screening assay.

    PubMed

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; AbouLaila, Mahmoud; Tuvshintulga, Bumduuren; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-08-30

    The validation of a fluorescence-based high-throughput screening (HTS) assay for determining the efficacies of large chemical libraries against Babesia divergens (bovine strain) in in vitro cultures was evaluated in this study. Hematocrits (HCTs) of 2.5%, 5%, and 10% were used for the in vitro culture at 1% parasitemia without daily replacement of the medium. Linearity and HTS assay results revealed that the best HCTs were 5% and 10%. The obtained IC50 values of diminazene aceturate, either by fluorescence-based HTS assay with and without daily replacement of medium or by fluorescence- and microscopy-based methods, did not differ significantly at 5% HCT. Actinonin and chloroquine diphosphate were the most effective drugs against the in vitro growth of B. divergens, followed by pyronaridine tetraphosphate- and luteolin-treated cultures. On contrary, tetracycline hydrochloride and (-)-epigallocatechin-3-gallate from green tea exhibited poor activity as compared with diminazene aceturate (positive control drug). The data indicated that 5% HCT without daily replacement of the culture medium mixed with bovine serum in vitro using a fluorescence-based HTS assay creates the best conditions for large-scale drug screening against B. divergens that infect cattle. PMID:27523944

  10. Development and Implementation of a High-Throughput Compound Screening Assay for Targeting Disrupted ER Calcium Homeostasis in Alzheimer's Disease

    PubMed Central

    Honarnejad, Kamran; Daschner, Alexander; Giese, Armin; Zall, Andrea; Schmidt, Boris; Szybinska, Aleksandra; Kuznicki, Jacek; Herms, Jochen

    2013-01-01

    Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer's disease (AD) pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER). Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease. PMID:24260442

  11. High-throughput microsomal stability assay for screening new chemical entities in drug discovery.

    PubMed

    Fonsi, Massimiliano; Orsale, Maria V; Monteagudo, Edith

    2008-10-01

    In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities. PMID:18812573

  12. Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

    EPA Science Inventory

    We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

  13. Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase.

    PubMed

    Meleza, Cesar; Thomasson, Bobbie; Ramachandran, Chidambaram; O'Neill, Jason W; Michelsen, Klaus; Lo, Mei-Chu

    2016-10-15

    Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH2, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay. PMID:27485270

  14. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    SciTech Connect

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer An endothelial cell apoptosis assay using FRET-based biosensor was developed. Black-Right-Pointing-Pointer The fluorescence of the cells changed from green to blue during apoptosis. Black-Right-Pointing-Pointer This method was developed into a high-throughput assay in 96-well plates. Black-Right-Pointing-Pointer This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z Prime factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  15. Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format.

    PubMed

    Klumpp, Martin; Boettcher, Andreas; Becker, Damaris; Meder, Gabriele; Blank, Jutta; Leder, Lukas; Forstner, Michael; Ottl, Johannes; Mayr, Lorenz M

    2006-09-01

    This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns. PMID:16760365

  16. A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels.

    PubMed

    Kruger, Wade; Gilbert, Daniel; Hawthorne, Rebecca; Hryciw, Deanne H; Frings, Stephan; Poronnik, Philip; Lynch, Joseph W

    2005-06-01

    There is a significant clinical need to identify novel ligands with high selectivity and potency for GABA(A), GABA(C) and glycine receptor Cl- channels. Two recently developed, yellow fluorescent protein variants (YFP-I152L and YFP-V163S) are highly sensitive to quench by small anions and are thus suited to reporting anionic influx into cells. The aim of this study was to establish the optimal conditions for using these constructs for high-throughput screening of GABA(A), GABA(C) and glycine receptors transiently expressed in HEK293 cells. We found that a 70% fluorescence reduction was achieved by quenching YFP-I152L with a 10 s influx of I- ions, driven by an external I- concentration of at least 50 mM. The fluorescence quench was rapid, with a mean time constant of 3 s. These responses were similar for all anion receptor types studied. We also show the assay is sufficiently sensitive to measure agonist and antagonist concentration-responses using either imaging- or photomultiplier-based detection systems. The robustness, sensitivity and low cost of this assay render it suited for high-throughput screening of transiently expressed anionic ligand-gated channels. PMID:15862914

  17. Development of FRET assay into quantitative and high-throughput screening technology platforms for protein-protein interactions.

    PubMed

    Song, Yang; Madahar, Vipul; Liao, Jiayu

    2011-04-01

    Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a very powerful tool in elucidating protein interactions in many cellular processes. Ubiquitination and SUMOylation are multi-step cascade reactions, involving multiple enzymes and protein-protein interactions. Here we report the development of dissociation constant (K (d)) determination for protein-protein interaction and cell-based high-throughput screening (HTS) assay in SUMOylation cascade using FRET technology. These developments are based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused with SUMO1 and Ubc9, respectively. The developments in theoretical and experimental procedures for protein interaction K (d) determination and cell-based HTS provide novel tools in affinity measurement and protein interaction inhibitor screening. The K (d) determined by FRET between SUMO1 and Ubc9 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The FRET-based HTS is pioneer in cell-based HTS. Both K (d) determination and cell-based HTS, carried out in 384-well plate format, provide powerful tools for large-scale and high-throughput applications. PMID:21174150

  18. Luciferase-Based, High-Throughput Assay for Screening and Profiling Transmission-Blocking Compounds against Plasmodium falciparum Gametocytes.

    PubMed

    Lucantoni, Leonardo; Fidock, David A; Avery, Vicky M

    2016-04-01

    The discovery of new antimalarial drugs able to target both the asexual and gametocyte stages ofPlasmodium falciparumis critical to the success of the malaria eradication campaign. We have developed and validated a robust, rapid, and cost-effective high-throughput reporter gene assay to identify compounds active against late-stage (stage IV and V) gametocytes. The assay, which is suitable for testing compound activity at incubation times up to 72 h, demonstrates excellent quality and reproducibility, with averageZ' values of 0.85 ± 0.01. We used the assay to screen more than 10,000 compounds from three chemically diverse libraries. The screening outcomes highlighted the opportunity to use collections of compounds with known activity against the asexual stages of the parasites as a starting point for gametocytocidal activity detection in order to maximize the chances of identifying gametocytocidal compounds. This assay extends the capabilities of our previously reported luciferase assay, which tested compounds against early-stage gametocytes, and opens possibilities to profile the activities of gametocytocidal compounds over the entire course of gametocytogenesis. PMID:26787698

  19. A Cell-based PDE4 Assay in 1536-well Plate format for High Throughput Screening

    PubMed Central

    Titus, Steven A.; Li, Xiao; Southall, Noel; Lu, Jianming; Inglese, James; Brasch, Michael; Austin, Christopher P.; Zheng, Wei

    2009-01-01

    The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3', 5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'-nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases including asthma, cardiovascular disease, ADHD, Parkinson’s disease, and Alzheimer’s disease. Though biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. Here we report the development and validation of a new cell-based PDE4 assay using a constitutively active GPCR as a driving force for cAMP production and a cyclic nucleotide gated (CNG) cation channel as a biosensor in 1536-well plates. PMID:18591513

  20. A Phenotypic High Throughput Screening Assay for the Identification of Pharmacoperones for the Gonadotropin Releasing Hormone Receptor

    PubMed Central

    Smith, Emery; Spicer, Timothy; Chase, Peter; Scampavia, Louis; Janovick, Jo Ann

    2014-01-01

    Abstract We describe a phenotypic high throughput screening (HTS) calcium flux assay designed to identify pharmacoperones for the gonadotropin releasing hormone receptor (GnRHR). Pharmacoperones are target-specific, small molecules that diffuse into cells, rescue misfolded protein mutants, and restore them to function. Rescue is based on correcting the trafficking of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function correctly. This approach identifies drugs with a significant degree of novelty, relying on cellular mechanisms that are not currently exploited. Development of such assays is important, since the extensive use of agonist/antagonist screens alone means that useful chemical structures may be present in existing libraries but have not been previously identified using existing methods. Our assay utilizes cell lines stably expressing a GnRHR mutant under the control of a tetracycline (OFF) transactivator. This allows us to quantitate the level of functional and properly trafficked G protein coupled receptors present in each test well. Furthermore, since we are able to turn receptor expression on and off, we can rapidly eliminate the majority of false positives from our screening results. Our data show that this approach is likely to be successful in identifying hits from large chemical libraries. PMID:24831790

  1. Quantitative High Throughput Screening Using a Live Cell cAMP Assay Identifies Small Molecule Agonists of the TSH Receptor

    PubMed Central

    Titus, Steve; Neumann, Susanne; Zheng, Wei; Southall, Noel; Michael, Sam; Klumpp, Carleen; Yasgar, Adam; Shinn, Paul; Thomas, Craig J.; Inglese, Jim; Gershengorn, Marvin C.; Austin, Christopher P.

    2009-01-01

    The thyroid stimulating hormone receptor (TSHR) belongs to the glycoprotein hormone receptor subfamily of seven-transmembrane spanning receptors. TSHR is expressed in thyroid follicular cells and is activated by TSH, which regulates growth and function of these cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small molecule agonist of the TSHR is available. To screen for novel TSHR agonists, we miniaturized a cell-based cAMP assay into 1536-well plate format. This assay uses a HEK293 cell line stably expressing the TSHR and a cyclic nucleotide gated ion channel (CNG), which functions as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal HTRF cAMP-based assay. 49 compounds in several structural classes have been confirmed as small molecule TSHR agonists that will serve as starting compounds for chemical optimization and studies of thyroid physiology in health and disease. PMID:18216391

  2. High throughput screening technologies for ion channels

    PubMed Central

    Yu, Hai-bo; Li, Min; Wang, Wei-ping; Wang, Xiao-liang

    2016-01-01

    Ion channels are involved in a variety of fundamental physiological processes, and their malfunction causes numerous human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Chronologically, HTS methods for ion channels include the ligand binding assay, flux-based assay, fluorescence-based assay, and automated electrophysiological assay. In this review we summarize the current HTS technologies for different ion channel classes and their applications. PMID:26657056

  3. Risk-Based High-Throughput Chemical Screening and Prioritization using Exposure Models and in Vitro Bioactivity Assays.

    PubMed

    Shin, Hyeong-Moo; Ernstoff, Alexi; Arnot, Jon A; Wetmore, Barbara A; Csiszar, Susan A; Fantke, Peter; Zhang, Xianming; McKone, Thomas E; Jolliet, Olivier; Bennett, Deborah H

    2015-06-01

    We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate daily intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models. PMID:25932772

  4. Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

    SciTech Connect

    Shin, Hyeong -Moo; Ernstoff, Alexi; Arnot, Jon A.; Wetmore, Barbara A.; Csiszar, Susan A.; Fantke, Peter; Zhang, Xianming; McKone, Thomas E.; Jolliet, Olivier; Bennett, Deborah H.

    2015-05-01

    We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate daily intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models.

  5. Risk-based high-throughput chemical screening and prioritization using exposure models and in vitro bioactivity assays

    DOE PAGESBeta

    Shin, Hyeong -Moo; Ernstoff, Alexi; Arnot, Jon A.; Wetmore, Barbara A.; Csiszar, Susan A.; Fantke, Peter; Zhang, Xianming; McKone, Thomas E.; Jolliet, Olivier; Bennett, Deborah H.

    2015-05-01

    We present a risk-based high-throughput screening (HTS) method to identify chemicals for potential health concerns or for which additional information is needed. The method is applied to 180 organic chemicals as a case study. We first obtain information on how the chemical is used and identify relevant use scenarios (e.g., dermal application, indoor emissions). For each chemical and use scenario, exposure models are then used to calculate a chemical intake fraction, or a product intake fraction, accounting for chemical properties and the exposed population. We then combine these intake fractions with use scenario-specific estimates of chemical quantity to calculate dailymore » intake rates (iR; mg/kg/day). These intake rates are compared to oral equivalent doses (OED; mg/kg/day), calculated from a suite of ToxCast in vitro bioactivity assays using in vitro-to-in vivo extrapolation and reverse dosimetry. Bioactivity quotients (BQs) are calculated as iR/OED to obtain estimates of potential impact associated with each relevant use scenario. Of the 180 chemicals considered, 38 had maximum iRs exceeding minimum OEDs (i.e., BQs > 1). For most of these compounds, exposures are associated with direct intake, food/oral contact, or dermal exposure. The method provides high-throughput estimates of exposure and important input for decision makers to identify chemicals of concern for further evaluation with additional information or more refined models.« less

  6. A High-Throughput Radiometric Kinase Assay.

    PubMed

    Duong-Ly, Krisna C; Peterson, Jeffrey R

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening of libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small-molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  7. High-throughput screening assay for the environmental water samples using cellular response profiles.

    PubMed

    Pan, Tianhong; Li, Haoran; Khare, Swanand; Huang, Biao; Yu Huang, Dorothy; Zhang, Weiping; Gabos, Stephan

    2015-04-01

    Chemical and physical analyses are commonly used as screening methods for the environmental water. However, these methods can only look for the targeted substance but may miss unexpected toxicants. Furthermore, the synergistic effects of mixture cannot be detected. In order to set up the assay criteria for determining various biological activities at a cellular level that could potentially lead to toxicity of environmental water samples, a novel test based on cellular response by using Real-Time Cellular Analyzer (RTCA) is proposed in this study. First, the water sample is diluted to a series of strengths (80%, 60%, 40%, 30%, 20% and 10%) to get the multi-concentration cellular response profile. Then, the area under the cellular response profile (AUCRP) is calculated. Comparing to the normal cell growth of negative control, a new biological activity index named Percentage of Effect (PoE) has been presented which reflects the cumulative inhibitory activity of cell growth over the log-phase. Finally, a synthetical index PoE50 is proposed to evaluate the intensity of biological activities in water samples. The biological experiment demonstrates the effectiveness of the proposed method. PMID:25637748

  8. A novel, sensitive assay for high-throughput molecular detection of plasmodia for active screening of malaria for elimination.

    PubMed

    Cheng, Zhibin; Sun, Xiaodong; Yang, Ye; Wang, Heng; Zheng, Zhi

    2013-01-01

    Although malaria remains one of the leading infectious diseases in the world, the decline in malaria transmission in some area makes it possible to consider elimination of the disease. As countries approach elimination, malaria diagnosis needs to change from diagnosing ill patients to actively detecting infections in all carriers, including asymptomatic and low-parasite-load patients. However, few of the current diagnostic methods have both the throughput and the sensitivity required. We adopted a sandwich RNA hybridization assay to detect genus Plasmodium 18S rRNA directly from whole-blood samples from Plasmodium falciparum and Plasmodium vivax patients without RNA isolation. We tested the assay with 202 febrile patients from areas where malaria is endemic, using 20 μl of each blood sample in a 96-well plate format with a 2-day enzyme-linked immunosorbent assay (ELISA)-like work flow. The results were compared with diagnoses obtained using microscopy, a rapid diagnostic test (RDT), and genus-specific real-time PCR. Our assay identified all 66 positive samples diagnosed by microscopy, including 49 poorly stored samples that underwent multiple freeze-thaw cycles due to resource limitation. The assay uncovered three false-negative samples by microscopy and four false-negative samples by RDT and agreed completely with real-time PCR diagnosis. There was no negative sample by our assay that would show a positive result when tested with other methods. The detection limit of our assay for P. falciparum was 0.04 parasite/μl. The assay's simple work flow, high throughput, and sensitivity make it suitable for active malaria screening. PMID:23100347

  9. Adaptation of the bivalve embryotoxicity assay for the high throughput screening of emerging contaminants in Mytilus galloprovincialis.

    PubMed

    Fabbri, Rita; Montagna, Michele; Balbi, Teresa; Raffo, Enrico; Palumbo, Franca; Canesi, Laura

    2014-08-01

    Emerging contaminants (such as Endocrine disrupting chemicals-EDCs, brominated and perfluorinated compounds-BFRs and PFCs, pharmaceuticals) are chemicals currently not included in regulatory monitoring programs, and whose fate and biological impacts are poorly understood. Assessment of ecosystem health with respect to these chemicals is of particular concern also in the marine environment: in this respect, data on the effects on early life stages are important to establish the sensitivity of marine species. In this work, the acute (48 h) bivalve embryo toxicity test was applied for screening the developmental effects of different emerging contaminants in the Mediterranean mussel Mytilus galloprovincialis. The assay was adapted to 96-microwell plates, and standardized in order to obtain to normal D-shaped larvae with acceptability of test results based on negative control and positive control (copper) comparable with those reported in literature for Mytilus spp. The effects of different model compounds representative of EDCs (Nonylphenol-NP and Bisphenol A-BPA), BFRs (Tetrabromobisphenol A-TBBPA), PFCs (perfluorooctanoid acid-PFOA and perfluorooctane sulphonate-PFOAS) and pharmaceuticals (Ibuprofen-IBU, Diclofenac-DCF, Bezafibrate-BEZA) in a wide concentration range (0.01-0.1-1-10-100-1000 μg/L) were evaluated. The assay proved as a sensitive tool for high throughput screening of emerging contaminants in a marine species, leading to production of significant amounts of data that may be useful for regulatory purposes. PMID:25081847

  10. A High-Throughput Fluorescence-Based Assay System for Appetite-Regulating Gene and Drug Screening

    PubMed Central

    Shimada, Yasuhito; Hirano, Minoru; Nishimura, Yuhei; Tanaka, Toshio

    2012-01-01

    The increasing number of people suffering from metabolic syndrome and obesity is becoming a serious problem not only in developed countries, but also in developing countries. However, there are few agents currently approved for the treatment of obesity. Those that are available are mainly appetite suppressants and gastrointestinal fat blockers. We have developed a simple and rapid method for the measurement of the feeding volume of Danio rerio (zebrafish). This assay can be used to screen appetite suppressants and enhancers. In this study, zebrafish were fed viable paramecia that were fluorescently-labeled, and feeding volume was measured using a 96-well microplate reader. Gene expression analysis of brain-derived neurotrophic factor (bdnf), knockdown of appetite-regulating genes (neuropeptide Y, preproinsulin, melanocortin 4 receptor, agouti related protein, and cannabinoid receptor 1), and the administration of clinical appetite suppressants (fluoxetine, sibutramine, mazindol, phentermine, and rimonabant) revealed the similarity among mechanisms regulating appetite in zebrafish and mammals. In combination with behavioral analysis, we were able to evaluate adverse effects on locomotor activities from gene knockdown and chemical treatments. In conclusion, we have developed an assay that uses zebrafish, which can be applied to high-throughput screening and target gene discovery for appetite suppressants and enhancers. PMID:23300705

  11. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents.

    PubMed

    Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

    2012-02-24

    In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z' factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening. PMID:22290227

  12. Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay.

    PubMed

    Sharma, Pooja; Jindal, Swati; Bala, Kiran; Kumari, Kirti; Niharika, Neha; Kaur, Jasvinder; Pandey, Gunjan; Pandey, Rinku; Russell, Robyn J; Oakeshott, John G; Lal, Rup

    2014-04-01

    Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified. PMID:23740574

  13. Origin and evolution of high throughput screening

    PubMed Central

    Pereira, D A; Williams, J A

    2007-01-01

    This article reviews the origin and evolution of high throughput screening (HTS) through the experience of an individual pharmaceutical company, revealing some of the mysteries of the early stages of drug discovery to the wider pharmacology audience. HTS in this company (Pfizer, Groton, USA) had its origin in natural products screening in 1986, by substituting fermentation broths with dimethyl sulphoxide solutions of synthetic compounds, using 96-well plates and reduced assay volumes of 50-100μl. A nominal 30mM source compound concentration provided high μM assay concentrations. Starting at 800 compounds each week, the process reached a steady state of 7200 compounds per week by 1989. Screening in the Applied Biotechnology and Screening Group was centralized with screens operating in lock-step to maximize efficiency. Initial screens were full files run in triplicate. Autoradiography and image analysis were introduced for 125I receptor ligand screens. Reverse transcriptase (RT) coupled with quantitative PCR and multiplexing addressed several targets in a single assay. By 1992 HTS produced ‘hits' as starting matter for approximately 40% of the Discovery portfolio. In 1995, the HTS methodology was expanded to include ADMET targets. ADME targets required each compound to be physically detected leading to the development of automated high throughput LC-MS. In 1996, 90 compounds/week were screened in microsomal, protein binding and serum stability assays. Subsequently, the mutagenic Ames assay was adapted to a 96-well plate liquid assay and novel algorithms permitted automated image analysis of the micronucleus assay. By 1999 ADME HTS was fully integrated into the discovery cycle. PMID:17603542

  14. Using in Vitro High Throughput Screening Assays to Identify Potential Endocrine-Disrupting Chemicals

    EPA Science Inventory

    Over the past 20 years, an increased focus on detecting environmental chemicals posing a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. EPA Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP, whic...

  15. High-Throughput Cell Toxicity Assays.

    PubMed

    Murray, David; McWilliams, Lisa; Wigglesworth, Mark

    2016-01-01

    Understanding compound-driven cell toxicity is vitally important for all drug discovery approaches. With high-throughput screening (HTS) being the key strategy to find hit and lead compounds for drug discovery projects in the pharmaceutical industry [1], an understanding of the cell toxicity profile of hit molecules from HTS activities is fundamentally important. Recently, there has been a resurgence of interest in phenotypic drug discovery and these cell-based assays are now being run in HTS labs on ever increasing numbers of compounds. As the use of cell assays increases the ability to measure toxicity of compounds on a large scale becomes increasingly important to ensure that false hits are not progressed and that compounds do not carry forward a toxic liability that may cause them to fail at later stages of a project. Here we describe methods employed in the AstraZeneca HTS laboratory to carry out very large scale cell toxicity screening. PMID:27317000

  16. Characterization of Diversity in Toxicity Mechanism Using In Vitro Cytotoxicity Assays in Quantitative High Throughput Screening

    PubMed Central

    Huang, Ruili; Southall, Noel; Cho, Ming-Hsuang; Xia, Menghang; Inglese, James; Austin, Christopher P.

    2009-01-01

    Assessing the potential health risks of environmental chemical compounds is an expensive undertaking which has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms. PMID:18281954

  17. High-throughput assay comparison and standardization for metal chelating capacity screening: A proposal and application.

    PubMed

    Santos, Jânio Sousa; Alvarenga Brizola, Vitor Rafael; Granato, Daniel

    2017-01-01

    Aiming to standardize the experimental protocols to assess the ability to chelate Fe(2+) and Cu(2+) using 96-well microplates, we analyzed Brazilian coffees (n=20) as a study-case in relation to their antioxidant activity using conventional methods (DPPH and FRAP assays) and correlated the results with the total phenolic content (TPC) using bivariate and multivariate statistical approaches. Complementarily, we assessed the repeatability, reproducibility, recovery, and linearity of both methods. Data showed that the proposed assays presented a good repeatability and reproducibility (<7% RSD) and mean recovery values of 96.66% and 98.91% for the iron and copper assays, respectively. Both methods were linear in the range of 0-100mg EDTA equivalents/L. Cu(2+)-chelating ability was significantly correlated to FRAP, DPPH, and TPC, while sparse (p<0.05) correlations were obtained with Fe(2+)-chelating ability. Overall, both micro assays can be used to assess the ability of plant-based extracts to chelate Fe(2+) and Cu(2+)in vitro. PMID:27507505

  18. A Call for Nominations of Quantitative High-Throughput Screening Assays from Relevant Human Toxicity Pathways

    EPA Science Inventory

    The National Research Council of the United States National Academies of Science has recently released a document outlining a long-range vision and strategy for transforming toxicity testing from largely whole animal-based testing to one based on in vitro assays. “Toxicity Testin...

  19. Evaluation of Compatibility of ToxCast High-Throughput/High-Content Screening Assays with Engineered Nanomaterials

    EPA Science Inventory

    High-throughput and high-content screens are attractive approaches for prioritizing nanomaterial hazards and informing targeted testing due to the impracticality of using traditional toxicological testing on the large numbers and varieties of nanomaterials. The ToxCast program a...

  20. Evaluating the Impact of Uncertainties in Clearance and Exposure When Prioritizing Chemicals Screened in High-Throughput Assays.

    PubMed

    Leonard, Jeremy A; Sobel Leonard, Ashley; Chang, Daniel T; Edwards, Stephen; Lu, Jingtao; Scholle, Steven; Key, Phillip; Winter, Maxwell; Isaacs, Kristin; Tan, Yu-Mei

    2016-06-01

    The toxicity-testing paradigm has evolved to include high-throughput (HT) methods for addressing the increasing need to screen hundreds to thousands of chemicals rapidly. Approaches that involve in vitro screening assays, in silico predictions of exposure concentrations, and pharmacokinetic (PK) characteristics provide the foundation for HT risk prioritization. Underlying uncertainties in predicted exposure concentrations or PK behaviors can significantly influence the prioritization of chemicals, though the impact of such influences is unclear. In the current study, a framework was developed to incorporate absorbed doses, PK properties, and in vitro dose-response data into a PK/pharmacodynamic (PD) model to allow for placement of chemicals into discrete priority bins. Literature-reported or predicted values for clearance rates and absorbed doses were used in the PK/PD model to evaluate the impact of their uncertainties on chemical prioritization. Scenarios using predicted absorbed doses resulted in a larger number of bin misassignments than those scenarios using predicted clearance rates, when comparing to bin placement using literature-reported values. Sensitivity of parameters on the model output of toxicological activity was examined across possible ranges for those parameters to provide insight into how uncertainty in their predicted values might impact uncertainty in activity. PMID:27124219

  1. High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.

    PubMed

    Tsotsou, Georgia Eleni; Cass, Anthony Edward George; Gilardi, Gianfranco

    2002-01-01

    A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts. PMID:11742743

  2. Bringing the light to high throughput screening: use of optogenetic tools for the development of recombinant cellular assays

    NASA Astrophysics Data System (ADS)

    Agus, Viviana; Di Silvio, Alberto; Rolland, Jean Francois; Mondini, Anna; Tremolada, Sara; Montag, Katharina; Scarabottolo, Lia; Redaelli, Loredana; Lohmer, Stefan

    2015-03-01

    The use of light-activated proteins represents a powerful tool to control biological processes with high spatial and temporal precision. These so called "optogenetic" technologies have been successfully validated in many recombinant systems, and have been widely applied to the study of cellular mechanisms in intact tissues or behaving animals; to do that, complex, high-intensity, often home-made instrumentations were developed to achieve the optimal power and precision of light stimulation. In our study we sought to determine if this optical modulation can be obtained also in a miniaturized format, such as a 384-well plate, using the instrumentations normally dedicated to fluorescence analysis in High Throughput Screening (HTS) activities, such as for example the FLIPR (Fluorometric Imaging Plate Reader) instrument. We successfully generated optogenetic assays for the study of different ion channel targets: the CaV1.3 calcium channel was modulated by the light-activated Channelrhodopsin-2, the HCN2 cyclic nucleotide gated (CNG) channel was modulated by the light activated bPAC adenylyl cyclase, and finally the genetically encoded voltage indicator ArcLight was efficiently used to measure potassium, sodium or chloride channel activity. Our results showed that stable, robust and miniaturized cellular assays can be developed using different optogenetic tools, and efficiently modulated by the FLIPR instrument LEDs in a 384-well format. The spatial and temporal resolution delivered by this technology might enormously advantage the early stages of drug discovery, leading to the identification of more physiological and effective drug molecules.

  3. A high-throughput differential filtration assay to screen and select detergents for membrane proteins

    PubMed Central

    Vergis, James M.; Purdy, Michael D.; Wiener, Michael C.

    2015-01-01

    Structural studies on integral membrane proteins are routinely performed on protein–detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and “protein-expensive” process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours. PMID:20667442

  4. High-throughput Screening of ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell (mESC) Assay Reveals Disruption of Potential Toxicity Pathways

    EPA Science Inventory

    Little information is available regarding the potential for many commercial chemicals to induce developmental toxicity. The mESC Adherent Cell Differentiation and Cytoxicity (ACDC) assay is a high-throughput screen used to close this data gap. Thus, ToxCast™ Phase I chemicals wer...

  5. Application of Targeted Functional Assays to Assess a Putative Vascular Disruption Developmental Toxicity Pathway Informed By ToxCast High-Throughput Screening Data

    EPA Science Inventory

    Chemical perturbation of vascular development is a putative toxicity pathway which may result in developmental toxicity. EPA’s high-throughput screening (HTS) ToxCast program contains assays which measure cellular signals and biological processes critical for blood vessel develop...

  6. New high-throughput screening protease assay based upon supramolecular self-assembly.

    SciTech Connect

    Whitten, David G.; Tang, Yanli; Zhou, Zhijun; Achyuthan, Komandoor E.

    2008-11-01

    We previously demonstrated that the supramolecular self-assembly of cyanines could be useful for developing fluorescent enzymatic assays. We took that concept a step further by synthesizing a covalent adduct of the tetrapeptide Asp-Glu-Val-Asp (DEVD) and a cyanine (DEVD-cyanine). The DEVD-cyanine due to its canonical sequence was recognized and hydrolyzed by the proteases, Caspase-3 and -7 in 96- or 384-microwell plate reactions. The catalytically liberated cyanine self-assembled upon scaffolds of carboxymethylamylose (CMA), carboxymethylcellulose (CMC), or a mixture of CMA and CMC resulting in a J aggregate exhibiting bright fluorescence at a 470 nm emission wavelength (optimum signal/background using excitation wavelengths of 415-440 nm). The fluorescence intensity increased with enzyme and substrate concentrations or reaction time and exhibited classical saturation profiles of a rectangular hyperbola. Saturation of the reaction was at 30 U/mL (1 {micro}g/mL) Caspase-3 and 250 {micro}M DEVD-cyanine. The reaction kinetics was linear between 1 and 20 min and saturated at 60 min. The affinity constant (Km) for DEVD-cyanine was 23 {micro}M, similar to those of previously reported values for other DEVD substrates of Caspase-3. Maximal fluorescence emission was observed by using a mixture of CMA and CMC scaffolds at 65 and 35 {micro}M, respectively. The reaction kinetics of Caspase-7 executed in a 384-well plate was similar to the reaction kinetics of Caspase-3 conducted in a 96-well plate. We believe that this is the first demonstration of a cyanine liberated from a covalent adduct due to protease action, leading to supramolecular self-assembly and the detection of protease activity.

  7. Big Data in Chemical Toxicity Research: The Use of High-Throughput Screening Assays To Identify Potential Toxicants

    PubMed Central

    2015-01-01

    High-throughput screening (HTS) assays that measure the in vitro toxicity of environmental compounds have been widely applied as an alternative to in vivo animal tests of chemical toxicity. Current HTS studies provide the community with rich toxicology information that has the potential to be integrated into toxicity research. The available in vitro toxicity data is updated daily in structured formats (e.g., deposited into PubChem and other data-sharing web portals) or in an unstructured way (papers, laboratory reports, toxicity Web site updates, etc.). The information derived from the current toxicity data is so large and complex that it becomes difficult to process using available database management tools or traditional data processing applications. For this reason, it is necessary to develop a big data approach when conducting modern chemical toxicity research. In vitro data for a compound, obtained from meaningful bioassays, can be viewed as a response profile that gives detailed information about the compound’s ability to affect relevant biological proteins/receptors. This information is critical for the evaluation of complex bioactivities (e.g., animal toxicities) and grows rapidly as big data in toxicology communities. This review focuses mainly on the existing structured in vitro data (e.g., PubChem data sets) as response profiles for compounds of environmental interest (e.g., potential human/animal toxicants). Potential modeling and mining tools to use the current big data pool in chemical toxicity research are also described. PMID:25195622

  8. Big data in chemical toxicity research: the use of high-throughput screening assays to identify potential toxicants.

    PubMed

    Zhu, Hao; Zhang, Jun; Kim, Marlene T; Boison, Abena; Sedykh, Alexander; Moran, Kimberlee

    2014-10-20

    High-throughput screening (HTS) assays that measure the in vitro toxicity of environmental compounds have been widely applied as an alternative to in vivo animal tests of chemical toxicity. Current HTS studies provide the community with rich toxicology information that has the potential to be integrated into toxicity research. The available in vitro toxicity data is updated daily in structured formats (e.g., deposited into PubChem and other data-sharing web portals) or in an unstructured way (papers, laboratory reports, toxicity Web site updates, etc.). The information derived from the current toxicity data is so large and complex that it becomes difficult to process using available database management tools or traditional data processing applications. For this reason, it is necessary to develop a big data approach when conducting modern chemical toxicity research. In vitro data for a compound, obtained from meaningful bioassays, can be viewed as a response profile that gives detailed information about the compound's ability to affect relevant biological proteins/receptors. This information is critical for the evaluation of complex bioactivities (e.g., animal toxicities) and grows rapidly as big data in toxicology communities. This review focuses mainly on the existing structured in vitro data (e.g., PubChem data sets) as response profiles for compounds of environmental interest (e.g., potential human/animal toxicants). Potential modeling and mining tools to use the current big data pool in chemical toxicity research are also described. PMID:25195622

  9. Development of multicellular tumor spheroid (MCTS) culture from breast cancer cell and a high throughput screening method using the MTT assay.

    PubMed

    Ho, Wan Yong; Yeap, Swee Keong; Ho, Chai Ling; Rahim, Raha Abdul; Alitheen, Noorjahan Banu

    2012-01-01

    In comparison to monolayer cells, MCTS has been claimed as more suitable candidate for studying drug penetration due to the high resemblance to solid tumors. However, the cultivation of MCTS is cumbersome, time consuming, and most technique fail to generate spheroids with uniform sizes. Therefore, the application of spheroid cultures in high throughput screening has been rather limiting. Besides, the lack of a well established screening protocol method that is applicable to spheroid could also be attributed to this limitation. Here we report a simple way of cultivating homogenous MCTS cultures with compact and rigid structure from the MCF-7 cells. Besides, we had also made some modifications to the standard MTT assay to realize high throughput screening of these spheroids. Using the modified protocol, tamoxifen showed cytotoxicity effect towards MCTS cultures from MCF-7 with high consistency. The results correlated well with the cultures' response assessed by LDH release assay but the latter assay was not ideal for detecting a wide range of cytotoxicity due to high basal background reading. The MTT assay emerged as a better indicator to apoptosis event in comparison to the LDH release assay. Therefore, the method for spheroid generation and the modified MTT assay we reported here could be potentially applied to high throughput screening for response of spheroid cultures generated from MCF-7 as well as other cancer cell lines towards cytotoxic stimuli. PMID:22970274

  10. High throughput protein production screening

    DOEpatents

    Beernink, Peter T.; Coleman, Matthew A.; Segelke, Brent W.

    2009-09-08

    Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

  11. Fully Bayesian Analysis of High-throughput Targeted Metabolomics Assays

    EPA Science Inventory

    High-throughput metabolomic assays that allow simultaneous targeted screening of hundreds of metabolites have recently become available in kit form. Such assays provide a window into understanding changes to biochemical pathways due to chemical exposure or disease, and are usefu...

  12. A High-Throughput Assay for Arylamine Halogenation Based on a Peroxidase-Mediated Quinone–Amine Coupling with Applications in the Screening of Enzymatic Halogenations

    PubMed Central

    Hosford, Joseph; Shepherd, Sarah A; Micklefield, Jason; Wong, Lu Shin

    2014-01-01

    Arylhalides are important building blocks in many fine chemicals, pharmaceuticals and agrochemicals, and there has been increasing interest in the development of more “green” halogenation methods based on enzyme catalysis. However, the screening and development of new enzymes for biohalogenation has been hampered by a lack of high-throughput screening methods. Described herein is the development of a colorimetric assay for detecting both chemical and enzymatic arylamine halogenation reactions in an aqueous environment. The assay is based on the unique UV/Vis spectrum created by the formation of an ortho-benzoquinone-amine adduct, which is produced by the peroxidase-catalysed benzoquinone generation, followed by Michael addition of either a halogenated or non-halogenated arylamine. This assay is sensitive, rapid and amenable to high-throughput screening platforms. We have also shown this assay to be easily coupled to a flavin-dependent halogenase, which currently lacks any convenient colorimetric assay, in a “one-pot” workflow. PMID:25319801

  13. Chemoenzymatic Synthesis of a Type 2 Blood Group A Tetrasaccharide and Development of High-throughput Assays Enables a Platform for Screening Blood Group Antigen-cleaving Enzymes.

    PubMed

    Kwan, David H; Ernst, Sabrina; Kötzler, Miriam P; Withers, Stephen G

    2015-08-01

    A facile enzymatic synthesis of the methylumbelliferyl β-glycoside of the type 2 A blood group tetrasaccharide in good yields is reported. Using this compound, we developed highly sensitive fluorescence-based high-throughput assays for both endo-β-galactosidase and α-N-acetylgalactosaminidase activity specific for the oligosaccharide structure of the blood group A antigen. We further demonstrate the potential to use this assay to screen the expressed gene products of metagenomic libraries in the search for efficient blood group antigen-cleaving enzymes. PMID:25964111

  14. The Design, Synthesis and Potential Utility of Fluorescence Probes that Target DFG-out Conformation of p38[alpha] for High Throughput Screening Binding Assay

    SciTech Connect

    Tecle, Haile; Feru, Frederic; Liu, Hu; Kuhn, Cyrille; Rennie, Glen; Morris, Mark; Shao, Jiangxing; Cheng, Alan C.; Gikunju, Diana; Miret, Juan; Coli, Rocco; Xi, Simon; Clugston, Susan L.; Low, Simon; Kazmirski, Steven; Ding, Yuan-Hua; Cao, Qing; Johnson, Theresa L.; Deshmukh, Gayatri D.; DiNitto, Jonathan P.; Wu, Joe C.; English, Jessie M.; Pfizer

    2010-10-18

    The design, synthesis and utility of fluorescence probes that bind to the DFG-out conformation of p38{alpha} kinase are described. Probes that demonstrate good affinity for p38{alpha}, have been identified and one of the probes, PF-04438255, has been successfully used in an high throughput screening (HTS) assay to identify two novel non-classical p38{alpha} inhibitors. In addition, a cascade activity assay was utilized to validate the selective binding of these non-classical kinase inhibitors to the unactive form of the enzyme.

  15. High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

    PubMed

    Islam, Md Koushikul; Baudin, Maria; Eriksson, Jonas; Öberg, Christopher; Habjan, Matthias; Weber, Friedemann; Överby, Anna K; Ahlm, Clas; Evander, Magnus

    2016-04-01

    Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection. PMID:26762502

  16. High-Throughput Screening in Primary Neurons

    PubMed Central

    Sharma, Punita; Ando, D. Michael; Daub, Aaron; Kaye, Julia A.; Finkbeiner, Steven

    2013-01-01

    Despite years of incremental progress in our understanding of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), there are still no disease-modifying therapeutics. The discrepancy between the number of lead compounds and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states. Our high-throughput screening (HTS) system in a primary neuron model can help address this unmet need. HTS allows scientists to assay thousands of conditions in a short period of time which can reveal completely new aspects of biology and identify potential therapeutics in the span of a few months when conventional methods could take years or fail all together. HTS in primary neurons combines the advantages of HTS with the biological relevance of intact, fully differentiated neurons which can capture the critical cellular events or homeostatic states that make neurons uniquely susceptible to disease-associated proteins. We detail methodologies of our primary neuron HTS assay workflow from sample preparation to data reporting. We also discuss our adaptation of our HTS system into high-content screening (HCS), a type of HTS that uses multichannel fluorescence images to capture biological events in situ, and is uniquely suited to study dynamical processes in living cells. PMID:22341232

  17. High-throughput screening in primary neurons.

    PubMed

    Sharma, Punita; Ando, D Michael; Daub, Aaron; Kaye, Julia A; Finkbeiner, Steven

    2012-01-01

    Despite years of incremental progress in our understanding of diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), there are still no disease-modifying therapeutics. The discrepancy between the number of lead compounds and approved drugs may partially be a result of the methods used to generate the leads and highlights the need for new technology to obtain more detailed and physiologically relevant information on cellular processes in normal and diseased states. Our high-throughput screening (HTS) system in a primary neuron model can help address this unmet need. HTS allows scientists to assay thousands of conditions in a short period of time which can reveal completely new aspects of biology and identify potential therapeutics in the span of a few months when conventional methods could take years or fail all together. HTS in primary neurons combines the advantages of HTS with the biological relevance of intact, fully differentiated neurons which can capture the critical cellular events or homeostatic states that make neurons uniquely susceptible to disease-associated proteins. We detail methodologies of our primary neuron HTS assay workflow from sample preparation to data reporting. We also discuss the adaptation of our HTS system into high-content screening (HCS), a type of HTS that uses multichannel fluorescence images to capture biological events in situ, and is uniquely suited to study dynamical processes in living cells. PMID:22341232

  18. Development of a high-throughput screening for nerve agent detoxifying materials using a fully-automated robot-assisted biological assay.

    PubMed

    Wille, T; Thiermann, H; Worek, F

    2010-04-01

    Developing improved medical countermeasures against chemical warfare agents (nerve agents) is urgently needed but time-consuming and costly. Here we introduce a robot-assisted liquid handling system with warming, cooling and incubating facilities to screen the detoxifying properties of biological and chemical materials against nerve agents. Two biological tests were established and plasma from various species, DFPase and three cyclodextrins were used as test materials. In test 1, plasma was mixed with sarin or VX and the inhibitory potency of the incubate was determined with human acetylcholinesterase (AChE) at 0, 30 and 60 min. In test 2, test materials and nerve agents were mixed and incubated. Between 0 and 40 min samples were taken and incubated for 3 min with AChE and the residual AChE inhibition was determined to enable the semi-quantitative evaluation of the detoxification kinetics. The automated assays proved to be highly reproducible. It was possible to pre-select detoxifying reagents with test 1 and to determine more detailed detoxifying kinetics with test 2. In conclusion, the automated assay may be considered as a versatile tool for the high-throughput screening of potential detoxifying materials against different nerve agents. With this two-step assay it is possible to screen effectively for detoxifying materials in a high-throughput system. PMID:19961920

  19. Quantitative RT-PCR assay for high-throughput screening (HTS) of drugs against the growth of Cryptosporidium parvum in vitro

    PubMed Central

    Zhang, Haili; Zhu, Guan

    2015-01-01

    Our laboratory has previously developed a qRT-PCR assay to assess drug efficacy on the growth of Cryptosporidium parvum in vitro by detecting the levels of parasite 18S rRNA. This approach displayed up to four orders of magnitude of linear dynamic range and was much less labor-intensive than the traditional microscopic methods. However, conventional qRT-PCR protocol is not very amendable to high-throughput analysis when total RNA needs to be purified by lengthy, multi-step procedures. Recently, several commercial reagents are available for preparing cell lysates that could be directly used in downstream qRT-PCR analysis (e.g., Ambion Cell-to-cDNA kit and Bio-Rad iScript sample preparation reagent). Using these reagents, we are able to adapt the qRT-PCR assay into high-throughput screening of drugs in vitro (i.e., 96-well and 384-well formats for the cultivation of parasites and qRT-PCR detection, respectively). This qRT-PCR protocol is able to give a >150-fold linear dynamic range using samples isolated from cells infected with various numbers of parasites. The new assay is also validated by the NIH-recommended intra-plate, inter-plate, and inter-day uniformity tests. The robustness and effectiveness of the assay are also confirmed by evaluating the anti-cryptosporidial efficacy of paromomycin and by a small scale screening of compounds. PMID:26441920

  20. High-Throughput Screening for Small Molecule Inhibitors of LARG-Stimulated RhoA Nucleotide Binding via a Novel Fluorescence Polarization Assay

    PubMed Central

    Evelyn, Chris R.; Ferng, Timothy; Rojas, Rafael J.; Larsen, Martha J.; Sondek, John; Neubig, Richard R.

    2009-01-01

    Guanine nucleotide-exchange factors (GEFs) stimulate guanine nucleotide exchange and the subsequent activation of Rho-family proteins in response to extracellular stimuli acting upon cytokine, tyrosine kinase, adhesion, integrin, and G-protein coupled receptors (GPCRs). Upon Rho activation, several downstream events occur, such as morphological and cytokskeletal changes, motility, growth, survival, and gene transcription. The RhoGEF Leukemia-Associated RhoGEF (LARG) is a member of the Regulators of G-protein Signaling Homology Domain (RH) family of GEFs originally identified as a result of chromosomal translocation in acute myeloid leukemia. Using a novel fluorescence polarization guanine nucleotide binding assay utilizing BODIPY-Texas Red-GTPγS (BODIPY-TR-GTPγS), we performed a ten-thousand compound high-throughput screen for inhibitors of LARG-stimulated RhoA nucleotide binding. Five compounds identified from the high-throughput screen were confirmed in a non-fluorescent radioactive guanine nucleotide binding assay measuring LARG-stimulated [35S] GTPγS binding to RhoA, thus ruling out non-specific fluorescent effects. All five compounds selectively inhibited LARG-stimulated RhoA [35S] GTPγS binding, but had little to no effect upon RhoA or Gαo [35S] GTPγS binding. Therefore, these five compounds should serve as promising starting points for the development of small molecule inhibitors of LARG-mediated nucleotide exchange as both pharmacological tools and therapeutics. In addition, the fluorescence polarization guanine nucleotide binding assay described here should serve as a useful approach for both high-throughput screening and general biological applications. PMID:19196702

  1. A Quantitative Toxicogenomics Assay for High-throughput and Mechanistic Genotoxicity Assessment and Screening of Environmental Pollutants.

    PubMed

    Lan, Jiaqi; Gou, Na; Rahman, Sheikh Mokhles; Gao, Ce; He, Miao; Gu, April Z

    2016-03-15

    The ecological and health concern of mutagenicity and carcinogenicity potentially associated with an overwhelmingly large and ever-increasing number of chemicals demands for cost-effective and feasible method for genotoxicity screening and risk assessment. This study proposed a genotoxicity assay using GFP-tagged yeast reporter strains, covering 38 selected protein biomarkers indicative of all the seven known DNA damage repair pathways. The assay was applied to assess four model genotoxic chemicals, eight environmental pollutants and four negative controls across six concentrations. Quantitative molecular genotoxicity end points were derived based on dose response modeling of a newly developed integrated molecular effect quantifier, Protein Effect Level Index (PELI). The molecular genotoxicity end points were consistent with multiple conventional in vitro genotoxicity assays, as well as with in vivo carcinogenicity assay results. Further more, the proposed genotoxicity end point PELI values quantitatively correlated with both comet assay in human cell and carcinogenicity potency assay in mice, providing promising evidence for linking the molecular disturbance measurements to adverse outcomes at a biological relevant level. In addition, the high-resolution DNA damaging repair pathway alternated protein expression profiles allowed for chemical clustering and classification. This toxicogenomics-based assay presents a promising alternative for fast, efficient and mechanistic genotoxicity screening and assessment of drugs, foods, and environmental contaminants. PMID:26855253

  2. High-throughput micro-plate HCL-vanillin assay for screening tannin content in sorghum grain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum contains tannin which is a phenolic compound that offers health promoting antioxidant capacity. The HCl-vanillin assay is a common and time consuming method for determining tannin content, but is not efficient for screening large sample sets as seen in association mapping panels or breeding ...

  3. A novel cell-based duplex high-throughput screening assay combining fluorescent Ca(2+) measurement with homogeneous time-resolved fluorescence technology.

    PubMed

    Kiss, László; Cselenyák, Attila; Varga, Ágnes; Visegrády, András

    2016-08-15

    Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca(2+)] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm. PMID:27235172

  4. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

    NASA Astrophysics Data System (ADS)

    Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

    2013-10-01

    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

  5. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

    PubMed Central

    Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

    2013-01-01

    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures. PMID:24141454

  6. Novel Cell-Based Hepatitis C Virus Infection Assay for Quantitative High-Throughput Screening of Anti-Hepatitis C Virus Compounds

    PubMed Central

    Hu, Zongyi; Lan, Keng-Hsin; He, Shanshan; Swaroop, Manju; Hu, Xin; Southall, Noel; Zheng, Wei

    2014-01-01

    Therapy 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. PMID:24277038

  7. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

    PubMed

    Ivanov, Delyan P; Parker, Terry L; Walker, David A; Alexander, Cameron; Ashford, Marianne B; Gellert, Paul R; Garnett, Martin C

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  8. Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

    PubMed Central

    Ivanov, Delyan P.; Parker, Terry L.; Walker, David A.; Alexander, Cameron; Ashford, Marianne B.; Gellert, Paul R.; Garnett, Martin C.

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  9. High-throughput screening methods for nitrilases.

    PubMed

    Xue, Ya-Ping; Yang, Yue-Kai; Lv, Sheng-Zhi; Liu, Zhi-Qiang; Zheng, Yu-Guo

    2016-04-01

    Nitrilases have been widely acknowledged as important alternatives to chemical catalysts, as they have been proved to transform an immense variety of nitriles under mild conditions and often in a stereoselective or regioselective manner. In the discovery of new nitrilases to establish viable industrial processes, screening plays an important role in identifying which subset of candidates contains a nitrilase of interest from a collection of organisms, clone banks, or enzyme libraries. However, the traditional methods for evaluating the nitrilases are a time-consuming, laborious, and costly process and have been regarded as a bottleneck in developing these nitrilases as industrial biocatalysts. In the past few years, a number of high-throughput screening methods have been developed for rapid evaluation and identification of nitrilases. Here, we review the various methodologies developed for high-throughput screening of nitrilases and focus on their advantages and limitations. PMID:26894402

  10. Imaging-Based High-Throughput Screening Assay To Identify New Molecules with Transmission-Blocking Potential against Plasmodium falciparum Female Gamete Formation

    PubMed Central

    Miguel-Blanco, Celia; Lelièvre, Joël; Delves, Michael J.; Bardera, Ana I.; Presa, Jesús L.; López-Barragán, María José; Ruecker, Andrea; Marques, Sara; Sinden, Robert E.

    2015-01-01

    In response to a call for the global eradication of malaria, drug discovery has recently been extended to identify compounds that prevent the onward transmission of the parasite, which is mediated by Plasmodium falciparum stage V gametocytes. Lately, metabolic activity has been used in vitro as a surrogate for gametocyte viability; however, as gametocytes remain relatively quiescent at this stage, their ability to undergo onward development (gamete formation) may be a better measure of their functional viability. During gamete formation, female gametocytes undergo profound morphological changes and express translationally repressed mRNA. By assessing female gamete cell surface expression of one such repressed protein, Pfs25, as the readout for female gametocyte functional viability, we developed an imaging-based high-throughput screening (HTS) assay to identify transmission-blocking compounds. This assay, designated the P. falciparum female gametocyte activation assay (FGAA), was scaled up to a high-throughput format (Z′ factor, 0.7 ± 0.1) and subsequently validated using a selection of 50 known antimalarials from diverse chemical families. Only a few of these agents showed submicromolar 50% inhibitory concentrations in the assay: thiostrepton, methylene blue, and some endoperoxides. To determine the best conditions for HTS, a robustness test was performed with a selection of the GlaxoSmithKline Tres Cantos Antimalarial Set (TCAMS) and the final screening conditions for this library were determined to be a 2 μM concentration and 48 h of incubation with gametocytes. The P. falciparum FGAA has been proven to be a robust HTS assay faithful to Plasmodium transmission-stage cell biology, and it is an innovative useful tool for antimalarial drug discovery which aims to identify new molecules with transmission-blocking potential. PMID:25801574

  11. A high-throughput screening-compatible homogeneous time-resolved fluorescence assay measuring the glycohydrolase activity of human poly(ADP-ribose) glycohydrolase.

    PubMed

    Stowell, Alexandra I J; James, Dominic I; Waddell, Ian D; Bennett, Neil; Truman, Caroline; Hardern, Ian M; Ogilvie, Donald J

    2016-06-15

    Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition. PMID:27036617

  12. A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

    PubMed Central

    Iantomasi, Raffaella; Veyron-Churlet, Romain; Deboosère, Nathalie; Landry, Valérie; Baulard, Alain; Brodin, Priscille

    2014-01-01

    Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb within host cells. PMID:24473237

  13. High throughput screening (HTS) for phototoxicity hazard using the in vitro 3T3 neutral red uptake assay.

    PubMed

    Jones, P A; King, A V

    2003-01-01

    Testing for phototoxic hazard is usually carried out for product ingredients intended for use on skin, which may be exposed to sunlight. Unilever currently uses the validated in vitro 3T3 Neutral Red Uptake phototoxicity test (NRU PT). This protocol involves 2-3 experiments, each taking 3 days to perform. One person can test up to seven test materials plus positive control at any one time, requiring approximately 0.5 g test material. Higher throughput is required where libraries of potential actives are being generated and screening for potential phototoxicants is required. A proposed HTS protocol would use the NRU PT, but only one concentration (10 microg/ml) in a single experiment. The validity of the HTS protocol was investigated by a retrospective examination of data from 86 materials previously tested. Phototoxic hazard predictions made using the conventional NRU PT were compared with those obtained if only data at 10 microg/ml were considered. A majority of 73 materials (84.9%) gave agreement in predictions between the two protocols; for 13 materials (15.1%) the assessments did not agree. There were no false positives; however, there were some false negatives, i.e., predicted as phototoxic from the conventional assay, but non-phototoxic at 10 microg/ml. As this protocol is intended for screening purposes only it is considered that this would be acceptable at this stage in material selection. One person could screen 128 test materials in 3 days, requiring <1 mg test material, giving a substantial increase in productivity. Any material selected for further development and inclusion in a formulation may require further confirmatory testing, e.g. using a human skin model assay for phototoxicity. PMID:14599466

  14. Cell-based assay system for high-throughput screening of anti-photo-aging agents in fibroblast transfectants.

    PubMed

    Lee, S; Shin, S; Jung, E; Park, D

    2016-08-01

    The matricellular protein CCN1 is significantly elevated in acutely ultraviolet-irradiated human skin and negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation. In this study, we established a stable cell line, termed CCN1-GFs, by transfection of the pAcGFP1-1-CCN1 promoter plasmid and examined its usefulness as a cell-based assay system for screening anti-aging ingredients. The promoter of the reporter plasmid pAcGFP1-1-CCN1 promoter was transfected into NIH3T3 cells using the Lipofectamine reagent. G418-resistant cells were selected and further cloned. To confirm whether AcGFP1-1-CCN1 promoter plasmid recombined in the NIH3T3 cells, the level of AcGFP1-1-CCN1 was measured by PCR analysis. To determine if NIH3T3 cells expressed the gene encoding green fluorescence protein in a CCN1 promoter-dependent manner, the reporter enzyme activities were assayed using a fluorimeter and flow cytometer. To determine if CCN1 inhibitor, which was selected through this system, exerted a direct effect on the downstream signal, mRNA expression of collagen1 and MMP1A was checked by using real-time PCR. UVB irradiation of CCN1-GFs resulted in increased CCN1 promoter activity. Treatment with retinoic acid, a CCN1 inhibitor, inhibited UV-induced CCN1 promoter activity. Subsequent use of this assay system to screen anti-aging ingredients revealed that CCN1-GFs treated with sclareol showed decreased levels of UVB-induced CCN1 expression. Sclareol attenuated UVB-induced photo-aging by an increase in collagen synthesis and decrease in MMP-1 activity. PMID:26281901

  15. Development of a differential scanning fluorimetry based high throughput screening assay for the discovery of affinity binders against an anthrax protein.

    PubMed

    Sorrell, Fiona J; Greenwood, Gemma K; Birchall, Kristian; Chen, Beining

    2010-09-01

    The anthrax protein protective antigen (PA) is responsible for cell-surface recognition and aids the delivery of the toxic anthrax enzymes into host cells. By targeting PA and preventing it from binding to host cells, it is hoped that the delivery of toxins into the cell will be inhibited. The current assay reported for PA is a low throughput functional assay. Here, the high throughput screening method using differential scanning fluorimetry (DSF) was developed and optimized to screen a number of libraries from various sources including a selection of FDA-approved drugs as well as hits selected by a virtual screening campaign. DSF is a rapid technique that uses fluorescence to monitor the thermal unfolding of proteins using a standard QPCR instrument. A positive shift in the calculated melting temperature (Tm), of the protein in the presence of a compound, relative to the Tm of the unbound protein, indicates that stabilization of the protein by ligand binding may have occurred. Optimization of the melting assay showed SYPRO Orange to be an ideal dye as a marker and lead to the reduction of DMSO concentration to <1% (v/v) in the final assay. The final assay volume was minimized to 25 L with 5 g protein per well of 96-well plate. In addition, a buffer, salt and additive screen lead to the selection of 10 mM HEPES-NaOH pH 7.5, 100 mM NaCl as the assay buffer. This method has been shown here to be useful as a primary method for the detection of small-molecule PA ligands, giving a hit rate of approximately 7%. These ligands can then be studied further using PA functional assays to confirm their biological activities before being selected as lead compounds for the treatment of anthrax. PMID:20376913

  16. Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

    PubMed

    Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph

    2016-01-01

    Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular

  17. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide.

    PubMed

    Motabar, Omid; Goldin, Ehud; Leister, William; Liu, Ke; Southall, Noel; Huang, Wenwei; Marugan, Juan J; Sidransky, Ellen; Zheng, Wei

    2012-01-01

    Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening. PMID:22033823

  18. A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

    PubMed Central

    Motabar, Omid; Goldin, Ehud; Leister, William; Liu, Ke; Southall, Noel; Huang, Wenwei; Marugan, Juan J.; Sidransky, Ellen

    2012-01-01

    Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening. PMID:22033823

  19. High throughput assays for analyzing transcription factors.

    PubMed

    Li, Xianqiang; Jiang, Xin; Yaoi, Takuro

    2006-06-01

    Transcription factors are a group of proteins that modulate the expression of genes involved in many biological processes, such as cell growth and differentiation. Alterations in transcription factor function are associated with many human diseases, and therefore these proteins are attractive potential drug targets. A key issue in the development of such therapeutics is the generation of effective tools that can be used for high throughput discovery of the critical transcription factors involved in human diseases, and the measurement of their activities in a variety of disease or compound-treated samples. Here, a number of innovative arrays and 96-well format assays for profiling and measuring the activities of transcription factors will be discussed. PMID:16834538

  20. High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid, 96-well microtiter assays were compared to conventional assays for quantifying total phenolic content, flavonoid content, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in sorghum grain. The 96-well assays exhibited a correlation of >0.9 to the conventional assays. The 96-well assays allowed for ...

  1. Screening assay of angiotensin-converting enzyme inhibitory activity from complex natural colourants and foods using high-throughput LC-MS/MS.

    PubMed

    Inoue, Koichi; Kitade, Marie; Hino, Tomoaki; Oka, Hisao

    2011-06-15

    Inhibition of angiotensin-converting enzyme (ACE) by various foods decreases the blood pressure. ACE inhibitors derived from natural components may be of therapeutic value in preventive medicine. In this study, we report a novel screening assay of ACE inhibitors from complex natural colourants and foods that employ solid phase extraction (SPE), high-throughput liquid chromatography (LC) separation, and stable isotope dilution electrospray tandem mass spectrometry (SID-ESI-MS/MS). When a target sample was subjected to N-Hippuryl-His-Leu (HHL) and ACE in phosphate buffer (pH 7.4), generated hippuric acid (HA) was extracted by SPE. LC/SID-ESI-MS/MS detection of HA allowed us to accurately identify the effects of complex substances such natural colourants and foods that inhibit the ACE of HHL. The major HA and HA-d5 fragment ions at m/z 180→105 and 185→110 in the multiple reaction monitoring (MRM) mode can quantify levels that are lower than other methods. The LC/SID-ESI-MS/MS method described here is a rapid, selective, sensitive, and highly reproducible method for the determination of HA in various samples. Based on the assay developed, all samples such as natural colourants, infant formula, soy paste, ketchup, mayonnaise, wheat flour, orange juice, supplement drink, tea, and coffee could be accurately measured for ACE inhibition in various matrices. High-throughput LC/SID-ESI-MS/MS assay has no limitations in the evaluation of inhibition activity in various natural samples such as colour, high-matrix, and processed foods. PMID:25213976

  2. High Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume By Exploiting Energy Transfer

    PubMed Central

    Miyata, Yoshinari; Chang, Lyra; Bainor, Anthony; McQuade, Thomas J.; Walczak, Christopher P.; Zhang, Yaru; Larsen, Martha J.; Kirchhoff, Paul; Gestwicki, Jason E.

    2011-01-01

    Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate-detection reagents, such as quinaldine red (QR). While such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70’s weak enzymatic activity have combined to create significant challenges in high throughput screening. To overcome these difficulties, we have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal. Biochem. 2005, 342:254–259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, we tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z′ ~ 0.6, CV ~8%) and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70-family members. PMID:20926844

  3. High-throughput Screening of ToxCast" Phase I Chemicals in an Embryonic Stem Cell Assay Reveals Potential Disruption of a Critical Developmental Signaling Pathway

    EPA Science Inventory

    Little is known about the developmental toxicity of the expansive chemical landscape in existence today. Significant efforts are being made to apply novel methods to predict developmental activity of chemicals utilizing high-throughput screening (HTS) and high-content screening (...

  4. HIGH-THROUGHPUT SCREENING ASSAY FOR THE IDENTIFICATION OF COMPOUNDS REGULATING SELF-RENEWAL AND DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS

    PubMed Central

    Desbordes, Sabrina C.; Placantonakis, Dimitris G.; Ciro, Anthony; Socci, Nicholas D.; Lee, Gabsang; Djaballah, Hakim; Studer, Lorenz

    2009-01-01

    Summary High-throughput screening (HTS) of chemical libraries has become a critical tool in basic biology and drug discovery. However, its implementation and the adaptation of high content assays to human embryonic stem cells (hESCs) have been hampered by multiple technical challenges. Here we present a strategy to adapt hESCs to HTS conditions, resulting in an assay suitable for the discovery of small molecules that drive hESC self-renewal or differentiation. Use of this new assay has led to the identification of several marketed drugs and natural compounds promoting short-term hESC maintenance and compounds directing early lineage choice during differentiation. Global gene expression analysis upon drug treatment defines known and novel pathways correlated to hESC self-renewal and differentiation. Our results demonstrate feasibility of hESC-based HTS and enhance the repertoire of chemical compounds for manipulating hESC fate. The availability of high content assays should accelerate progress in basic and translational hESC biology. PMID:18522853

  5. High throughput adjustable 96-well plate assay for androgen receptor binding: a practical approach for EDC screening using the chimpanzee AR.

    PubMed

    Hartig, P C; Cardon, M C; Blystone, C R; Gray, L E; Wilson, V S

    2008-09-26

    The issue as to whether natural and man-made chemicals interfere with endocrine function has raised concerns. This interference could be biologically significant even at very low doses if the chemicals interact deleteriously with hormone receptors at low concentrations. Therefore, the United States Environmental Protection Agency (USEPA) Office of Coordination and Policy (OSCP) requested that a nonhuman mammalian androgen receptor binding assay be developed for possible use in their Endocrine Disruptor Screening Program (EDSP). Ideally, this assay would be high throughput, not use animals as a source of receptor protein, easily deployed throughout the scientific community, utilize reagents available to both the public and private sector, and have the potential for future automation. We developed a highly modified 96-well plate assay which meets these criteria. It employs a baculovirus expressed recombinant primate androgen receptor which is publically available and exploits the unique ability of some mammalian androgen receptors to remain biologically active after guanidine hydrochloride (GdnHCl) solubilization. This GdnHCl treated receptor remains soluble and requires no additional purification prior to use. We provide a very detailed description of the assay protocol itself, and similarly detailed method for producing and solubilizing the receptor. PMID:18691642

  6. High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

    PubMed

    Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

    2014-01-17

    The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains. PMID:24134695

  7. High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases

    PubMed Central

    Lieberman, Ori J.; Orr, Mona W.; Wang, Yan; Lee, Vincent T.

    2013-01-01

    The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains. PMID:24134695

  8. Development and Implementation of a High-Throughput High-Content Screening Assay to Identify Inhibitors of Androgen Receptor Nuclear Localization in Castration-Resistant Prostate Cancer Cells.

    PubMed

    Johnston, Paul A; Nguyen, Minh M; Dar, Javid A; Ai, Junkui; Wang, Yujuan; Masoodi, Khalid Z; Shun, Tongying; Shinde, Sunita; Camarco, Daniel P; Hua, Yun; Huryn, Donna M; Wilson, Gabriela Mustata; Lazo, John S; Nelson, Joel B; Wipf, Peter; Wang, Zhou

    2016-05-01

    Patients with castration-resistant prostate cancer (CRPC) can be treated with abiraterone, a potent inhibitor of androgen synthesis, or enzalutamide, a second-generation androgen receptor (AR) antagonist, both targeting AR signaling. However, most patients relapse after several months of therapy and a majority of patients with relapsed CRPC tumors express the AR target gene prostate-specific antigen (PSA), suggesting that AR signaling is reactivated and can be targeted again to inhibit the relapsed tumors. Novel small molecules capable of inhibiting AR function may lead to urgently needed therapies for patients resistant to abiraterone, enzalutamide, and/or other previously approved antiandrogen therapies. Here, we describe a high-throughput high-content screening (HCS) campaign to identify small-molecule inhibitors of AR nuclear localization in the C4-2 CRPC cell line stably transfected with GFP-AR-GFP (2GFP-AR). The implementation of this HCS assay to screen a National Institutes of Health library of 219,055 compounds led to the discovery of 3 small molecules capable of inhibiting AR nuclear localization and function in C4-2 cells, demonstrating the feasibility of using this cell-based phenotypic assay to identify small molecules targeting the subcellular localization of AR. Furthermore, the three hit compounds provide opportunities to develop novel AR drugs with potential for therapeutic intervention in CRPC patients who have relapsed after treatment with antiandrogens, such as abiraterone and/or enzalutamide. PMID:27187604

  9. Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

    PubMed Central

    2004-01-01

    DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development. PMID:15283677

  10. A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors.

    PubMed

    Vedel, Line; Bräuner-Osborne, Hans; Mathiesen, Jesper Mosolff

    2015-08-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein-coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or Gi-coupled GPCRs, which leads to increased or decreased cAMP levels, respectively. Here we describe a real-time Förster resonance energy transfer (FRET)-based cAMP high-throughput screening (HTS) assay for identification and characterization of Gs-coupled GPCR ligands and phosphodiesterase (PDE) inhibitors in living cells. We used the β2-adrenergic receptor (β(2)AR) as a representative Gs-coupled receptor and characterized two cell lines with different expression levels. Low receptor expression allowed detection of desensitization kinetics and delineation of partial agonism, whereas high receptor expression resulted in prolonged signaling and enabled detection of weak partial agonists and/or ligands with low potency, which is highly advantageous in large HTS settings and hit identification. In addition, the assay enabled detection of β(2)AR inverse agonists and PDE inhibitors. High signal-to-noise ratios were also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS. PMID:25851033

  11. High Throughput Screening For Hazard and Risk of Environmental Contaminants

    EPA Science Inventory

    High throughput toxicity testing provides detailed mechanistic information on the concentration response of environmental contaminants in numerous potential toxicity pathways. High throughput screening (HTS) has several key advantages: (1) expense orders of magnitude less than an...

  12. Time-correlated single photon counting: an advancing technique in a plate reader for assay development and high throughput screening

    NASA Astrophysics Data System (ADS)

    Näther, Dirk U.; Fenske, Roger; Hurteaux, Reynald; Majno, Sandra; Smith, S. Desmond

    2006-10-01

    A new plate reader (Nanotaurus) has been developed by Edinburgh Instruments that has the principle design features of a confocal microscope and utilises the technique of Time Correlated Single Photon Counting for data acquisition. The advantages of Fluorescence Lifetime Measurements in the nanosecond time scale and analysis methods to recover lifetime parameters are discussed based on experimental data. First working assays using changes of lifetime parameters are presented that clearly demonstrate the advantages of the new instrument for biochemical assays and show strong promise for cell-based assays, by utilising the independence of lifetime parameters from sample volume and concentration.

  13. High Throughput siRNA Screening Using Reverse Transfection.

    PubMed

    von Schantz, Carina; Saarela, Jani

    2016-01-01

    RNA interference (RNAi) is a commonly used technique to knockdown gene function. Here, we describe a high throughput screening method for siRNA mediated gene silencing of the breast cancer cell line MDA-MB-231 using reverse transfection. Furthermore, we describe the setup for two separate methods for detecting viable and dead cells using either homogenous assays or image-based analysis. PMID:27581282

  14. A high-throughput chemically induced inflammation assay in zebrafish

    PubMed Central

    2010-01-01

    Background Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. Results Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. Conclusions This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148. PMID:21176202

  15. Validation of a High-Throughput Screening Assay for Identification of Adjunctive and Directly Acting Antimicrobials Targeting Carbapenem-Resistant Enterobacteriaceae.

    PubMed

    Smith, Kenneth P; Kirby, James E

    2016-04-01

    We describe development and validation of a high-throughput screen (HTS) for identifying small molecules that restore the efficacy of carbapenems (adjunctives) and/or directly inhibit growth of carbapenem-resistant Enterobacteriaceae (CRE). Our HTS assay is based on a screen-counterscreen approach using a representative multidrug-resistant CRE strain, Klebsiella pneumoniae BIDMC12A. Specifically, we tested the ability of small molecules to inhibit bacterial growth in the presence (screen) or absence (counterscreen) of meropenem, a representative carbapenem antibiotic. Primary screening of 11,698 known bioactive compounds identified 14 with adjunctive activity and 79 with direct antimicrobial effect. Secondary screening identified triclosan as a strongly synergistic meropenem adjunctive (fractional inhibitory concentration = 0.48) and confirmed azidothymidine (AZT) (minimal inhibitory concentration [MIC] = 4 μg mL(-1)), NH125 (MIC = 4 μg mL(-1)), diphenyleneiodonium chloride (MIC = 8 μg mL(-1)), and spectinomycin (MIC = 32 μg mL(-1)) as potent direct antimicrobials. Spectrum of activity of AZT and spectinomycin was tested against a collection of 103 representative Enterobacteriaceae strains (≈50% CRE). AZT, a nucleoside analog used to treat human immunodeficiency virus, demonstrated an MIC50 of 2 μg mL(-1). Spectinomycin, an antibiotic used to treat gonorrhea, had an MIC50 of 32 μg mL(-1). Therefore, a significant percentage of CRE strains appeared relatively susceptible to these antimicrobials. These data identified AZT and spectinomycin as available agents warranting further study for potential treatment of multidrug-resistant CRE infection. Our results provide proof of principle and impetus for performing a large-scale HTS for discovery of novel, small-molecule adjunctives and antibacterial agents directly targeting CRE. PMID:27045615

  16. High throughput screening and structure-activity relationship study of potential α2A-adrenoceptor agonists by LANCETM cAMP assay.

    PubMed

    Yang, Huan; He, Ling; Yan, Ming; He, Jian-Guo; Yu, Tao

    2013-06-28

    G protein-coupled receptors (GPCRs) are signaling molecules with a wide variety of skills. Members of this large family of membrane protein have been shown to regulate the activities of the different signaling pathways of the ligand specific manner. α2-adrenoceptors (α2-ARs) are one of the GPCRs and the stimulation of them could modulate many classical effects such as hypotension, bradycardia, etc. Recently, α2A-AR is more and more important for its role in the therapeutic applications in central nervous system (CNS) diseases.High throughput screening of α2A-AR agonists was established by LANCETM cAMP assay from a compound library of 80,000 small-molecule compounds to find out potential human α2A-adrenoceptor (α2A-AR) agonists that might have therapeutic effect in CNS diseases. From the preliminary and secondary screening, 37 compounds were identified as α2AAR agonists, and six compounds among them presented more pronounced α2A-AR stimulating activity than guanfacine, a selective α2A-AR agonist. The study provided referred data for the development of potent α2A-AR agonists and suggested that the existence of the parent structure (1, 2, 4-benzothiadiazine 1, 1-dioxide) bodes well for pharmaceutical development of α2A-AR agonists. PMID:23514320

  17. Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells

    PubMed Central

    Couturier, Cyril; Deprez, Benoit

    2012-01-01

    Each step of the cell life and its response or adaptation to its environment are mediated by a network of protein/protein interactions termed “interactome.” Our knowledge of this network keeps growing due to the development of sensitive techniques devoted to study these interactions. The bioluminescence resonance energy transfer (BRET) technique was primarily developed to allow the dynamic monitoring of protein/protein interactions (PPI) in living cells, and has widely been used to study receptor activation by intra- or extra-molecular conformational changes within receptors and activated complexes in mammal cells. Some interactions are described as crucial in human pathological processes, and a new class of drugs targeting them has recently emerged. The BRET method is well suited to identify inhibitors of PPI and here is described why and how to set up and optimize a high throughput screening assay based on BRET to search for such inhibitory compounds. The different parameters to take into account when developing such BRET assays in mammal cells are reviewed to give general guidelines: considerations on the targeted interaction, choice of BRET version, inducibility of the interaction, kinetic of the monitored interaction, and of the BRET reading, influence of substrate concentration, number of cells and medium composition used on the Z′ factor, and expected interferences from colored or fluorescent compounds. PMID:22973258

  18. High-throughput fluorescence screening assay for the identification and comparison of antimicrobial peptides' activity on various yeast species.

    PubMed

    Kodedová, Marie; Sychrová, Hana

    2016-09-10

    New antifungal compounds that circumvent the resistance of the pathogen by directly damaging yeast cell surface structures are promising agents for the treatment of fungal infections, due to their different mechanism of action from current clinically used antifungal drugs. We present here a rapid and cost-effective fluorescence method suitable for identifying new potent drugs that directly target yeast cell surface structures, causing cell permeabilization and thus bypassing the multidrug resistance mechanisms of pathogens. The fluorescence assay enabled us to detect with high sensitivity damage to the Candida plasma membrane (its hyperpolarization and permeabilization) as a result of short-term exposure to the antifungal compounds. Results can be obtained in 1-2h with minimal effort and consumption of the tested compounds, also 96 samples can be analysed simultaneously. We used this method to study antimicrobial peptides isolated from the venom of bees and their synthetic analogs, compare the potency of the peptides and determine their minimal effective concentrations. The antimicrobial peptides were able to kill yeast cells at low concentrations within a 15-min treatment, the LL-III peptide exhibited a broad spectrum of antifungal activity on various Saccharomyces, pathogenic Candida and osmotolerant yeast species. PMID:27369550

  19. Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity

    PubMed Central

    Nühs, Andrea; De Rycker, Manu; Manthri, Sujatha; Comer, Eamon; Scherer, Christina A.; Schreiber, Stuart L.; Ioset, Jean-Robert; Gray, David W.

    2015-01-01

    Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery. PMID:26407168

  20. Development and Validation of a Novel Leishmania donovani Screening Cascade for High-Throughput Screening Using a Novel Axenic Assay with High Predictivity of Leishmanicidal Intracellular Activity.

    PubMed

    Nühs, Andrea; De Rycker, Manu; Manthri, Sujatha; Comer, Eamon; Scherer, Christina A; Schreiber, Stuart L; Ioset, Jean-Robert; Gray, David W

    2015-09-01

    Visceral leishmaniasis is an important parasitic disease of the developing world with a limited arsenal of drugs available for treatment. The existing drugs have significant deficiencies so there is an urgent need for new and improved drugs. In the human host, Leishmania are obligate intracellular parasites which poses particular challenges in terms of drug discovery. To achieve sufficient throughput and robustness, free-living parasites are often used in primary screening assays as a surrogate for the more complex intracellular assays. We and others have found that such axenic assays have a high false positive rate relative to the intracellular assays, and that this limits their usefulness as a primary platform for screening of large compound collections. While many different reasons could lie behind the poor translation from axenic parasite to intracellular parasite, we show here that a key factor is the identification of growth slowing and cytostatic compounds by axenic assays in addition to the more desirable cytocidal compounds. We present a screening cascade based on a novel cytocidal-only axenic amastigote assay, developed by increasing starting density of cells and lowering the limit of detection, and show that it has a much improved translation to the intracellular assay. We propose that this assay is an improved primary platform in a new Leishmania screening cascade designed for the screening of large compound collections. This cascade was employed to screen a diversity-oriented-synthesis library, and yielded two novel antileishmanial chemotypes. The approach we have taken may have broad relevance to anti-infective and anti-parasitic drug discovery. PMID:26407168

  1. Data Analysis for High-Throughput RNAi Screening.

    PubMed

    Azorsa, David O; Turnidge, Megan A; Arora, Shilpi

    2016-01-01

    High-throughput RNA interference (HT-RNAi) screening is an effective technology to help identify important genes and pathways involved in a biological process. Analysis of high-throughput RNAi screening data is a critical part of this technology, and many analysis methods have been described. Here, we summarize the workflow and types of analyses commonly used in high-throughput RNAi screening. PMID:27581298

  2. A High-Throughput Cidality Screen for Mycobacterium Tuberculosis

    PubMed Central

    Kaur, Parvinder; Ghosh, Anirban; Krishnamurthy, Ramya Vadageri; Bhattacharjee, Deepa Gagwani; Achar, Vijayashree; Datta, Santanu; Narayanan, Shridhar; Anbarasu, Anand; Ramaiah, Sudha

    2015-01-01

    Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well ‘spot-assay’ for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process. PMID:25693161

  3. High Throughput Danio Rerio Energy Expenditure Assay.

    PubMed

    Williams, Savannah Y; Renquist, Benjamin J

    2016-01-01

    Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers). PMID:26863590

  4. High Throughput Screening and Selection Methods for Directed Enzyme Evolution

    PubMed Central

    2015-01-01

    Successful evolutionary enzyme engineering requires a high throughput screening or selection method, which considerably increases the chance of obtaining desired properties and reduces the time and cost. In this review, a series of high throughput screening and selection methods are illustrated with significant and recent examples. These high throughput strategies are also discussed with an emphasis on compatibility with phenotypic analysis during directed enzyme evolution. Lastly, certain limitations of current methods, as well as future developments, are briefly summarized. PMID:26074668

  5. Identification of novel anti-hepatitis C virus agents by a quantitative high throughput screen in a cell-based infection assay.

    PubMed

    Hu, Zongyi; Hu, Xin; He, Shanshan; Yim, Hyung Joon; Xiao, Jingbo; Swaroop, Manju; Tanega, Cordelle; Zhang, Ya-qin; Yi, Guanghui; Kao, C Cheng; Marugan, Juan; Ferrer, Marc; Zheng, Wei; Southall, Noel; Liang, T Jake

    2015-12-01

    Hepatitis C virus (HCV) poses a major health threat to the world. The recent development of direct-acting antivirals (DAAs) against HCV has markedly improved the response rate of HCV and reduced the side effects in comparison to the interferon-based therapy. Despite this therapeutic advance, there is still a need to develop new inhibitors that target different stages of the HCV life cycle because of various limitations of the current regimens. In this study, we performed a quantitative high throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of ∼350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial ∼3000 molecules that showed inhibitory activity for further structural and functional analyses. We were able to assign the majority of these compounds to specific stage(s) in the HCV life cycle. Three of them are direct inhibitors of NS3/4A protease. Most of the compounds appear to act on novel targets in HCV life cycle. Four compounds with novel structure and excellent drug-like properties, three targeting HCV entry and one targeting HCV assembly/secretion, were advanced for further development as lead hits. These compounds represent diverse chemotypes that are potential lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. In addition, they represent novel molecular probes to explore the complex interactions between HCV and the cells. PMID:26515788

  6. High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.

    PubMed

    Bompiani, Kristin M; Caglič, Dejan; Krutein, Michelle C; Benoni, Galit; Hrones, Morgan; Lairson, Luke L; Bian, Haiyan; Smith, Garry R; Dickerson, Tobin J

    2016-08-01

    Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 μM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified. PMID:27314875

  7. Integration of Dosimetry, Exposure and High-Throughput Screening Data in Chemical Toxicity Assessment

    EPA Science Inventory

    High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, c...

  8. High-Throughput RNA Interference Screening: Tricks of the Trade

    PubMed Central

    Nebane, N. Miranda; Coric, Tatjana; Whig, Kanupriya; McKellip, Sara; Woods, LaKeisha; Sosa, Melinda; Sheppard, Russell; Rasmussen, Lynn; Bjornsti, Mary-Ann; White, E. Lucile

    2016-01-01

    The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z′ factor, which incorporates both standard deviation and signal window. A Z′ factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z′ factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z′ factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality. PMID:23616418

  9. Towards high throughput screening of nanoparticle flotation collectors.

    PubMed

    Abarca, Carla; Yang, Songtao; Pelton, Robert H

    2015-12-15

    To function as flotation collectors for mineral processing, polymeric nanoparticles require a delicate balance of surface properties to give mineral-specific deposition and colloidal stability in high ionic strength alkaline media, while remaining sufficiently hydrophobic to promote flotation. Combinatorial nanoparticle surface modification, in conjunction with high throughput screening, is a promising approach for nanoparticle development. However, efficient automated screening assays are required to reject ineffective particles without having to undergo time consuming flotation testing. Herein we demonstrate that determining critical coagulation concentrations of sodium carbonate in combination with measuring the advancing water contact angle of nanoparticle-saturated glass surfaces can be used to screen ineffective nanoparticles. Finally, none of our first nanoparticle library based on poly(ethylene glycol) methyl ether methacrylate (PEG-methacrylate) were effective flotation collectors because the nanoparticles were too hydrophilic. PMID:26319325

  10. A High-Throughput Yeast Halo Assay for Bioactive Compounds.

    PubMed

    Bray, Walter; Lokey, R Scott

    2016-01-01

    When a disk of filter paper is impregnated with a cytotoxic or cytostatic drug and added to solid medium seeded with yeast, a visible clear zone forms around the disk whose size depends on the concentration and potency of the drug. This is the traditional "halo" assay and provides a convenient, if low-throughput, read-out of biological activity that has been the mainstay of antifungal and antibiotic testing for decades. Here, we describe a protocol for a high-throughput version of the halo assay, which uses an array of 384 pins to deliver ∼200 nL of stock solutions from compound plates onto single-well plates seeded with yeast. Using a plate reader in the absorbance mode, the resulting halos can be quantified and the data archived in the form of flat files that can be connected to compound databases with standard software. This assay has the convenience associated with the visual readout of the traditional halo assay but uses far less material and can be automated to screen thousands of compounds per day. PMID:27587777

  11. A Robotic Platform for Quantitative High-Throughput Screening

    PubMed Central

    Michael, Sam; Auld, Douglas; Klumpp, Carleen; Jadhav, Ajit; Zheng, Wei; Thorne, Natasha; Austin, Christopher P.; Inglese, James

    2008-01-01

    Abstract High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1,536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently developed quantitative HTS (qHTS) paradigm, which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay. The practical implementation of qHTS for cell-based and biochemical assays across libraries of > 100,000 compounds (e.g., between 700,000 and 2,000,000 sample wells tested) requires maximal efficiency and miniaturization and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully integrated and automated screening system for qHTS at the National Institutes of Health's Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has led to the generation of over 6 million CRCs from > 120 assays in the last 3 years and is a technology that can be widely implemented to increase efficiency of screening and lead generation. PMID:19035846

  12. High-throughput screening of microbial adaptation to environmental stress.

    PubMed

    Bélanger, Pier-Anne; Beaudin, Julie; Roy, Sébastien

    2011-05-01

    We developed a microwell plate, high-throughput, screening method aimed at quantitating the tolerance of a panel of Gram-positive and Gram-negative bacteria to metals (Frankia sp., Escherichia coli, Cupriavidus metallidurans, Rhizobium leguminosarum, and Streptomyces scabies). Microbial viability was quantified using MTS; a tetrazolium salt converted to a water-soluble formazan through microbial reduction. In this paper, we present the stepwise development of the method, highlighting the main elements underlying its reliability, and compare results obtained with literature. We conclude the method is well suited to efficiently screen bacteria, including those that are filamentous and slow-growing, without the need for large amounts of inoculum which may not always be available. The method allows testing of compound gradients with sufficient replicates to generate statistically robust results, and is transposable to other types of cell proliferation assays such as those for antimicrobial susceptibility, and chemoresistance. PMID:21315114

  13. Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing

    EPA Science Inventory

    In vitro high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. ...

  14. High Throughput Assays and Exposure Science (ISES annual meeting)

    EPA Science Inventory

    High throughput screening (HTS) data characterizing chemical-induced biological activity has been generated for thousands of environmentally-relevant chemicals by the US inter-agency Tox21 and the US EPA ToxCast programs. For a limited set of chemicals, bioactive concentrations r...

  15. Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators.

    PubMed

    Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

    2016-01-01

    The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the high basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR. PMID:27518621

  16. Evolving the EPA Endocrine Disruptor Screening Program: The case for and against using high-throughput screening assays in EDSP Tier 1

    EPA Science Inventory

    Testing has begun as part of the EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 battery of 11 in vitro and in vivo tests. A recognized issue with the EDSP is that the current Tier 1 screening battery is highly resource intensive in terms of cost, time and animal usage fo...

  17. High-throughput screening for modulators of cellular contractile force†

    PubMed Central

    Park, Chan Young; Zhou, Enhua H.; Tambe, Dhananjay; Chen, Bohao; Lavoie, Tera; Dowell, Maria; Simeonov, Anton; Maloney, David J.; Marinkovic, Aleksandar; Tschumperlin, Daniel J.; Burger, Stephanie; Frykenberg, Matthew; Butler, James P.; Stamer, W. Daniel; Johnson, Mark; Solway, Julian; Fredberg, Jeffrey J.

    2015-01-01

    When cellular contractile forces are central to pathophysiology, these forces comprise a logical target of therapy. Nevertheless, existing high-throughput screens are limited to upstream signalling intermediates with poorly defined relationships to such a physiological endpoint. Using cellular force as the target, here we report a new screening technology and demonstrate its applications using human airway smooth muscle cells in the context of asthma and Schlemm's canal endothelial cells in the context of glaucoma. This approach identified several drug candidates for both asthma and glaucoma. We attained rates of 1000 compounds per screening day, thus establishing a force-based cellular platform for high-throughput drug discovery. PMID:25953078

  18. A High-Throughput Screen for Antibiotic Drug Discovery

    PubMed Central

    Scanlon, Thomas C.; Dostal, Sarah M.; Griswold, Karl E.

    2014-01-01

    We describe an ultra-high-throughput screening platform enabling discovery and/or engineering of natural product antibiotics. The methodology involves creation of hydrogel-in-oil emulsions in which recombinant microorganisms are co-emulsified with bacterial pathogens; antibiotic activity is assayed by use of a fluorescent viability dye. We have successfully utilized both bulk emulsification and microfluidic technology for the generation of hydrogel microdroplets that are size-compatible with conventional flow cytometry. Hydrogel droplets are ~25 pL in volume, and can be synthesized and sorted at rates exceeding 3,000 drops/s. Using this technique, we have achieved screening throughputs exceeding 5 million clones/day. Proof-of-concept experiments demonstrate efficient selection of antibiotic-secreting yeast from a vast excess of negative controls. In addition, we have successfully used this technique to screen a metagenomic library for secreted antibiotics that kill the human pathogen Staphylococcus aureus. Our results establish the practical utility of the screening platform, and we anticipate that the accessible nature of our methods will enable others seeking to identify and engineer the next generation of antibacterial biomolecules. PMID:23955804

  19. Cheminformatics Aspects of High Throughput Screening: from Robots to Models: Symposium Summary

    PubMed Central

    Tseng, Y. Jane; Martin, Eric; Bologa, Cristian; Shelat, Anang A.

    2014-01-01

    The “Cheminformatics aspects of high throughput screening (HTS): from robots to models” symposium was part of the Computers in Chemistry (COMP) technical program at the American Chemical Society National Meeting in Denver, Colorado during the fall of 2011. This symposium brought together researchers from high throughput screening centersand molecular modelers from academia and industry to discuss the integration of currently available high throughput screening data and assays with computational analysis. The topics discussed at this symposium covered the data-infrastructure at various academic, hospital, and NIH-funded high throughput screening centers, the cheminformatics and molecular modeling methods used in real world examples to guide screening and hit-finding, and how academic and non-profit organizations can benefit from current high throughput screening cheminformatics resources. Specifically, this article also covers the remarks and discussions in the open panel discussion in thesymposium and summarizes the following talks on “Accurate Kinase virtual screening: biochemical, cellular and selectivity”, “Selective, privileged and promiscuous chemical patterns in high-throughput screening” and “Visualizing and exploring relationships among HTS hits using network graphs”. PMID:23636795

  20. Workflow for High Throughput Screening of Gas Sensing Materials

    PubMed Central

    Koplin, Tobias J.; Siemons, Maike; Océn-Valéntin, César; Sanders, Daniel; Simon, Ulrich

    2006-01-01

    The workflow of a high throughput screening setup for the rapid identification of new and improved sensor materials is presented. The polyol method was applied to prepare nanoparticular metal oxides as base materials, which were functionalised by surface doping. Using multi-electrode substrates and high throughput impedance spectroscopy (HT-IS) a wide range of materials could be screened in a short time. Applying HT-IS in search of new selective gas sensing materials a NO2-tolerant NO sensing material with reduced sensitivities towards other test gases was identified based on iridium doped zinc oxide. Analogous behaviour was observed for iridium doped indium oxide.

  1. Substrate independent ATPase activity may complicate high throughput screening.

    PubMed

    Tuntland, Micheal L; Fung, L W-M

    2016-10-01

    Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening. PMID:27430931

  2. Compound Management for Quantitative High-Throughput Screening

    PubMed Central

    Yasgar, Adam; Shinn, Paul; Jadhav, Ajit; Auld, Douglas; Michael, Sam; Zheng, Wei; Austin, Christopher P.; Inglese, James; Simeonov, Anton

    2008-01-01

    An efficient and versatile Compound Management operation is essential for the success of all downstream processes in high-throughput screening (HTS) and small molecule lead development. Staff, equipment, and processes need to be not only reliable, but remain flexible and prepared to incorporate paradigm changes. In the present report, we describe a system and associated processes which enable handling of compounds for both screening and follow-up purposes at the NIH Chemical Genomics Center (NCGC), a recently-established HTS and probe development center within the Molecular Libraries Initiative of the NIH Roadmap. Our screening process, termed quantitative HTS (qHTS), involves assaying the complete compound library, currently containing >200,000 members, at a series of dilutions to construct a full concentration-response profile. As such, Compound Management at the NCGC has been uniquely tasked to prepare, store, register, and track a vertically-developed plate dilution series (i.e., inter-plate titrations) in the 384-well format. These are compressed into a series of 1,536-well plates and are registered to track all subsequent plate storage. Here, we present details on the selection of equipment to enable automated, reliable and parallel compound manipulation in 384- and 1,536-well formats, protocols for preparation of inter-plate dilution series for qHTS, as well as qHTS-specific processes and issues. PMID:18496600

  3. Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings

    PubMed Central

    Virto, Juan M.; Holgado, Olaia; Diez, Maria; Izpisua Belmonte, Juan Carlos; Callol-Massot, Carles

    2012-01-01

    The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism. PMID:22615792

  4. ToxCast Assay Network (TCAN) Viewer: A Visualization Tool for High-throughput Assay Chemical Data (SOT)

    EPA Science Inventory

    USEPA’s ToxCast program has generated high-throughput bioactivity screening (HTS) data on thousands of chemicals. The ToxCast program has described and annotated the HTS assay battery with respect to assay design and target information (e.g., gene target). Recent stakeholder and ...

  5. A Protocol for a High-Throughput Multiplex Cell Viability Assay.

    PubMed

    Gilbert, Daniel F; Boutros, Michael

    2016-01-01

    High-throughput cell viability assays are broadly used in RNAi and small molecule screening experiments to identify compounds that selectively kill cancer cells or as counter screens to exclude the compounds that have a generic effect on cell growth. While there are several assaying techniques available, cellular fitness is often assessed on the basis of one single and often rather indirect physiological indicator. This can lead to inconsistencies and poor correspondence between cell viability screening experiments, conducted under comparable conditions but with different viability indicators. Multiplexing, i.e., the combination of different individual assaying techniques in one experiment and subsequent comparative analysis of multiparametric data can decrease inter-assay variability and increase dataset concordance. Here, we describe a protocol for a multiplexing approach for high-throughput cell viability screening to address the issues encountered in the classical strategy using a single fitness indicator described above. The method combines a biochemical, luminescence-based approach and two fluorescence-based assay types. The biochemical method assesses cellular fitness by quantifying intracellular ATP concentration. Calcein labeling reflects cell fitness through membrane integrity and indirect measurement of ATP-dependent enzymatic esterase activity. Hoechst DNA stain correlates cell fitness with cellular DNA content. The presented multiplexing approach is suitable for low, medium and high-throughput screening and has the potential to decrease inter-assay variability and increase dataset concordance as well as reproducibility of experimental results. PMID:27581285

  6. In Vitro High Throughput Screening, What Next? Lessons from the Screening for Aurora Kinase Inhibitors

    PubMed Central

    Hoang, Thi-My-Nhung; Vu, Hong-Lien; Le, Ly-Thuy-Tram; Nguyen, Chi-Hung; Molla, Annie

    2014-01-01

    Based on in vitro assays, we performed a High Throughput Screening (HTS) to identify kinase inhibitors among 10,000 small chemical compounds. In this didactic paper, we describe step-by-step the approach to validate the hits as well as the major pitfalls encountered in the development of active molecules. We propose a decision tree that could be adapted to most in vitro HTS. PMID:24833340

  7. High-throughput screening, predictive modeling and computational embryology

    EPA Science Inventory

    High-throughput screening (HTS) studies are providing a rich source of data that can be applied to profile thousands of chemical compounds for biological activity and potential toxicity. EPA’s ToxCast™ project, and the broader Tox21 consortium, in addition to projects worldwide,...

  8. High-throughput screening, predictive modeling and computational embryology - Abstract

    EPA Science Inventory

    High-throughput screening (HTS) studies are providing a rich source of data that can be applied to chemical profiling to address sensitivity and specificity of molecular targets, biological pathways, cellular and developmental processes. EPA’s ToxCast project is testing 960 uniq...

  9. Environmental Impact on Vascular Development Predicted by High Throughput Screening

    EPA Science Inventory

    Understanding health risks to embryonic development from exposure to environmental chemicals is a significant challenge given the diverse chemical landscape and paucity of data for most of these compounds. High throughput screening (HTS) in EPA’s ToxCastTM project provides vast d...

  10. High-throughput assays for DNA gyrase and other topoisomerases.

    PubMed

    Maxwell, Anthony; Burton, Nicolas P; O'Hagan, Natasha

    2006-01-01

    We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format. PMID:16936317

  11. High-throughput automated refolding screening of inclusion bodies

    PubMed Central

    Vincentelli, Renaud; Canaan, Stéphane; Campanacci, Valérie; Valencia, Christel; Maurin, Damien; Frassinetti, Frédéric; Scappucini-Calvo, Loréna; Bourne, Yves; Cambillau, Christian; Bignon, Christophe

    2004-01-01

    One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no “universal” refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals. PMID:15388864

  12. Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay

    PubMed Central

    Lordkipanidzé, Marie; Lowe, Gillian C.; Kirkby, Nicholas S.; Chan, Melissa V.; Lundberg, Martina H.; Morgan, Neil V.; Bem, Danai; Nisar, Shaista P.; Leo, Vincenzo C.; Jones, Matthew L.; Mundell, Stuart J.; Daly, Martina E.; Mumford, Andrew D.; Warner, Timothy D.; Watson, Steve P.; Watson, Steve P.; Mumford, Andrew D.; Mundell, Stuart J.; Gissen, Paul; Daly, Martina E.; Lester, Will; Clark, Justin; Williams, Mike; Motwani, Jayashree; Marshall, Dianne; Nyatanga, Priscilla; Mann, Pat; Kirwan, Julie; Wilde, Jonathan; Dunkley, Tracey; Greenway, April; Makris, Michael; Pavord, Sue; Dattani, Rashesh; Grimley, Gerry Dolan Charlotte; Stokley, Simone; Astwood, Emma; Chang, Cherry; Foros, Merri; Trower, Linda; Thachil, Jecko; Hay, Charlie; Pike, Gill; Will, Andrew; Grainger, John; Foulkes, Matt; Fareh, Mona; Talks, Kate; Biss, Tina; Kesteven, Patrick; Hanley, John; Vowles, Julie; Basey, Lesley; Barnes, Michelle; Collins, Peter; Rayment, Rachel; Alikhan, Raza; Morris, Ana Guerrero Rebecca; Mansell, Dianne; Toh, Cheng Hock; Martlew, Vanessa; Murphy, Elaine; Lachmann, Robin; Rose, Peter; Chapman, Oliver; Lokare, Anand; Marshall, Kathryn; Khan, Naseem; Keeling, David; Giangrande, Paul; Austin, Steve; Bevan, David; Alamelu, Jayanthi

    2014-01-01

    Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167. PMID:24408324

  13. Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay.

    PubMed

    Lordkipanidzé, Marie; Lowe, Gillian C; Kirkby, Nicholas S; Chan, Melissa V; Lundberg, Martina H; Morgan, Neil V; Bem, Danai; Nisar, Shaista P; Leo, Vincenzo C; Jones, Matthew L; Mundell, Stuart J; Daly, Martina E; Mumford, Andrew D; Warner, Timothy D; Watson, Steve P

    2014-02-20

    Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167. PMID:24408324

  14. Developing High-Throughput HIV Incidence Assay with Pyrosequencing Platform

    PubMed Central

    Park, Sung Yong; Goeken, Nolan; Lee, Hyo Jin; Bolan, Robert; Dubé, Michael P.

    2014-01-01

    ABSTRACT Human immunodeficiency virus (HIV) incidence is an important measure for monitoring the epidemic and evaluating the efficacy of intervention and prevention trials. This study developed a high-throughput, single-measure incidence assay by implementing a pyrosequencing platform. We devised a signal-masking bioinformatics pipeline, which yielded a process error rate of 5.8 × 10−4 per base. The pipeline was then applied to analyze 18,434 envelope gene segments (HXB2 7212 to 7601) obtained from 12 incident and 24 chronic patients who had documented HIV-negative and/or -positive tests. The pyrosequencing data were cross-checked by using the single-genome-amplification (SGA) method to independently obtain 302 sequences from 13 patients. Using two genomic biomarkers that probe for the presence of similar sequences, the pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 of 24 chronic subjects (96% specificity). One misclassified subject's chronic infection was correctly classified by conducting the same analysis with SGA data. The biomarkers were statistically associated across the two platforms, suggesting the assay's reproducibility and robustness. Sampling simulations showed that the biomarkers were tolerant of sequencing errors and template resampling, two factors most likely to affect the accuracy of pyrosequencing results. We observed comparable biomarker scores between AIDS and non-AIDS chronic patients (multivariate analysis of variance [MANOVA], P = 0.12), indicating that the stage of HIV disease itself does not affect the classification scheme. The high-throughput genomic HIV incidence marks a significant step toward determining incidence from a single measure in cross-sectional surveys. IMPORTANCE Annual HIV incidence, the number of newly infected individuals within a year, is the key measure of monitoring the epidemic's rise and decline. Developing reliable assays differentiating recent from chronic

  15. Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.

    PubMed

    Sun, Ying; Wang, Zidao; Tao, Jiali; Wang, Yi; Wu, Andong; Yang, Ziwen; Wang, Kaimei; Shi, Liqiao; Chen, Yu; Guo, Deyin

    2014-04-01

    The 5'-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5'-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. Our previous work showed that SARS coronavirus (SARS-CoV) non-structural protein 14 represents a structurally novel and unique guanine-N7-methyltransferase (N7-MTase) that is able to functionally complement yeast cellular N7-MTase. In the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. The MTase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed N7-MTase in the yeast system. However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect within a cellular context. These results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. The yeast system was applied to the screening of 3000 natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human N7-MTase. PMID:24530452

  16. Promises and Pitfalls of High-Throughput Biological Assays.

    PubMed

    Finak, Greg; Gottardo, Raphael

    2016-01-01

    This chapter discusses some of the pitfalls encountered when performing biomedical research involving high-throughput "omics" data and presents some strategies and guidelines that researchers should follow when undertaking such studies. We discuss common errors in experimental design and data analysis that lead to irreproducible and non-replicable research and provide some guidelines to avoid these common mistakes so that researchers may have confidence in study outcomes, even if the results are negative. We discuss the importance of ranking and prespecifying hypotheses, performing power analysis, careful experimental design, and preplanning of statistical analyses in order to avoid the "fishing expedition" data analysis strategy, which is doomed to fail. The impact of multiple testing on false-positive rates is discussed, particularly in the context of the analysis of high-throughput data, and methods to correct for it are presented, as well as approaches to detect and correct for experimental biases and batch effects, which often plague high-throughput assays. We highlight the importance of sharing data and analysis code to facilitate reproducibility and present tools and software that are appropriate for this purpose. PMID:27115636

  17. A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors.

    PubMed

    McDonald, O B; Chen, W J; Ellis, B; Hoffman, C; Overton, L; Rink, M; Smith, A; Marshall, C J; Wood, E R

    1999-03-15

    We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein. PMID:10075822

  18. Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents.

    PubMed

    Lim, Kah Tee; Zahari, Zuriati; Amanah, Azimah; Zainuddin, Zafarina; Adenan, Mohd Ilham

    2016-03-01

    To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications. PMID:26772786

  19. Virtual High-Throughput Screening To Identify Novel Activin Antagonists.

    PubMed

    Zhu, Jie; Mishra, Rama K; Schiltz, Gary E; Makanji, Yogeshwar; Scheidt, Karl A; Mazar, Andrew P; Woodruff, Teresa K

    2015-07-23

    Activin belongs to the TGFβ superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin βA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHβ transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex's binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFβ superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFβ receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases. PMID:26098096

  20. Virtual High-Throughput Screening To Identify Novel Activin Antagonists

    PubMed Central

    Zhu, Jie; Mishra, Rama K.; Schiltz, Gary E.; Makanji, Yogeshwar; Scheidt, Karl A.; Mazar, Andrew P.; Woodruff, Teresa K.

    2015-01-01

    Activin belongs to the TGFβ superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin βA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHβ transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complex’s binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFβ superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFβ receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases. PMID:26098096

  1. High-throughput screening to identify inhibitors of lysine demethylases

    PubMed Central

    Gale, Molly; Yan, Qin

    2015-01-01

    Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several high-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the high-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors. PMID:25687466

  2. Creation of a small high-throughput screening facility.

    PubMed

    Flak, Tod

    2009-01-01

    The creation of a high-throughput screening facility within an organization is a difficult task, requiring a substantial investment of time, money, and organizational effort. Major issues to consider include the selection of equipment, the establishment of data analysis methodologies, and the formation of a group having the necessary competencies. If done properly, it is possible to build a screening system in incremental steps, adding new pieces of equipment and data analysis modules as the need grows. Based upon our experience with the creation of a small screening service, we present some guidelines to consider in planning a screening facility. PMID:19551356

  3. Development of a Multiplex Assay for Studying Functional Selectivity of Human Serotonin 5-HT2A Receptors and Identification of Active Compounds by High-Throughput Screening.

    PubMed

    Iglesias, Alba; Lage, Sonia; Cadavid, Maria Isabel; Loza, Maria Isabel; Brea, José

    2016-09-01

    G protein-coupled receptors (GPCRs) exist as collections of conformations in equilibrium, and the efficacy of drugs has been proposed to be associated with their absolute and relative affinities for these different conformations. The serotonin 2A (5-HT2A) receptor regulates multiple physiological functions, is involved in the pathophysiology of schizophrenia, and serves as an important target of atypical antipsychotic drugs. This receptor was one of the first GPCRs for which the functional selectivity phenomenon was observed, with its various ligands exerting differential effects on the phospholipase A2 (PLA2) and phospholipase C (PLC) signaling pathways. We aimed to develop a multiplex functional assay in 96-well plates for the simultaneous measurement of the PLA2 and PLC pathways coupled to 5-HT2A receptors; this approach enables the detection of either functional selectivity or cooperativity phenomena in early drug screening stages. The suitability of the method for running screening campaigns was tested using the Prestwick Chemical Library, and 22 confirmed hits with activities of more than 90% were identified; 11 of these hits produced statistically significant differences between the two effector pathways. Thus, we have developed a miniaturized multiplex assay in 96-well plates to measure functional selectivity for 5-HT2A receptors in the early stages of the drug discovery process. PMID:27095818

  4. A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel

    PubMed Central

    Titus, Steven A.; Beacham, Daniel; Shahane, Sampada A.; Southall, Noel; Xia, Menghang; Huang, Ruili; Hooten, Elizabeth; Zhao, Yong; Shou, Louie; Austin, Christopher P.; Zheng, Wei

    2009-01-01

    Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel. PMID:19583963

  5. High-Throughput Screening Based Identification of Paramyxovirus Inhibitors

    PubMed Central

    Yoon, Jeong-Jeong; Chawla, Dhruv; Paal, Tanja; Ndungu, Maina; Du, Yuhong; Kurtkaya, Serdar; Sun, Aiming; Snyder, James P; Plemper, Richard K

    2008-01-01

    Paramyxoviruses are negative strand non-segmented RNA viruses. Several members of this family constitute major human pathogens that, collectively, are responsible for major morbidity and mortality worldwide. In an effort to ultimately develop novel therapeutics against measles virus (MV), a prominent member of the paramyxovirus family, we report a high-throughput screening protocol that allows hit identification using non-recombinant primary MV strains as targets. Implementation of the assay has yielded 60 hit candidates from a 137,500-entry library. Counterscreening and generation of dose-response curves narrows this pool to 35 compounds with active concentrations ≤15.3 μM against the MV-Alaska strain and specificity indices ranging from 36 to >500. Library mining for structural analogs of several confirmed hits combined with re-testing of identified candidates reveals a low false-negative rate and, thus, a high accuracy of primary hit identification. Eleven of the confirmed hits were found to interfere with the viral entry machinery, while the remaining 24 compounds target post-entry steps of the viral life cycle. Activity testing against selected members of the paramyxovirus family reveals three patterns of activity: 1) exclusively MV-specific blockers; 2) inhibitors of MV and related viruses of the same genus; 3) broader-range inhibitors with activity against a different paramyxovirinae genus. Representatives of the last class may open avenues for the development of broad-range paramyxovirus inhibitors through hit-to-lead chemistry. PMID:18626114

  6. High-throughput screening of phosphodiesterase activity in living cells.

    PubMed

    Rich, Thomas C; Karpen, Jeffrey W

    2005-01-01

    Phosphodiesterases (PDEs) hydrolyze the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine 5'-monophosphate (cGMP) and play a crucial role in the termination and spatial segregation of cyclic nucleotide signals. Despite a wealth of molecular information, very little is known about how PDEs regulate cAMP and cGMP signals in living cells because conventional methods lack the necessary spatial and temporal resolution. We present here a sensitive optical method for monitoring cAMP levels and PDE activity near the membrane, using cyclic nucleotide-gated (CNG) ion channels as sensors. These channels are directly opened by the binding of cyclic nucleotides and allow cations to cross the membrane. The olfactory channel A subunit (CNGA2) has been genetically modified to improve its cAMP sensitivity and specificity. Channel activity is assessed by measuring Ca2+ influx using standard fluorometric techniques. In addition to studying PDEs in their native setting, the approach should be particularly useful in high-throughput screening assays to test for compounds that affect PDE activity, as well as the activities of the many G protein-coupled receptors that cause changes in intracellular cAMP. PMID:15988054

  7. A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme

    PubMed Central

    Bullard-Feibelman, Kristen M.; Fuller, Benjamin P.; Geiss, Brian J.

    2016-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that causes severe and debilitating disease symptoms. Alarmingly, transmission rates of CHIKV have increased dramatically over the last decade resulting in 1.7 million suspected cases in the Western hemisphere alone. There are currently no antivirals for treatment of CHIKV infection and novel anti-alphaviral compounds are badly needed. nsP1 is the alphavirus protein responsible for the methyltransferase and guanylyltransferase activities necessary for formation of the 5’ type 0 cap structure added to newly formed viral RNA. Formation of this cap depends on nsP1 binding GTP and transferring a methylated GMP to nascent viral RNA. We have developed a fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time. Determining the relative affinities of 15 GTP analogs for nsP1 GTP revealed important structural aspects of GTP that will inform identification of inhibitors able to outcompete GTP for the nsP1 binding site. Validation of the assay for HTS was completed and a secondary orthogonal assay that measures guanylation activity was developed in order to evaluate hits from future drug screens. This platform provides an avenue for identification of potent nsP1 inhibitors, which would potentially provide compounds capable of treating disease caused by CHIKV infection. PMID:27427769

  8. Reconfigurable microfluidic dilution for high-throughput quantitative assays.

    PubMed

    Fan, Jinzhen; Li, Baoqing; Xing, Siyuan; Pan, Tingrui

    2015-06-21

    This paper reports a reconfigurable microfluidic dilution device for high-throughput quantitative assays, which can easily produce discrete logarithmic/binary concentration profiles ranging from 1 to 100-fold dilution in parallel from a fixed sample volume (e.g., 10 μL) without any assistance of continuous fluidic pump or robotic automation. The integrated dilution generation chip consists of switchable distribution and collection channels, metering reservoirs, reaction chambers, and pressure-activatable Laplace valves. Following the sequential loading of a sample, a diluent, and a detection reagent into their individual metering chambers, the top microfluidic layer can be reconfigured to collect the metered chemicals into the reaction chambers in parallel, where detection will be conducted. To facilitate mixing and reaction in the microchambers, two acoustic microstreaming actuation mechanisms have been investigated for easy integrability and accessibility. Furthermore, the microfluidic dilution generator has been characterized by both colorimetric and fluorescent means. A further demonstration of the generic usage of the quantitative dilution chip has utilized the commonly available bicinchoninic acid (BCA) assay to analyse the protein concentrations of human tissue extracts. In brief, the microfluidic dilution generator offers a high-throughput high-efficiency quantitative analytical alternative to conventional quantitative assay platforms, by simple manipulation of a minute amount of chemicals in a compact microfluidic device with minimal equipment requirement, which can serve as a facile tool for biochemical and biological analyses in regular laboratories, point-of-care settings and low-resource environments. PMID:25994379

  9. Image-based high-throughput screening for inhibitors of angiogenesis.

    PubMed

    Evensen, Lasse; Link, Wolfgang; Lorens, James B

    2013-01-01

    Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling. PMID:23027002

  10. Development and Application of a High Throughput Protein Unfolding Kinetic Assay

    PubMed Central

    Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

    2016-01-01

    The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

  11. Self-Assembled Cell Microarray (SAMcell) for High-Throughput RNAi Screening.

    PubMed

    Zhang, Hanshuo; Li, Juan

    2016-01-01

    RNAi has now become a valuable research tool for cell-based high-throughput screening. However, traditional RNAi high-throughput methods are based on multi-well plates, relying on expensive instruments and complicated operations. In this chapter, we describe a method termed self-assembled cell microarray (SAMcell), which integrates micro-fabrication, reverse transfection, and RNAi technologies and allows for cell behavior investigations to be performed directly on the cell chip. This method has been successfully employed to perform large-scale functional screening assays to identify gene modulators of cell migration, cell proliferation, and cellular apoptosis. PMID:27581287

  12. Predictive Model of Rat Reproductive Toxicity from ToxCast High Throughput Screening

    EPA Science Inventory

    The EPA ToxCast research program uses high throughput screening for bioactivity profiling and predicting the toxicity of large numbers of chemicals. ToxCast Phase‐I tested 309 well‐characterized chemicals in over 500 assays for a wide range of molecular targets and cellular respo...

  13. Three-Dimensional Spheroid Cell Culture Model for Target Identification Utilizing High-Throughput RNAi Screens.

    PubMed

    Iles, LaKesla R; Bartholomeusz, Geoffrey A

    2016-01-01

    The intrinsic limitations of 2D monolayer cell culture models have prompted the development of 3D cell culture model systems for in vitro studies. Multicellular tumor spheroid (MCTS) models closely simulate the pathophysiological milieu of solid tumors and are providing new insights into tumor biology as well as differentiation, tissue organization, and homeostasis. They are straightforward to apply in high-throughput screens and there is a great need for the development of reliable and robust 3D spheroid-based assays for high-throughput RNAi screening for target identification and cell signaling studies highlighting their potential in cancer research and treatment. In this chapter we describe a stringent standard operating procedure for the use of MCTS for high-throughput RNAi screens. PMID:27581289

  14. High throughput screening of starch structures using carbohydrate microarrays.

    PubMed

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Motawia, Mohammed Saddik; Shaik, Shahnoor Sultana; Mikkelsen, Maria Dalgaard; Krunic, Susanne Langgaard; Fangel, Jonatan Ulrik; Willats, William George Tycho; Blennow, Andreas

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches. PMID:27468930

  15. High throughput screening of starch structures using carbohydrate microarrays

    PubMed Central

    Tanackovic, Vanja; Rydahl, Maja Gro; Pedersen, Henriette Lodberg; Motawia, Mohammed Saddik; Shaik, Shahnoor Sultana; Mikkelsen, Maria Dalgaard; Krunic, Susanne Langgaard; Fangel, Jonatan Ulrik; Willats, William George Tycho; Blennow, Andreas

    2016-01-01

    In this study we introduce the starch-recognising carbohydrate binding module family 20 (CBM20) from Aspergillus niger for screening biological variations in starch molecular structure using high throughput carbohydrate microarray technology. Defined linear, branched and phosphorylated maltooligosaccharides, pure starch samples including a variety of different structures with variations in the amylopectin branching pattern, amylose content and phosphate content, enzymatically modified starches and glycogen were included. Using this technique, different important structures, including amylose content and branching degrees could be differentiated in a high throughput fashion. The screening method was validated using transgenic barley grain analysed during development and subjected to germination. Typically, extreme branching or linearity were detected less than normal starch structures. The method offers the potential for rapidly analysing resistant and slowly digested dietary starches. PMID:27468930

  16. A high-throughput assay for DNA topoisomerases and other enzymes, based on DNA triplex formation.

    PubMed

    Burrell, Matthew R; Burton, Nicolas P; Maxwell, Anthony

    2010-01-01

    We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase. PMID:19997889

  17. Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.

    PubMed

    Pantel, Jacques; Williams, Savannah Y; Mi, Dehui; Sebag, Julien; Corbin, Jackie D; Weaver, C David; Cone, Roger D

    2011-06-11

    The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z'=0.50). A pilot screen run on the Microsource Spectrum compound library (n=2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β(2)AR agonists - the β(2)AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor. PMID:21296065

  18. Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay

    PubMed Central

    Pantel, Jacques; Williams, Savannah Y.; Mi, Dehui; Sebag, Julien; Corbin, Jackie D.; Weaver, C. David; Cone, Roger D.

    2011-01-01

    The melanocortin MC4 receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC4 receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC4 receptor, we created HEK293 cell lines coexpressing the human melanocortin MC4 receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z’=0.50). A pilot screen run on the Microsource Spectrum compound library (n= 2,000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β2AR agonists –the β2AR being endogenously expressed in HEK293 cells-, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of an HTS for allosteric modulators for a Gs protein coupled receptor. PMID:21296065

  19. High-throughput screening with micro-x-ray fluorescence

    NASA Astrophysics Data System (ADS)

    Havrilla, George J.; Miller, Thomasin C.

    2005-06-01

    Micro-x-ray fluorescence (MXRF) is a useful characterization tool for high-throughput screening of combinatorial libraries. Due to the increasing threat of use of chemical warfare (CW) agents both in military actions and against civilians by terrorist extremists, there is a strong push to improve existing methods and develop means for the detection of a broad spectrum of CW agents in a minimal amount of time to increase national security. This paper describes a combinatorial high-throughput screening technique for CW receptor discovery to aid in sensor development. MXRF can screen materials for elemental composition at the mesoscale level (tens to hundreds of micrometers). The key aspect of this work is the use of commercial MXRF instrumentation coupled with the inherent heteroatom elements within the target molecules of the combinatorial reaction to provide rapid and specific identification of lead species. The method is demonstrated by screening an 11-mer oligopeptide library for selective binding of the degradation products of the nerve agent VX. The identified oligopeptides can be used as selective molecular receptors for sensor development. The MXRF screening method is nondestructive, requires minimal sample preparation or special tags for analysis, and the screening time depends on the desired sensitivity.

  20. High-throughput screening with micro-x-ray fluorescence

    SciTech Connect

    Havrilla, George J.; Miller, Thomasin C.

    2005-06-15

    Micro-x-ray fluorescence (MXRF) is a useful characterization tool for high-throughput screening of combinatorial libraries. Due to the increasing threat of use of chemical warfare (CW) agents both in military actions and against civilians by terrorist extremists, there is a strong push to improve existing methods and develop means for the detection of a broad spectrum of CW agents in a minimal amount of time to increase national security. This paper describes a combinatorial high-throughput screening technique for CW receptor discovery to aid in sensor development. MXRF can screen materials for elemental composition at the mesoscale level (tens to hundreds of micrometers). The key aspect of this work is the use of commercial MXRF instrumentation coupled with the inherent heteroatom elements within the target molecules of the combinatorial reaction to provide rapid and specific identification of lead species. The method is demonstrated by screening an 11-mer oligopeptide library for selective binding of the degradation products of the nerve agent VX. The identified oligopeptides can be used as selective molecular receptors for sensor development. The MXRF screening method is nondestructive, requires minimal sample preparation or special tags for analysis, and the screening time depends on the desired sensitivity.

  1. High-throughput protein analysis integrating bioinformatics and experimental assays.

    PubMed

    del Val, Coral; Mehrle, Alexander; Falkenhahn, Mechthild; Seiler, Markus; Glatting, Karl-Heinz; Poustka, Annemarie; Suhai, Sandor; Wiemann, Stefan

    2004-01-01

    The wealth of transcript information that has been made publicly available in recent years requires the development of high-throughput functional genomics and proteomics approaches for its analysis. Such approaches need suitable data integration procedures and a high level of automation in order to gain maximum benefit from the results generated. We have designed an automatic pipeline to analyse annotated open reading frames (ORFs) stemming from full-length cDNAs produced mainly by the German cDNA Consortium. The ORFs are cloned into expression vectors for use in large-scale assays such as the determination of subcellular protein localization or kinase reaction specificity. Additionally, all identified ORFs undergo exhaustive bioinformatic analysis such as similarity searches, protein domain architecture determination and prediction of physicochemical characteristics and secondary structure, using a wide variety of bioinformatic methods in combination with the most up-to-date public databases (e.g. PRINTS, BLOCKS, INTERPRO, PROSITE SWISSPROT). Data from experimental results and from the bioinformatic analysis are integrated and stored in a relational database (MS SQL-Server), which makes it possible for researchers to find answers to biological questions easily, thereby speeding up the selection of targets for further analysis. The designed pipeline constitutes a new automatic approach to obtaining and administrating relevant biological data from high-throughput investigations of cDNAs in order to systematically identify and characterize novel genes, as well as to comprehensively describe the function of the encoded proteins. PMID:14762202

  2. Evaluation of high-throughput assays for in vitro drug susceptibility testing of Tritrichomonas foetus trophozoites.

    PubMed

    Bader, Chris; Jesudoss Chelladurai, Jeba; Thompson, Kylie; Hall, Cindy; Carlson, Steve A; Brewer, Matthew T

    2016-06-15

    Tritrichomonas foetus is a sexually transmitted protozoan parasite that causes abortions in cattle and results in severe economic losses. In the United States, there are no safe and effective treatments for this parasite and infected animals are typically culled. In order to expedite drug discovery efforts, we investigated in vitro trophozoite killing assays amenable to high-throughput screening in 96 well plate formats. We evaluated the reduction of resorufin, incorporation of propidium iodide, and a luminescence-based ATP detection assay. Of these methods, reduction of resorufin was found to be the most reliable predictor of trophozoite concentrations. We further validated this method by conducting dose-response experiments suitable for calculation of EC50 values for two established compounds with known activity against trophozoites in vitro, namely, metronidazole and ronidazole. Our results demonstrate that the resorufin method is suitable for high-throughput screening and could be used to enhance efforts targeting new treatments for bovine trichomoniasis. PMID:27198774

  3. High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment

    PubMed Central

    Ankam, Soneela; Teo, Benjamin KK; Kukumberg, Marek; Yim, Evelyn KF

    2013-01-01

    Stem cells in vivo are housed within a functional microenvironment termed the “stem cell niche.” As the niche components can modulate stem cell behaviors like proliferation, migration and differentiation, evaluating these components would be important to determine the most optimal platform for their maintenance or differentiation. In this review, we have discussed methods and technologies that have aided in the development of high throughput screening assays for stem cell research, including enabling technologies such as the well-established multiwell/microwell plates and robotic spotting, and emerging technologies like microfluidics, micro-contact printing and lithography. We also discuss the studies that utilized high throughput screening platform to investigate stem cell response to extracellular matrix, topography, biomaterials and stiffness gradients in the stem cell niche. The combination of the aforementioned techniques could lay the foundation for new perspectives in further development of high throughput technology and stem cell research. PMID:23899508

  4. Development of a High-Throughput Functional Screen Using Nanowell-Assisted Cell Patterning.

    PubMed

    Ozkumur, Ayca Yalcin; Goods, Brittany A; Love, J Christopher

    2015-09-01

    Living-cell-based screens can facilitate lead discovery of functional therapeutics of interest. A versatile and scalable method is reported that uses dense arrays of nanowells for imparting defined patterns on monolayers of cells. It is shown that this approach can coordinate a multi-component biological assay by designing and implementing a high-throughput, functional nanoliter-scale neutralization assay to identify neutralizing antibodies against HIV. PMID:26121321

  5. Microfluidic cell chips for high-throughput drug screening.

    PubMed

    Chi, Chun-Wei; Ahmed, Ah Rezwanuddin; Dereli-Korkut, Zeynep; Wang, Sihong

    2016-05-01

    The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell-drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers. PMID:27071838

  6. High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Adult Schistosoma mansoni.

    PubMed

    Mansour, Nuha R; Paveley, Ross; Gardner, J Mark F; Bell, Andrew S; Parkinson, Tanya; Bickle, Quentin

    2016-04-01

    An estimated 600 million people are affected by the helminth disease schistosomiasis caused by parasites of the genus Schistosoma. There is currently only one drug recommended for treating schistosomiasis, praziquantel (PZQ), which is effective against adult worms but not against the juvenile stage. In an attempt to identify improved drugs for treating the disease, we have carried out high throughput screening of a number of small molecule libraries with the aim of identifying lead compounds with balanced activity against all life stages of Schistosoma. A total of almost 300,000 compounds were screened using a high throughput assay based on motility of worm larvae and image analysis of assay plates. Hits were screened against juvenile and adult worms to identify broadly active compounds and against a mammalian cell line to assess cytotoxicity. A number of compounds were identified as promising leads for further chemical optimization. PMID:27128493

  7. High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Adult Schistosoma mansoni

    PubMed Central

    Gardner, J. Mark F.; Bell, Andrew S.; Parkinson, Tanya; Bickle, Quentin

    2016-01-01

    An estimated 600 million people are affected by the helminth disease schistosomiasis caused by parasites of the genus Schistosoma. There is currently only one drug recommended for treating schistosomiasis, praziquantel (PZQ), which is effective against adult worms but not against the juvenile stage. In an attempt to identify improved drugs for treating the disease, we have carried out high throughput screening of a number of small molecule libraries with the aim of identifying lead compounds with balanced activity against all life stages of Schistosoma. A total of almost 300,000 compounds were screened using a high throughput assay based on motility of worm larvae and image analysis of assay plates. Hits were screened against juvenile and adult worms to identify broadly active compounds and against a mammalian cell line to assess cytotoxicity. A number of compounds were identified as promising leads for further chemical optimization. PMID:27128493

  8. High-throughput screening to enhance oncolytic virus immunotherapy.

    PubMed

    Allan, K J; Stojdl, David F; Swift, S L

    2016-01-01

    High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs) are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms - including those based on herpes simplex virus, reovirus, and vaccinia virus - have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. PMID:27579293

  9. High-throughput screening to enhance oncolytic virus immunotherapy

    PubMed Central

    Allan, KJ; Stojdl, David F; Swift, SL

    2016-01-01

    High-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. Although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. Oncolytic viruses (OVs) are biotherapeutic agents with an inherent specificity for treating malignant disease. Certain OV platforms – including those based on herpes simplex virus, reovirus, and vaccinia virus – have shown success against solid tumors in advanced clinical trials. Yet, many of these OVs have only undergone minimal engineering to solidify tumor specificity, with few extra modifications to manipulate additional factors. Several aspects of the interaction between an OV and a tumor-bearing host have clear value as targets to improve therapeutic outcomes. At the virus level, these include delivery to the tumor, infectivity, productivity, oncolysis, bystander killing, spread, and persistence. At the host level, these include engaging the immune system and manipulating the tumor microenvironment. Here, we review the chemical- and genome-based high-throughput screens that have been performed to manipulate such parameters during OV infection and analyze their impact on therapeutic efficacy. We further explore emerging themes that represent key areas of focus for future research. PMID:27579293

  10. A novel high-throughput nematicidal assay using embryo cells and larvae of Caenorhabditis elegans.

    PubMed

    Lai, Yiling; Xiang, Meichun; Liu, Shuchun; Li, Erwei; Che, Yongsheng; Liu, Xingzhong

    2014-04-01

    Human health safety and environmental concerns have resulted in the widespread deregistration of several agronomic important nematicides. New and safer nematicides are urgently needed. However, a high-throughput bioassay for screening potential nematicides has not been established. We developed a two-step high-throughput nematicidal screening method to combine a cell-based MTS colorimetric assay with Caenorhabditis elegans embryo cells for preliminary cytotoxicity screening (step 1) followed by in vitro larval assay for nematicidal activity (step 2). Based on three conventional nematicides' test, high correlations were obtained between cell viability and larval viability and "r" values were 0.78 for Avermectin, 0.95 for Fosthiazate, and 0.65 for Formaldehyde solution. Further assays with 60 fungal secondary metabolites (extracts, fractions and pure compounds) also demonstrated the high correlation between cell viability and larval viability (r=0.60) and between the C. elegans cell viability and the juvenile viability of soybean cyst nematode Heterodera glycines (r=0.48) and pine wood nematode Bursaphelenchus xylophilus (r=0.56). Six metabolites with high cytotoxicity have performed high larval mortality with a LC50 range of 6.8-500μg/ml. These results indicate that the proposed two-step screening assay represents an efficient and labor-saving method for screening natural nematicidal products. PMID:24594258

  11. A high-throughput assay of NK cell activity in whole blood and its clinical application

    SciTech Connect

    Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  12. Application of a High-throughput Fluorescent Acetyltransferase Assay to Identify Inhibitors of Homocitrate Synthase

    PubMed Central

    Bulfer, Stacie L.; McQuade, Thomas J.; Larsen, Martha J.; Trievel, Raymond C.

    2011-01-01

    Homocitrate synthase (HCS) catalyzes the first step of L-lysine biosynthesis in fungi by condensing acetyl-Coenzyme A and 2-oxoglutarate to form 3R-homocitrate and Coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust, fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of ~41,000 small molecules. Following confirmation, counter screens, and dose-response analysis, we prioritized over 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases and enzymes involved in lipid metabolism. PMID:21073853

  13. Droplet microfluidic technology for single-cell high-throughput screening.

    PubMed

    Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

    2009-08-25

    We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses. PMID:19617544

  14. Droplet microfluidic technology for single-cell high-throughput screening

    PubMed Central

    Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J. Brian; Rothberg, Jonathan M.; Link, Darren R.; Perrimon, Norbert; Samuels, Michael L.

    2009-01-01

    We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses. PMID:19617544

  15. A High Throughput Mechanical Screening Device for Cartilage Tissue Engineering

    PubMed Central

    Mohanraj, Bhavana; Hou, Chieh; Meloni, Greg R.; Cosgrove, Brian D.; Dodge, George R.; Mauck, Robert L.

    2014-01-01

    Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying ‘hits’, or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput. PMID:24275442

  16. Evaluation of High-throughput Genotoxicity Assays Used in Profiling the US EPA ToxCast Chemicals

    EPA Science Inventory

    Three high-throughput screening (HTS) genotoxicity assays-GreenScreen HC GADD45a-GFP (Gentronix Ltd.), CellCiphr p53 (Cellumen Inc.) and CellSensor p53RE-bla (Invitrogen Corp.)-were used to analyze the collection of 320 predominantly pesticide active compounds being tested in Pha...

  17. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles.

    PubMed

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  18. High Throughput Screening Method to Explore Protein Interactions with Nanoparticles

    PubMed Central

    Nasir, Irem; Fatih, Warda; Svensson, Anja; Radu, Dennis; Linse, Sara; Cabaleiro Lago, Celia; Lundqvist, Martin

    2015-01-01

    The interactions of biological macromolecules with nanoparticles underlie a wide variety of current and future applications in the fields of biotechnology, medicine and bioremediation. The same interactions are also responsible for mediating potential biohazards of nanomaterials. Some applications require that proteins adsorb to the nanomaterial and that the protein resists or undergoes structural rearrangements. This article presents a screening method for detecting nanoparticle-protein partners and conformational changes on time scales ranging from milliseconds to days. Mobile fluorophores are used as reporters to study the interaction between proteins and nanoparticles in a high-throughput manner in multi-well format. Furthermore, the screening method may reveal changes in colloidal stability of nanomaterials depending on the physicochemical conditions. PMID:26313757

  19. Fully automatized high-throughput enzyme library screening using a robotic platform.

    PubMed

    Dörr, Mark; Fibinger, Michael P C; Last, Daniel; Schmidt, Sandy; Santos-Aberturas, Javier; Böttcher, Dominique; Hummel, Anke; Vickers, Clare; Voss, Moritz; Bornscheuer, Uwe T

    2016-07-01

    A fully automatized robotic platform has been established to facilitate high-throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer-Villiger monooxygenase, transaminase, dehalogenase and acylase in the high-throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods. Biotechnol. Bioeng. 2016;113: 1421-1432. © 2016 Wiley Periodicals, Inc. PMID:26724475

  20. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

    PubMed

    Sun, Yiyi; Zang, Zhihe; Zhong, Ling; Wu, Min; Su, Qing; Gao, Xiurong; Zan, Wang; Lin, Dong; Zhao, Yan; Zhang, Zhonglin

    2013-01-01

    Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

  1. Identification of Adiponectin Receptor Agonist Utilizing a Fluorescence Polarization Based High Throughput Assay

    PubMed Central

    Sun, Yiyi; Zang, Zhihe; Zhong, Ling; Wu, Min; Su, Qing; Gao, Xiurong; Zan, Wang; Lin, Dong; Zhao, Yan; Zhang, Zhonglin

    2013-01-01

    Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (-)-arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases. PMID:23691032

  2. NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening

    PubMed Central

    Boute, Nicolas; Lowe, Peter; Berger, Sven; Malissard, Martine; Robert, Alain; Tesar, Michael

    2016-01-01

    Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a “swiss army knife solution” for today and future high throughput antibody drug screenings. PMID:26924984

  3. NanoLuc Luciferase - A Multifunctional Tool for High Throughput Antibody Screening.

    PubMed

    Boute, Nicolas; Lowe, Peter; Berger, Sven; Malissard, Martine; Robert, Alain; Tesar, Michael

    2016-01-01

    Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a "swiss army knife solution" for today and future high throughput antibody drug screenings. PMID:26924984

  4. High-throughput screening of binary catalysts for oxygen electroreduction

    NASA Astrophysics Data System (ADS)

    Liu, Jing Hua; Jeon, Min Ku; Woo, Seong Ihl

    2006-01-01

    A series of Pt based and non-Pt catalysts for proton exchange membrane fuel cell (PEMFC) and direct methanol fuel cell (DMFC) have been evaluated towards oxygen reduction, by high-throughput optical screening. Fluorescein was first used as pH indicator for detecting pH change of the electrolyte in the vicinity of cathode caused by oxygen reduction. Arrays of catalyst spot comprised of binary catalysts and pure Pt were prepared by using robotic micro-dispenser. The analysis of fluorescence images has showed that some of Pt based catalysts including PtBi, PtCu, PtSe, PtTe and PtIr, as well as RuFe, as a non-Pt catalyst, exhibited higher activities and methanol tolerance than pure Pt. Moreover, acceptable stability of these catalysts at high potential in acid environment suits them to the requirements of cathode catalyst in PEMFC or DMFC.

  5. Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System.

    PubMed

    Kim, Haseong; Kwon, Kil Koang; Seong, Wonjae; Lee, Seung-Goo

    2016-01-01

    The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library(1). We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification. PMID:27584951

  6. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    SciTech Connect

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  7. High-throughput Saccharification assay for lignocellulosic materials.

    PubMed

    Gomez, Leonardo D; Whitehead, Caragh; Roberts, Philip; McQueen-Mason, Simon J

    2011-01-01

    Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest (1). In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification (2). These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system. This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2

  8. A Quantitative High-Throughput Screening Data Analysis Pipeline for Activity Profiling.

    PubMed

    Huang, Ruili

    2016-01-01

    The US Tox21 program has developed in vitro assays to test large collections of environmental chemicals in a quantitative high-throughput screening (qHTS) format, using triplicate 15-dose titrations to generate over 50 million data points to date. Counter screens are also employed to minimize interferences from non-target-specific assay artifacts, such as compound auto fluorescence and cytotoxicity. New data analysis approaches are needed to integrate these data and characterize the activities observed from these assays. Here, we describe a complete analysis pipeline that evaluates these qHTS data for technical quality in terms of signal reproducibility. We integrate signals from repeated assay runs, primary readouts, and counter screens to produce a final call on on-target compound activity. PMID:27518629

  9. High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.

    PubMed

    Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Botana, Luis M

    2013-12-01

    The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL(-1). The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg(-1) of shellfish meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in shellfish meat in order to reduce the number of samples to be processed through bioassays or analytical methods. PMID:23827412

  10. High-throughput screening of solid-state catalyst libraries

    NASA Astrophysics Data System (ADS)

    Senkan, Selim M.

    1998-07-01

    Combinatorial synthesis methods allow the rapid preparation and processing of large libraries of solid-state materials. The use of these methods, together with the appropriate screening techniques, has recently led to the discovery of materials with promising superconducting, magnetoresistive, luminescent and dielectric properties. Solid-state catalysts, which play an increasingly important role in the chemical and oil industries, represent another class of material amenable to combinatorial synthesis. Yet typically, catalyst discovery still involves inefficient trial-and-error processes, because catalytic activity is inherently difficult to screen. In contrast to superconductivity, magnetoresistivity and dielectric properties, which can be tested by contact probes, or luminescence, which can be observed directly, the assessment of catalytic activity requires the unambiguous detection of a specific product molecule above a small catalyst site on a large library. Screening by in situ infrared thermography and microprobe sampling mass spectrometry, have been suggested, but the first method, while probing activity, provides no information on reaction products, whereas the second is difficult to implement because it requires the transport of minute gas samples from each library site to the detection system. Here I describe the use of laser-induced resonance-enhanced multiphoton ionization for sensitive, selective and high-throughput screening of a library of solid-state catalysts that activate the dehydrogenation of cyclohexane to benzene. I show that benzene, the product molecule, can be selectively photoionized in the vicinity of the catalytic sites, and that the detection of the resultant photoions by an array of microelectrodes provides information on the activity of individual sites. Adaptation of this technique for the screening of other catalytic reactions and larger libraries with smaller site size seems feasible, thus opening up the possibility of exploiting

  11. High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility

    PubMed Central

    Herington, Jennifer L.; Swale, Daniel R.; Brown, Naoko; Shelton, Elaine L.; Choi, Hyehun; Williams, Charles H.; Hong, Charles C.; Paria, Bibhash C.; Denton, Jerod S.; Reese, Jeff

    2015-01-01

    The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility. PMID:26600013

  12. Silicon microphysiometer for high-throughput drug screening

    NASA Astrophysics Data System (ADS)

    Verhaegen, Katarina; Baert, Christiaan; Puers, Bob; Sansen, Willy; Simaels, Jeannine; Van Driessche, Veerle; Hermans, Lou; Mertens, Robert P.

    1999-06-01

    We report on a micromachined silicon chip that is capable of providing a high-throughput functional assay based on calorimetry. A prototype twin microcalorimeter based on the Seebeck effect has been fabricated by IC technology and micromachined postprocessing techniques. A biocompatible liquid rubber membrane supports two identical 0.5 X 2 cm2 measurement chambers, situated at the cold and hot junction of a 666-junction aluminum/p+-polysilicon thermopile. The chambers can house up to 106 eukaryotic cells cultured to confluence. The advantage of the device over microcalorimeters on the market, is the integration of the measurement channels on chip, rendering microvolume reaction vessels, ranging from 10 to 600 (mu) l, in the closest possible contact with the thermopile sensor (no springs are needed). Power and temperature sensitivity of the sensor are 23 V/W and 130 mV/K, respectively. The small thermal inertia of the microchannels results in the short response time of 70 s, when filled with 50 (mu) l of water. Biological experiments were done with cultured kidney cells of Xenopus laevis (A6). The thermal equilibration time of the device is 45 min. Stimulation of transport mechanisms by reducing bath osmolality by 50% increased metabolism by 20%. Our results show that it is feasible to apply this large-area, small- volume whole-cell biosensor for drug discovery, where the binding assays that are commonly used to provide high- throughput need to be complemented with a functional assay. Solutions are brought onto the sensor by a simple pipette, making the use of an industrial microtiterplate dispenser feasible on a nx96-array of the microcalorimeter biosensor. Such an array of biosensors has been designed based on a new set of requirements as set forth by people in the field as this project moved on. The results obtained from the prototype large-area sensor were used to obtain an accurate model of the calorimeter, checked for by the simulation software ANSYS. At

  13. High-throughput optical screening of cellular mechanotransduction

    NASA Astrophysics Data System (ADS)

    Compton, Jonathan L.; Luo, Justin C.; Ma, Huan; Botvinick, Elliot; Venugopalan, Vasan

    2014-09-01

    We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated microcavitation bubbles without requiring flow chambers or microfluidics. These microcavitation bubbles expose adherent cells to a microtsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1 mm2. We demonstrate microtsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation, resulting in Ca2+ release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microtsunami-induced Ca2+ signalling by introducing a known inhibitor to this pathway. The imaging of Ca2+ signalling and its modulation by exogenous molecules demonstrates the capacity to initiate and assess cellular mechanosignalling in real time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry.

  14. High-throughput optical screening of cellular mechanotransduction.

    PubMed

    Compton, Jonathan L; Luo, Justin C; Ma, Huan; Botvinick, Elliot; Venugopalan, Vasan

    2014-09-01

    We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated micro-cavitation bubbles (μCBs) without requiring flow chambers or microfluidics. These μCBs expose adherent cells to a microTsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1mm(2). We demonstrate microTsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation resulting in Ca(2+) release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microTsunami-induced Ca(2+) signalling by introducing a known inhibitor to this pathway. The imaging of Ca(2+) signalling, and its modulation by exogenous molecules, demonstrates the capacity to initiate and assess cellular mechanosignalling in real-time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry. PMID:25309621

  15. High-throughput optical screening of cellular mechanotransduction

    PubMed Central

    Compton, Jonathan L.; Luo, Justin C.; Ma, Huan; Botvinick, Elliot; Venugopalan, Vasan

    2014-01-01

    We introduce an optical platform for rapid, high-throughput screening of exogenous molecules that affect cellular mechanotransduction. Our method initiates mechanotransduction in adherent cells using single laser-microbeam generated micro-cavitation bubbles (μCBs) without requiring flow chambers or microfluidics. These μCBs expose adherent cells to a microTsunami, a transient microscale burst of hydrodynamic shear stress, which stimulates cells over areas approaching 1mm2. We demonstrate microTsunami-initiated mechanosignalling in primary human endothelial cells. This observed signalling is consistent with G-protein-coupled receptor stimulation resulting in Ca2+ release by the endoplasmic reticulum. Moreover, we demonstrate the dose-dependent modulation of microTsunami-induced Ca2+ signalling by introducing a known inhibitor to this pathway. The imaging of Ca2+ signalling, and its modulation by exogenous molecules, demonstrates the capacity to initiate and assess cellular mechanosignalling in real-time. We utilize this capability to screen the effects of a set of small molecules on cellular mechanotransduction in 96-well plates using standard imaging cytometry. PMID:25309621

  16. Towards Prebiotic Catalytic Amyloids Using High Throughput Screening

    PubMed Central

    Friedmann, Michael P.; Torbeev, Vladimir; Zelenay, Viviane; Sobol, Alexander; Greenwald, Jason; Riek, Roland

    2015-01-01

    Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-β-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications. PMID:26650386

  17. Filtration improves the performance of a high-throughput screen for anti-mycobacterial compounds.

    PubMed

    Cheng, Nancy; Porter, Melissa A; Frick, Lloyd W; Nguyen, Yvonne; Hayden, Jennifer D; Young, Ellen F; Braunstein, Miriam S; Hull-Ryde, Emily A; Janzen, William P

    2014-01-01

    The tendency for mycobacteria to aggregate poses a challenge for their use in microplate based assays. Good dispersions have been difficult to achieve in high-throughput screening (HTS) assays used in the search for novel antibacterial drugs to treat tuberculosis and other related diseases. Here we describe a method using filtration to overcome the problem of variability resulting from aggregation of mycobacteria. This method consistently yielded higher reproducibility and lower variability than conventional methods, such as settling under gravity and vortexing. PMID:24788852

  18. Institutional Profile: The Sheffield RNAi screening facility: a service for high-throughput, genome-wide Drosophila RNAi screens.

    PubMed

    Brown, Stephen

    2010-12-01

    The Sheffield RNAi Screening Facility (SRSF) was established in November 2008, as Britain's first Drosophila RNAi screening centre, funded by the University of Sheffield, Biomedical Sciences Department and the Wellcome Trust. The SRSF was formed to service the needs of research groups wanting to carry out high-throughput RNAi screens with Drosophila cells. The rationale for the SRSF is to provide RNAi libraries and the specialist equipment and expertise to do such screens. The facility supports both plate reader assays, high-content microscopy as well as the equipment needed to process these samples in a high-throughput fashion. The SRSF can either be used to identify genes involved in disease representing future drug targets, or to identify genes involved in drug resistance and efficacy. PMID:21428803

  19. High throughput screening of electrocatalysts for fuel cell applications

    NASA Astrophysics Data System (ADS)

    Prochaska, Mark; Jin, Jing; Rochefort, Dominic; Zhuang, Lin; DiSalvo, Francis J.; Abruña, Héctor D.; van Dover, R. B.

    2006-05-01

    We describe methodologies for the generation and screening of combinatorial libraries of electrocatalyst materials for fuel cell applications, generated by cosputtering of three elements onto a Si substrate coated with a Ta adhesion underlayer. Screening was carried out via a fluorescence assay as well as by scanning electrochemical microscopy. Whereas the former provided rapid qualitative screening with limited spatial resolution, the latter provided high spatial resolution. The fluorescence screening method was tested on Pt, PtBi, PtPb, and PtRu nanoparticles, while both methods were tested on a film containing a Pt-Bi-Pb ternary composition spread.

  20. High throughput assay of diffusion through Cx43 gap junction channels with a microfluidic chip.

    PubMed

    Bathany, Cédric; Beahm, Derek; Felske, James D; Sachs, Frederick; Hua, Susan Z

    2011-02-01

    This paper describes a microfluidic-based assay capable of measuring gap-junction mediated dye diffusion in cultured cells. The technique exploits multistream laminar flow to selectively expose cells to different environments, enabling continuous loading of cells in one compartment while monitoring, in real time, dye diffusion into cells of a neighboring compartment. A simple one-dimensional diffusion model fit to the data extracted the diffusion coefficient of four different dyes, 5-(6)-carboxyfluorescein, 5-chloromethylfluorescein, Oregon green 488 carboxylic acid, and calcein. Different inhibitors were assayed for their ability to reduce dye coupling. The chip can screen multiple inhibitors in parallel in the same cell preparation, demonstrating its potential for high throughput. The technique provides a convenient method to measure gap junction mediated diffusion and a screen for drugs that affect gap junction communication. PMID:21182279

  1. Towards high throughput screening of electrochemical stability of battery electrolytes.

    PubMed

    Borodin, Oleg; Olguin, Marco; Spear, Carrie E; Leiter, Kenneth W; Knap, Jaroslaw

    2015-09-01

    High throughput screening of solvents and additives with potential applications in lithium batteries is reported. The initial test set is limited to carbonate and phosphate-based compounds and focused on their electrochemical properties. Solvent stability towards first and second reduction and oxidation is reported from density functional theory (DFT) calculations performed on isolated solvents surrounded by implicit solvent. The reorganization energy is estimated from the difference between vertical and adiabatic redox energies and found to be especially important for the accurate prediction of reduction stability. A majority of tested compounds had the second reduction potential higher than the first reduction potential indicating that the second reduction reaction might play an important role in the passivation layer formation. Similarly, the second oxidation potential was smaller for a significant subset of tested molecules than the first oxidation potential. A number of potential sources of errors introduced during screening of the electrolyte electrochemical properties were examined. The formation of lithium fluoride during reduction of semifluorinated solvents such as fluoroethylene carbonate and the H-transfer during oxidation of solvents were found to shift the electrochemical potential by 1.5-2 V and could shrink the electrochemical stability window by as much as 3.5 V when such reactions are included in the screening procedure. The initial oxidation reaction of ethylene carbonate and dimethyl carbonate at the surface of the completely de-lithiated LiNi0.5Mn1.5O4 high voltage spinel cathode was examined using DFT. Depending on the molecular orientation at the cathode surface, a carbonate molecule either exhibited deprotonation or was found bound to the transition metal via its carbonyl oxygen. PMID:26266636

  2. Towards high throughput screening of electrochemical stability of battery electrolytes

    NASA Astrophysics Data System (ADS)

    Borodin, Oleg; Olguin, Marco; Spear, Carrie E.; Leiter, Kenneth W.; Knap, Jaroslaw

    2015-09-01

    High throughput screening of solvents and additives with potential applications in lithium batteries is reported. The initial test set is limited to carbonate and phosphate-based compounds and focused on their electrochemical properties. Solvent stability towards first and second reduction and oxidation is reported from density functional theory (DFT) calculations performed on isolated solvents surrounded by implicit solvent. The reorganization energy is estimated from the difference between vertical and adiabatic redox energies and found to be especially important for the accurate prediction of reduction stability. A majority of tested compounds had the second reduction potential higher than the first reduction potential indicating that the second reduction reaction might play an important role in the passivation layer formation. Similarly, the second oxidation potential was smaller for a significant subset of tested molecules than the first oxidation potential. A number of potential sources of errors introduced during screening of the electrolyte electrochemical properties were examined. The formation of lithium fluoride during reduction of semifluorinated solvents such as fluoroethylene carbonate and the H-transfer during oxidation of solvents were found to shift the electrochemical potential by 1.5-2 V and could shrink the electrochemical stability window by as much as 3.5 V when such reactions are included in the screening procedure. The initial oxidation reaction of ethylene carbonate and dimethyl carbonate at the surface of the completely de-lithiated LiNi0.5Mn1.5O4 high voltage spinel cathode was examined using DFT. Depending on the molecular orientation at the cathode surface, a carbonate molecule either exhibited deprotonation or was found bound to the transition metal via its carbonyl oxygen.

  3. Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

    PubMed

    Camattari, Andrea; Weinhandl, Katrin; Gudiminchi, Rama K

    2014-01-01

    The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant. PMID:24744029

  4. High-throughput microtitre plate-based assay for DNA topoisomerases.

    PubMed

    Taylor, James A; Burton, Nicolas P; Maxwell, Anthony

    2012-01-01

    We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of DNA topoisomerases. The assay utilizes intermolecular triplex formation between an immobilized triplex-forming oligo (TFO) and a triplex-forming region inserted into the plasmid substrate (pNO1), and capitalizes on the observation that supercoiled DNA forms triplexes more readily than relaxed DNA. Thus, supercoiled DNA is preferentially retained by the TFO under triplex-forming conditions while relaxed DNA can be washed away. Due to its high speed of sample analysis and reduced sample handling over conventional gel-based techniques, this assay can be used to screen chemical libraries for novel inhibitors of topoisomerases. PMID:22130995

  5. A high throughput screening for rarely transcribed differentially expressed genes.

    PubMed Central

    von Stein, O D; Thies, W G; Hofmann, M

    1997-01-01

    A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined. PMID:9185570

  6. Advances in High Throughput Screening of Biomass Recalcitrance (Poster)

    SciTech Connect

    Turner, G. B.; Decker, S. R.; Tucker, M. P.; Law, C.; Doeppke, C.; Sykes, R. W.; Davis, M. F.; Ziebell, A.

    2012-06-01

    This was a poster displayed at the Symposium. Advances on previous high throughput screening of biomass recalcitrance methods have resulted in improved conversion and replicate precision. Changes in plate reactor metallurgy, improved preparation of control biomass, species-specific pretreatment conditions, and enzymatic hydrolysis parameters have reduced overall coefficients of variation to an average of 6% for sample replicates. These method changes have improved plate-to-plate variation of control biomass recalcitrance and improved confidence in sugar release differences between samples. With smaller errors plant researchers can have a higher degree of assurance more low recalcitrance candidates can be identified. Significant changes in plate reactor, control biomass preparation, pretreatment conditions and enzyme have significantly reduced sample and control replicate variability. Reactor plate metallurgy significantly impacts sugar release aluminum leaching into reaction during pretreatment degrades sugars and inhibits enzyme activity. Removal of starch and extractives significantly decreases control biomass variability. New enzyme formulations give more consistent and higher conversion levels, however required re-optimization for switchgrass. Pretreatment time and temperature (severity) should be adjusted to specific biomass types i.e. woody vs. herbaceous. Desalting of enzyme preps to remove low molecular weight stabilizers and improved conversion levels likely due to water activity impacts on enzyme structure and substrate interactions not attempted here due to need to continually desalt and validate precise enzyme concentration and activity.

  7. Adaptation to high throughput batch chromatography enhances multivariate screening.

    PubMed

    Barker, Gregory A; Calzada, Joseph; Herzer, Sibylle; Rieble, Siegfried

    2015-09-01

    High throughput process development offers unique approaches to explore complex process design spaces with relatively low material consumption. Batch chromatography is one technique that can be used to screen chromatographic conditions in a 96-well plate. Typical batch chromatography workflows examine variations in buffer conditions or comparison of multiple resins in a given process, as opposed to the assessment of protein loading conditions in combination with other factors. A modification to the batch chromatography paradigm is described here where experimental planning, programming, and a staggered loading approach increase the multivariate space that can be explored with a liquid handling system. The iterative batch chromatography (IBC) approach is described, which treats every well in a 96-well plate as an individual experiment, wherein protein loading conditions can be varied alongside other factors such as wash and elution buffer conditions. As all of these factors are explored in the same experiment, the interactions between them are characterized and the number of follow-up confirmatory experiments is reduced. This in turn improves statistical power and throughput. Two examples of the IBC method are shown and the impact of the load conditions are assessed in combination with the other factors explored. PMID:25914370

  8. High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei

    PubMed Central

    MacGregor, Paula; Ivens, Alasdair; Shave, Steven; Collie, Iain; Gray, David; Auer, Manfred

    2014-01-01

    In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite's infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo. Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes. PMID:24442893

  9. A comprehensive statistical analysis of predicting in vivo hazard using high-throughput in vitro screening.

    PubMed

    Thomas, Russell S; Black, Michael B; Li, Lili; Healy, Eric; Chu, Tzu-Ming; Bao, Wenjun; Andersen, Melvin E; Wolfinger, Russell D

    2012-08-01

    Over the past 5 years, increased attention has been focused on using high-throughput in vitro screening for identifying chemical hazards and prioritizing chemicals for additional in vivo testing. The U.S. Environmental Protection Agency's ToxCast program has generated a significant amount of high-throughput screening data allowing a broad-based assessment of the utility of these assays for predicting in vivo responses. In this study, a comprehensive cross-validation model comparison was performed to evaluate the predictive performance of the more than 600 in vitro assays from the ToxCast phase I screening effort across 60 in vivo endpoints using 84 different statistical classification methods. The predictive performance of the in vitro assays was compared and combined with that from chemical structure descriptors. With the exception of chronic in vivo cholinesterase inhibition, the overall predictive power of both the in vitro assays and the chemical descriptors was relatively low. The predictive power of the in vitro assays was not significantly different from that of the chemical descriptors and aggregating the assays based on genes reduced predictive performance. Prefiltering the in vitro assay data outside the cross-validation loop, as done in some previous studies, significantly biased estimates of model performance. The results suggest that the current ToxCast phase I assays and chemicals have limited applicability for predicting in vivo chemical hazards using standard statistical classification methods. However, if viewed as a survey of potential molecular initiating events and interpreted as risk factors for toxicity, the assays may still be useful for chemical prioritization. PMID:22543276

  10. High Throughput Screening for Drugs that Modulate Intermediate Filament Proteins

    PubMed Central

    Sun, Jingyuan; Groppi, Vincent E.; Gui, Honglian; Chen, Lu; Xie, Qing; Liu, Li

    2016-01-01

    Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green-fluorescent-protein-tagged keratin 18 (K18) R90C in A549 cells. The readout is drug ‘hits’ that convert the dot-like keratin filament distribution, due to the R90C mutation, to a wildtype-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. ‘Hits’ of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant-IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients. PMID:26795471

  11. Hypoxia-sensitive reporter system for high-throughput screening.

    PubMed

    Tsujita, Tadayuki; Kawaguchi, Shin-ichi; Dan, Takashi; Baird, Liam; Miyata, Toshio; Yamamoto, Masayuki

    2015-01-01

    The induction of anti-hypoxic stress enzymes and proteins has the potential to be a potent therapeutic strategy to prevent the progression of ischemic heart, kidney or brain diseases. To realize this idea, small chemical compounds, which mimic hypoxic conditions by activating the PHD-HIF-α system, have been developed. However, to date, none of these compounds were identified by monitoring the transcriptional activation of hypoxia-inducible factors (HIFs). Thus, to facilitate the discovery of potent inducers of HIF-α, we have developed an effective high-throughput screening (HTS) system to directly monitor the output of HIF-α transcription. We generated a HIF-α-dependent reporter system that responds to hypoxic stimuli in a concentration- and time-dependent manner. This system was developed through multiple optimization steps, resulting in the generation of a construct that consists of the secretion-type luciferase gene (Metridia luciferase, MLuc) under the transcriptional regulation of an enhancer containing 7 copies of 40-bp hypoxia responsive element (HRE) upstream of a mini-TATA promoter. This construct was stably integrated into the human neuroblastoma cell line, SK-N-BE(2)c, to generate a reporter system, named SKN:HRE-MLuc. To improve this system and to increase its suitability for the HTS platform, we incorporated the next generation luciferase, Nano luciferase (NLuc), whose longer half-life provides us with flexibility for the use of this reporter. We thus generated a stably transformed clone with NLuc, named SKN:HRE-NLuc, and found that it showed significantly improved reporter activity compared to SKN:HRE-MLuc. In this study, we have successfully developed the SKN:HRE-NLuc screening system as an efficient platform for future HTS. PMID:25746387

  12. High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

    PubMed Central

    Wu, Jing; Pugh, Roberta; Laughlin, Richard C.; Andrews-Polymenis, Helene; McClelland, Michael; Bäumler, Andreas J.; Adams, L. Garry

    2014-01-01

    Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions. PMID:25146526

  13. Adapting the medaka embryo assay to a high-throughput approach for developmental toxicity testing.

    PubMed

    Oxendine, Sharon L; Cowden, John; Hinton, David E; Padilla, Stephanie

    2006-09-01

    Chemical exposure during embryonic development may cause persistent effects, yet developmental toxicity data exist for very few chemicals. Current testing procedures are time consuming and costly, underlining the need for rapid and low cost screening strategies. While in vitro methods are useful for screening, these methods do not replicate all the intricacies of embryonic development and should ideally be complemented by an in vivo screening strategy. In this study, we modify a medaka fish embryo assay to meet the requirements of high-throughput, developmental toxicant testing in vivo. The Japanese medaka (Oryzias latipes) offers several advantages over traditional mammalian model systems, including economic husbandry, high fecundity, and rapid ex utero development. In most studies where fish eggs are exposed to a chemical, the exposure takes place in a common vessel, with many embryos being exposed to the same solution. This type of design is not amenable to high-throughput methodology, does not allow the investigator to follow the same embryo throughout gestation, and may confound statistical analysis of the results. Therefore, we developed a 96-well microtiter plate method to facilitate exposure of individual medaka embryos in single wells and compared this approach to the common vessel method using the industrial solvent dimethyl sulfoxide (DMSO) as the test compound. At lower DMSO concentrations (0% or 1%), the 96-well microtiter plate assay replicated results obtained using the common vessel exposure method. There was, however, increased lethality and decreased hatching rate in the bottle-reared embryos treated with the higher DMSO concentrations (5% or 10%). Because the embryos reared in the 96-well microtiter plates never showed increased adverse effects (as compared to the bottle-reared embryos) at any DMSO concentration, we conclude that the 96-well microtiter plate assay provides a rapid and efficient alternative for developmental toxicity screens that

  14. A Microfluidic Platform for High-Throughput Screening of Small Mutant Libraries.

    PubMed

    Lim, Ji Won; Shin, Kwang Soo; Moon, Jaemin; Lee, Sung Kuk; Kim, Taesung

    2016-05-17

    The screening and isolation of target microorganisms from mutated recombinant libraries are crucial for the advancement of synthetic biology and metabolic engineering. However, conventional screening tools present several limitations in throughput, cost, and labor. Herein, we describe a novel microfluidic high-throughput screening (HTS) platform with several advantages. The platform utilizes a fluid array to compartmentalize bacterial cells in well-ordered separated microwells and allows long-term cell culture with high throughput. The platform enables the extraction of selected target cells from the fluid array for additional culture and postanalysis by using a capillary-driven sample relocation method. To confirm the feasibility of the platform, we demonstrated two different types of HTS methods based on the levels of reporter gene expression and cellular growth rate difference. For the reporter gene-based HTS, a spike recovery approach was taken to demonstrate that target cells are successfully screened out from a mixture containing nontarget cells by repeating the culture and extraction processes. Additionally, the same platform allowed us to screen and sort target cells according to their cellular growth rate difference, which seems hard in conventional screening methods. Hence, the platform could be used for various microbiological assays, including the detection of cell-excreted metabolites, microbial biosensors, and other HTS systems. PMID:27104360

  15. A human cDNA library for high-throughput protein expression screening.

    PubMed

    Büssow, K; Nordhoff, E; Lübbert, C; Lehrach, H; Walter, G

    2000-04-01

    We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses. PMID:10777659

  16. Analysis of image-based phenotypic parameters for high throughput gene perturbation assays.

    PubMed

    Song, Mee; Jeong, Euna; Lee, Tae-Kyu; Tsoy, Yury; Kwon, Yong-Jun; Yoon, Sukjoon

    2015-10-01

    Although image-based phenotypic assays are considered a powerful tool for siRNA library screening, the reproducibility and biological implications of various image-based assays are not well-characterized in a systematic manner. Here, we compared the resolution of high throughput assays of image-based cell count and typical cell viability measures for cancer samples. It was found that the optimal plating density of cells was important to obtain maximal resolution in both types of assays. In general, cell counting provided better resolution than the cell viability measure in diverse batches of siRNAs. In addition to cell count, diverse image-based measures were simultaneously collected from a single screening and showed good reproducibility in repetitions. They were classified into a few functional categories according to biological process, based on the differential patterns of hit (i.e., siRNAs) prioritization from the same screening data. The presented systematic analyses of image-based parameters provide new insight to a multitude of applications and better biological interpretation of high content cell-based assays. PMID:26256799

  17. Sensitive high-throughput screening for the detection of reducing sugars.

    PubMed

    Mellitzer, Andrea; Glieder, Anton; Weis, Roland; Reisinger, Christoph; Flicker, Karlheinz

    2012-01-01

    The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2. PMID:21538898

  18. High-throughput assay and engineering of self-cleaving ribozymes by sequencing

    PubMed Central

    Kobori, Shungo; Nomura, Yoko; Miu, Anh; Yokobayashi, Yohei

    2015-01-01

    Self-cleaving ribozymes are found in all domains of life and are believed to play important roles in biology. Additionally, self-cleaving ribozymes have been the subject of extensive engineering efforts for applications in synthetic biology. These studies often involve laborious assays of multiple individual variants that are either designed rationally or discovered through selection or screening. However, these assays provide only a limited view of the large sequence space relevant to the ribozyme function. Here, we report a strategy that allows quantitative characterization of greater than 1000 ribozyme variants in a single experiment. We generated a library of predefined ribozyme variants that were converted to DNA and analyzed by high-throughput sequencing. By counting the number of cleaved and uncleaved reads of every variant in the library, we obtained a complete activity profile of the ribozyme pool which was used to both analyze and engineer allosteric ribozymes. PMID:25829176

  19. Testing Tuberculosis Drug Efficacy in a Zebrafish High-Throughput Translational Medicine Screen

    PubMed Central

    Ordas, Anita; Raterink, Robert-Jan; Cunningham, Fraser; Jansen, Hans J.; Wiweger, Malgorzata I.; Jong-Raadsen, Susanne; Bos, Sabine; Bates, Robert H.; Barros, David; Meijer, Annemarie H.; Vreeken, Rob J.; Ballell-Pages, Lluís; Dirks, Ron P.

    2014-01-01

    The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans. PMID:25385118

  20. High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions

    PubMed Central

    2015-01-01

    We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch–ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets. PMID:25233437

  1. High throughput kinetic Vibrio fischeri bioluminescence inhibition assay for study of toxic effects of nanoparticles.

    PubMed

    Mortimer, M; Kasemets, K; Heinlaan, M; Kurvet, I; Kahru, A

    2008-08-01

    Despite of the growing production and use of nanoparticles (NPs) in various applications, current regulations, including EC new chemical policy REACH, fail to address the environmental, health, and safety risks posed by NPs. This paper shows that kinetic Vibrio fischeri luminescence inhibition test--Flash Assay--that up to now was mainly used for toxicity analysis of solid and colored environmental samples (e.g. sediments, soil suspensions), is a powerful tool for screening the toxic properties of NPs. To demonstrate that Flash Assay (initially designed for a tube luminometer) can also be adapted to a microplate format for high throughput toxicity screening of NPs, altogether 11 chemicals were comparatively analyzed. The studied chemicals included bulk and nanosized CuO and ZnO, polyethylenimine (PEI) and polyamidoamine dendrimer generations 2 and 5 (PAMAM G2 and G5). The results showed that EC50 values of 30-min Flash Assay in tube and microplate formats were practically similar and correlated very well (log-logR2=0.98), classifying all analyzed chemicals, except nano CuO (that was more toxic in cuvette format), analogously when compared to the risk phrases of the EC Directive 93/67/EEC for ranking toxicity of chemicals for aquatic organisms. The 30-min EC50 values of nanoscale organic cationic polymers (PEI and dendrimers) ranged from 215 to 775 mg/l. Thirty-minute EC50 values of metal oxides varied largely, ranging from approximately 4 mg/l (bulk and nano ZnO) to approximately 100 mg/l (nano CuO) and approximately 4000 mg/l (bulk CuO). Thus, considering an excellent correlation between both formats, 96-well microplate Flash Assay can be successfully used for high throughput evaluation of harmful properties of chemicals (including organic and inorganic NPs) to bacteria. PMID:18400463

  2. New Compound Sets Identified from High Throughput Phenotypic Screening Against Three Kinetoplastid Parasites: An Open Resource

    PubMed Central

    Peña, Imanol; Pilar Manzano, M.; Cantizani, Juan; Kessler, Albane; Alonso-Padilla, Julio; Bardera, Ana I.; Alvarez, Emilio; Colmenarejo, Gonzalo; Cotillo, Ignacio; Roquero, Irene; de Dios-Anton, Francisco; Barroso, Vanessa; Rodriguez, Ana; Gray, David W.; Navarro, Miguel; Kumar, Vinod; Sherstnev, Alexander; Drewry, David H.; Brown, James R.; Fiandor, Jose M.; Julio Martin, J.

    2015-01-01

    Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host–pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions. PMID:25740547

  3. A Novel High-Throughput 1536-well Notch1 γ-Secretase AlphaLISA Assay

    PubMed Central

    Chau, De-ming; Shum, David; Radu, Constantin; Bhinder, Bhavneet; Gin, David; Gilchrist, M. Lane; Djaballah, Hakim; Li, Yue-Ming

    2013-01-01

    The Notch pathway plays a crucial role in cell fate decisions through controlling various cellular processes. Overactive Notch signal contributes to cancer development from leukemias to solid tumors. γ-Secretase is an intramembrane protease responsible for the final proteolytic step of Notch that releases the membrane-tethered Notch fragment for signaling. Therefore, γ-secretase is an attractive drug target in treating Notch-mediated cancers. However, the absence of high-throughput γ-secretase assay using Notch substrate has limited the identification and development of γ-secretase inhibitors that specifically target the Notch signaling pathway. Here, we report on the development of a 1536-well γ-secretase assay using a biotinylated recombinant Notch1 substrate. We effectively assimilated and miniaturized this newly developed Notch1 substrate with the AlphaLISA detection technology and demonstrated its robustness with a calculated Z’ score of 0.66. We further validated this optimized assay by performing a pilot screening against a chemical library consisting of ~5,600 chemicals and identified known γ-secretase inhibitors e.g. DAPT, and Calpeptin; as well as a novel γ-secretase inhibitor referred to as KD-I-085. This assay is the first reported 1536-well AlphaLISA format and represents a novel high-throughput Notch1-γ-secretase assay, which provides an unprecedented opportunity to discover Notch-selective γ-secretase inhibitors that can be potentially used for the treatment of cancer and other human disorders. PMID:23448293

  4. A quantitative and high-throughput assay of human papillomavirus DNA replication.

    PubMed

    Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

    2015-01-01

    Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof. PMID:25348316

  5. High throughput assay for evaluation of reactive carbonyl scavenging capacity☆

    PubMed Central

    Vidal, N.; Cavaille, J.P.; Graziani, F.; Robin, M.; Ouari, O.; Pietri, S.; Stocker, P.

    2014-01-01

    Many carbonyl species from either lipid peroxidation or glycoxidation are extremely reactive and can disrupt the function of proteins and enzymes. 4-hydroxynonenal and methylglyoxal are the most abundant and toxic lipid-derived reactive carbonyl species. The presence of these toxics leads to carbonyl stress and cause a significant amount of macromolecular damages in several diseases. Much evidence indicates trapping of reactive carbonyl intermediates may be a useful strategy for inhibiting or decreasing carbonyl stress-associated pathologies. There is no rapid and convenient analytical method available for the assessment of direct carbonyl scavenging capacity, and a very limited number of carbonyl scavengers have been identified to date, their therapeutic potential being highlighted only recently. In this context, we have developed a new and rapid sensitive fluorimetric method for the assessment of reactive carbonyl scavengers without involvement glycoxidation systems. Efficacy of various thiol- and non-thiol-carbonyl scavenger pharmacophores was tested both using this screening assay adapted to 96-well microplates and in cultured cells. The scavenging effects on the formation of Advanced Glycation End-product of Bovine Serum Albumin formed with methylglyoxal, 4-hydroxynonenal and glucose-glycated as molecular models were also examined. Low molecular mass thiols with an α-amino-β-mercaptoethane structure showed the highest degree of inhibitory activity toward both α,β-unsaturated aldehydes and dicarbonyls. Cysteine and cysteamine have the best scavenging ability toward methylglyoxal. WR-1065 which is currently approved for clinical use as a protective agent against radiation and renal toxicity was identified as the best inhibitor of 4-hydroxynonenal. PMID:24688895

  6. An Automatic Quality Control Pipeline for High-Throughput Screening Hit Identification.

    PubMed

    Zhai, Yufeng; Chen, Kaisheng; Zhong, Yang; Zhou, Bin; Ainscow, Edward; Wu, Ying-Ta; Zhou, Yingyao

    2016-09-01

    The correction or removal of signal errors in high-throughput screening (HTS) data is critical to the identification of high-quality lead candidates. Although a number of strategies have been previously developed to correct systematic errors and to remove screening artifacts, they are not universally effective and still require fair amount of human intervention. We introduce a fully automated quality control (QC) pipeline that can correct generic interplate systematic errors and remove intraplate random artifacts. The new pipeline was first applied to ~100 large-scale historical HTS assays; in silico analysis showed auto-QC led to a noticeably stronger structure-activity relationship. The method was further tested in several independent HTS runs, where QC results were sampled for experimental validation. Significantly increased hit confirmation rates were obtained after the QC steps, confirming that the proposed method was effective in enriching true-positive hits. An implementation of the algorithm is available to the screening community. PMID:27313114

  7. Current in vitro high throughput screening approaches to assess nuclear receptor activation.

    PubMed

    Raucy, Judy L; Lasker, Jerome M

    2010-11-01

    The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluorescently-labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient- and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening. PMID:21189134

  8. High-Throughput Giardia lamblia Viability Assay Using Bioluminescent ATP Content Measurements▿

    PubMed Central

    Chen, Catherine Z.; Kulakova, Liudmila; Southall, Noel; Marugan, Juan J.; Galkin, Andrey; Austin, Christopher P.; Herzberg, Osnat; Zheng, Wei

    2011-01-01

    The human pathogen Giardia lamblia is an anaerobic protozoan parasite that causes giardiasis, one of the most common diarrheal diseases worldwide. Although several drugs are available for the treatment of giardiasis, drug resistance has been reported and is likely to increase, and recurrent infections are common. The search for new drugs that can overcome the drug-resistant strains of Giardia is an unmet medical need. New drug screen methods can facilitate the drug discovery process and aid with the identification of new drug targets. Using a bioluminescent ATP content assay, we have developed a phenotypic drug screen method to identify compounds that act against the actively growing trophozoite stage of the parasite. This assay is homogeneous, robust, and suitable for high-throughput screening of large compound collections. A screen of 4,096 pharmacologically active small molecules and approved drugs revealed 43 compounds with selective anti-Giardia properties, including 32 previously reported and 11 novel anti-Giardia agents. The most potent novel compound was fumagillin, which showed 50% inhibitory concentrations of 10 nM against the WB isolate and 2 nM against the GS isolate. PMID:21078930

  9. High Throughput Screen Identifies Small Molecule Inhibitors Specific for Mycobacterium tuberculosis Phosphoserine Phosphatase*

    PubMed Central

    Arora, Garima; Tiwari, Prabhakar; Mandal, Rahul Shubhra; Gupta, Arpit; Sharma, Deepak; Saha, Sudipto; Singh, Ramandeep

    2014-01-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening. PMID:25037224

  10. High throughput screen identifies small molecule inhibitors specific for Mycobacterium tuberculosis phosphoserine phosphatase.

    PubMed

    Arora, Garima; Tiwari, Prabhakar; Mandal, Rahul Shubhra; Gupta, Arpit; Sharma, Deepak; Saha, Sudipto; Singh, Ramandeep

    2014-09-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis makes identification and validation of newer drug targets a global priority. Phosphoserine phosphatase (PSP), a key essential metabolic enzyme involved in conversion of O-phospho-l-serine to l-serine, was characterized in this study. The M. tuberculosis genome harbors all enzymes involved in l-serine biosynthesis including two PSP homologs: Rv0505c (SerB1) and Rv3042c (SerB2). In the present study, we have biochemically characterized SerB2 enzyme and developed malachite green-based high throughput assay system to identify SerB2 inhibitors. We have identified 10 compounds that were structurally different from known PSP inhibitors, and few of these scaffolds were highly specific in their ability to inhibit SerB2 enzyme, were noncytotoxic against mammalian cell lines, and inhibited M. tuberculosis growth in vitro. Surface plasmon resonance experiments demonstrated the relative binding for these inhibitors. The two best hits identified in our screen, clorobiocin and rosaniline, were bactericidal in activity and killed intracellular bacteria in a dose-dependent manner. We have also identified amino acid residues critical for these SerB2-small molecule interactions. This is the first study where we validate that M. tuberculosis SerB2 is a druggable and suitable target to pursue for further high throughput assay system screening. PMID:25037224

  11. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  12. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

    PubMed Central

    Fox, Jennifer T.; Sakamuru, Srilatha; Huang, Ruili; Teneva, Nedelina; Simmons, Steven O.; Xia, Menghang; Tice, Raymond R.; Austin, Christopher P.; Myung, Kyungjae

    2012-01-01

    Human ATAD5 is a biomarker for identifying genotoxic compounds because ATAD5 protein levels increase posttranscriptionally in response to DNA damage. We screened over 4,000 compounds with a cell-based quantitative high-throughput ATAD5-luciferase assay detecting genotoxic compounds. We identified 22 antioxidants, including resveratrol, genistein, and baicalein, that are currently used or investigated for the treatment of cardiovascular disease, type 2 diabetes, osteopenia, osteoporosis, and chronic hepatitis, as well as for antiaging. Treatment of dividing cells with these compounds induced DNA damage and resulted in cell death. Despite their genotoxic effects, resveratrol, genistein, and baicalein did not cause mutagenesis, which is a major side effect of conventional anticancer drugs. Furthermore, resveratrol and genistein killed multidrug-resistant cancer cells. We therefore propose that resveratrol, genistein, and baicalein are attractive candidates for improved chemotherapeutic agents. PMID:22431602

  13. High throughput screening of hydrolytic enzymes from termites using a natural substrate derived from sugarcane bagasse

    PubMed Central

    2011-01-01

    Background The description of new hydrolytic enzymes is an important step in the development of techniques which use lignocellulosic materials as a starting point for fuel production. Sugarcane bagasse, which is subjected to pre-treatment, hydrolysis and fermentation for the production of ethanol in several test refineries, is the most promising source of raw material for the production of second generation renewable fuels in Brazil. One problem when screening hydrolytic activities is that the activity against commercial substrates, such as carboxymethylcellulose, does not always correspond to the activity against the natural lignocellulosic material. Besides that, the macroscopic characteristics of the raw material, such as insolubility and heterogeneity, hinder its use for high throughput screenings. Results In this paper, we present the preparation of a colloidal suspension of particles obtained from sugarcane bagasse, with minimal chemical change in the lignocellulosic material, and demonstrate its use for high throughput assays of hydrolases using Brazilian termites as the screened organisms. Conclusions Important differences between the use of the natural substrate and commercial cellulase substrates, such as carboxymethylcellulose or crystalline cellulose, were observed. This suggests that wood feeding termites, in contrast to litter feeding termites, might not be the best source for enzymes that degrade sugarcane biomass. PMID:22081987

  14. Novel Acoustic Loading of a Mass Spectrometer: Toward Next-Generation High-Throughput MS Screening.

    PubMed

    Sinclair, Ian; Stearns, Rick; Pringle, Steven; Wingfield, Jonathan; Datwani, Sammy; Hall, Eric; Ghislain, Luke; Majlof, Lars; Bachman, Martin

    2016-02-01

    High-throughput, direct measurement of substrate-to-product conversion by label-free detection, without the need for engineered substrates or secondary assays, could be considered the "holy grail" of drug discovery screening. Mass spectrometry (MS) has the potential to be part of this ultimate screening solution, but is constrained by the limitations of existing MS sample introduction modes that cannot meet the throughput requirements of high-throughput screening (HTS). Here we report data from a prototype system (Echo-MS) that uses acoustic droplet ejection (ADE) to transfer femtoliter-scale droplets in a rapid, precise, and accurate fashion directly into the MS. The acoustic source can load samples into the MS from a microtiter plate at a rate of up to three samples per second. The resulting MS signal displays a very sharp attack profile and ions are detected within 50 ms of activation of the acoustic transducer. Additionally, we show that the system is capable of generating multiply charged ion species from simple peptides and large proteins. The combination of high speed and low sample volume has significant potential within not only drug discovery, but also other areas of the industry. PMID:26721821

  15. Droplet Electrospray Ionization Mass Spectrometry for High Throughput Screening for Enzyme Inhibitors

    PubMed Central

    2015-01-01

    High throughput screening (HTS) is important for identifying molecules with desired properties. Mass spectrometry (MS) is potentially powerful for label-free HTS due to its high sensitivity, speed, and resolution. Segmented flow, where samples are manipulated as droplets separated by an immiscible fluid, is an intriguing format for high throughput MS because it can be used to reliably and precisely manipulate nanoliter volumes and can be directly coupled to electrospray ionization (ESI) MS for rapid analysis. In this study, we describe a “MS Plate Reader” that couples standard multiwell plate HTS workflow to droplet ESI-MS. The MS plate reader can reformat 3072 samples from eight 384-well plates into nanoliter droplets segmented by an immiscible oil at 4.5 samples/s and sequentially analyze them by MS at 2 samples/s. Using the system, a label-free screen for cathepsin B modulators against 1280 chemicals was completed in 45 min with a high Z-factor (>0.72) and no false positives (24 of 24 hits confirmed). The assay revealed 11 structures not previously linked to cathepsin inhibition. For even larger scale screening, reformatting and analysis could be conducted simultaneously, which would enable more than 145 000 samples to be analyzed in 1 day. PMID:25137241

  16. High throughput screening of ferroelectric thin film libraries

    NASA Astrophysics Data System (ADS)

    Schroeter, Christian; Wessler, Berit; Schoenecker, Andreas; Keitel, Uwe; Eng, Lukas M.

    2006-12-01

    High throughput methods can significantly speed up the search for advanced materials in a multidimensional configuration space, hence keeping innovation cycles short. In the search for improved materials, high throughput methods are wanted to optimize composition and processing of promising systems, and to find candidate compounds. Such a method is described here which is applicable to the development of ferroelectric thin films. Libraries with samples of varying chemical composition were produced via the sol-gel route on structured and metallized silicon wafers. To determine the permittivity of the films, automated measurements of film thickness and capacity were established. Furthermore, ferroelectric hysterisis measurements were performed on samples with a particularly high permittivity. This high throughput route, which allows for synthesis and characterization of over hundred samples per day, was proved and tested by means of lead zirconate titanate as a standard material. It was possible to obtain films with remarkable high permittivity and low coercive field at optimal lead zirconate/lead titanate ratio and by compensating for lead loss during processing by finding the optimal lead excess added to the precursor solutions.

  17. A Novel High-Throughput Cell-Based Assay Aimed at Identifying Inhibitors of DNA Metabolism in Bacteria

    PubMed Central

    Fan, Jun; de Jonge, Boudewijn L. M.; MacCormack, Kathy; Sriram, Shubha; McLaughlin, Robert E.; Plant, Helen; Preston, Marian; Fleming, Paul R.; Albert, Robert; Foulk, Melinda

    2014-01-01

    Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening. PMID:25246396

  18. Perspectives on Validation of High-Throughput Assays Supporting 21st Century Toxicity Testing1

    PubMed Central

    Judson, Richard; Kavlock, Robert; Martin, Matt; Reif, David; Houck, Keith; Knudsen, Thomas; Richard, Ann; Tice, Raymond R.; Whelan, Maurice; Xia, Menghang; Huang, Ruili; Austin, Christopher; Daston, George; Hartung, Thomas; Fowle, John R.; Wooge, William; Tong, Weida; Dix, David

    2014-01-01

    Summary In vitro, high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals, but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. Here we discuss streamlining the validation process, specifically for prioritization applications in which HTS assays are used to identify a high-concern subset of a collection of chemicals. The high-concern chemicals could then be tested sooner rather than later in standard guideline bioassays. The streamlined validation process would continue to ensure the reliability and relevance of assays for this application. We discuss the following practical guidelines: (1) follow current validation practice to the extent possible and practical; (2) make increased use of reference compounds to better demonstrate assay reliability and relevance; (3) deemphasize the need for cross-laboratory testing, and; (4) implement a web-based, transparent and expedited peer review process. PMID:23338806

  19. High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria

    PubMed Central

    2016-01-01

    In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented synthesis (DOS)-derived compounds. Of the compounds screened, we obtain hit rates of 12–13 and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 μM and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages. PMID:27275010

  20. High-Throughput Screening for Ligands of the HEPN Domain of Sacsin

    PubMed Central

    Li, Xinlu; Ménade, Marie; Kozlov, Guennadi; Hu, Zheping; Dai, Zheng; McPherson, Peter S.; Brais, Bernard; Gehring, Kalle

    2015-01-01

    Sacsin is a large protein implicated in the neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), which features the loss of Purkinje neurons in the cerebellum. Although the domain architecture of sacsin suggests that it is a neuronal chaperone assisting in protein quality control, the precise function of sacsin remains elusive. Using fluorescence polarization (FP) assays, we confirmed that the HEPN domain of sacsin binds to nucleotides with low micromolar affinities. FP competition assays with a variety of nucleotides and nucleotide analogs revealed that the binding is primarily mediated by the phosphate groups of nucleotides. A high-throughput screen subsequently identified novel small molecule ligands of HEPN, providing new chemical probes for cell culture studies and drug development. Together, the results are consistent with the HEPN domain contributing to the functional activity of sacsin by binding to nucleotides or other multiply charged anionic compounds in neurons. PMID:26366743

  1. Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels.

    PubMed

    Su, Zhenwei; Brown, Emily C; Wang, Weiwei; MacKinnon, Roderick

    2016-05-17

    K(+) channels, a superfamily of ∼80 members, control cell excitability, ion homeostasis, and many forms of cell signaling. Their malfunctions cause numerous diseases including neuronal disorders, cardiac arrhythmia, diabetes, and asthma. Here we present a novel liposome flux assay (LFA) that is applicable to most K(+) channels. It is robust, low cost, and high throughput. Using LFA, we performed small molecule screens on three different K(+) channels and identified new activators and inhibitors for biological research on channel function and for medicinal development. We further engineered a hERG (human ether-à-go-go-related gene) channel, which, when used in LFA, provides a highly sensitive (zero false negatives on 50 hERG-sensitive drugs) and highly specific (zero false positives on 50 hERG-insensitive drugs), low-cost hERG safety assay. PMID:27091997

  2. Experiment and Modeling of Spatially Indexed Microbead Arrays for High-Throughput Screening Applications

    NASA Astrophysics Data System (ADS)

    Leary, Thomas; Maldarelli, Charles; Couzis, Alexander

    2011-11-01

    The development of platforms for multiplexed, high throughput screening of the binding interactions of target biomolecules against a library of potential binding probes enables progress in many areas in medicine and biology. Formats in which probes are linked to microbeads arrayed in a microfluidic channel offer high sensitivity, reduced reagent consumption and are easily parallelized for multiplexed detection. This presentation describes a microfluidically assembled microbead array in which beads are streamed through a channel with an array of wells inscribed in the floor of the channel. The beads are captured in the wells via gravity. We demonstrate that an array of beads displaying different receptors can be assembled in this format, indexed by sequential depostion and used for a prototype assay. Solutions for a two dimensional mass transfer model of the conjugation of the probe to the receptor on the bead surface identify kinetically limited regimes which are used to measure the binding kinetics of the prototype assay.

  3. Development of a high-throughput purification method and a continuous assay system for chlorophyllase.

    PubMed

    Arkus, Kiani A J; Jez, Joseph M

    2006-06-01

    In the degradation of chlorophyll, chlorophyllase catalyzes the initial hydrolysis of the phytol moiety from the pigment. Since chlorophyll degradation is a defining feature of plant senescence, compounds inhibiting chlorophyllase activity may delay senescence, thereby improving shelf life and appearance of plant products. Here we describe the development of a 96-well plate-based purification and assay system for measuring chlorophyllase activity. Integrated lysis and immobilized metal affinity chromatography plates were used for purifying recombinant hexahistidine-tagged Triticum aestivum (wheat) chlorophyllase from Escherichia coli. Chlorophyllase assays using chlorophyll as a substrate showed that the immobilized fusion protein displayed kinetic parameters similar to those of recombinant enzyme purified by affinity chromatography; however, the need to extract reaction products from a multiwell plate limits the value of this assay for high-throughput screening applications. Replacing chlorophyll with p-nitrophenyl-ester substrates eliminates the extraction step and allows for continuous measurement of chlorophyllase activity in a multiwell plate format. Determination of steady state kinetic constants, pH rate profile, the inhibitory effects of metal ions and esterase inhibitors, and the effect of functional group-modifying reagents validated the utility of the plate-based system. The combined purification and assay system provides a convenient and rapid method for the assessment of chlorophyllase activity. PMID:16643837

  4. A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica.

    PubMed

    Bordes, Florence; Fudalej, Franck; Dossat, Valérie; Nicaud, Jean-Marc; Marty, Alain

    2007-09-01

    Development of a high-throughput eukaryotic screening procedure is important to increase success in obtaining improved enzymes through directed enzyme evolution. This procedure was developed for the yeast Yarrowia lipolytica which becomes the second eukaryotic host for this purpose. The extracellular lipase Lip2 was used as expressed enzyme but this system will be easily adjusted for other enzymes. We adapted and optimized the protocol for protein expression by Y. lipolytica in 96-well microplates. Yeast transformation efficiency and expression cassette insertion were increased by constructing a strain containing a zeta docking platform for targeted integration into the genome. The coefficient of variance of the full process was reduced from 36.3% to 18.9%. The main part of the variability (11.7%) arises from the specific lipase enzyme assay whereas the coefficient of variance concerning transformation, growth and expression steps represents only 7.2%. The rate of clone with no activity was reduced from 5.8% to 0.2%. Both transformation efficiency and variability are then compatible with high-throughput screening in the yeast Y. lipolytica. PMID:17669530

  5. High-Throughput Carrier Screening Using TaqMan Allelic Discrimination

    PubMed Central

    Fedick, Anastasia; Su, Jing; Jalas, Chaim; Northrop, Lesley; Devkota, Batsal; Ekstein, Josef; Treff, Nathan R.

    2013-01-01

    Members of the Ashkenazi Jewish community are at an increased risk for inheritance of numerous genetic diseases such that carrier screening is medically recommended. This paper describes the development and evaluation of 30 TaqMan allelic discrimination qPCR assays for 29 mutations on 2 different high-throughput platforms. Four of these mutations are in the GBA gene and are successfully examined using short amplicons due to the qualitative nature of TaqMan allelic discrimination. Two systems were tested for their reliability (call rate) and consistency with previous diagnoses (diagnostic accuracy) indicating a call rate of 99.04% and a diagnostic accuracy of 100% (+/−0.00%) from one platform, and a call rate of 94.66% and a diagnostic accuracy of 93.35% (+/−0.29%) from a second for 9,216 genotypes. Results for mutations tested at the expected carrier frequency indicated a call rate of 97.87% and a diagnostic accuracy of 99.96% (+/−0.05%). This study demonstrated the ability of a high throughput qPCR methodology to accurately and reliably genotype 29 mutations in parallel. The universally applicable nature of this technology provides an opportunity to increase the number of mutations that can be screened simultaneously, and reduce the cost and turnaround time for accommodating newly identified and clinically relevant mutations. PMID:23555759

  6. A Quantitative High-Throughput Screen Identifies Potential Epigenetic Modulators of Gene Expression

    PubMed Central

    Johnson, Ronald L.; Huang, Wenwei; Jadhav, Ajit; Austin, Christopher P.; Inglese, James; Martinez, Elisabeth D.

    2008-01-01

    Epigenetic regulation of gene expression is essential in embryonic development and contributes to cancer pathology. We used a cell-based imaging assay that measures derepression of a silenced GFP reporter to identify novel classes of compounds involved in epigenetic regulation. This Locus Derepression (LDR) assay was screened against a 69,137-member chemical library using quantitative high-throughput screening (qHTS), a titration-response method that assays compounds at multiple concentrations. From structure-activity relationships of the 411 actives recovered from the qHTS, six distinct chemical series were chosen for further study. Forty-eight qHTS actives and analogs were counter screened using the parental line of the LDR cells, which lack the GFP reporter. Three series, 8-hydroxy quinoline, quinoline-8-thiol and 1,3,5-thiadiazinane-2-thione, were not fluorescent and re-confirmed activity in the LDR cells. The three active series did not inhibit histone deacetylase activity in nuclear extracts or reactivate the expression of the densely methylated p16 gene in cancer cells. However, one series induced expression of the methylated CDH13 gene and inhibited the viability of several lung cancer lines at submicromolar concentrations. These results suggest that the identified small molecules act on epigenetic or transcriptional components and validate our approach of using a cell-based imaging assay in conjunction with qHTS. PMID:18211814

  7. A Robust and Adaptable High Throughput Screening Method to Study Host-Microbiota Interactions in the Human Intestine

    PubMed Central

    de Wouters, Tomas; Ledue, Florence; Nepelska, Malgorzata; Doré, Joël; Blottière, Hervé M.; Lapaque, Nicolas

    2014-01-01

    The intestinal microbiota has many beneficial roles for its host. However, the precise mechanisms developed by the microbiota to influence the host intestinal cell responses are only partially known. The complexity of the ecosystem and our inability to culture most of these micro-organisms have led to the development of molecular approaches such as functional metagenomics, i.e. the heterologous expression of a metagenome in order to identify functions. This elegant strategy coupled to high throughput screening allowed to identify novel enzymes from different ecosystems where culture methods have not yet been adapted to isolate the candidate microorganisms. We have proposed to use this functional metagenomic approach in order to model the microbiota’s interaction with the host by combining this heterologous expression with intestinal reporter cell lines. The addition of the cellular component to this functional metagenomic approach introduced a second important source of variability resulting in a novel challenge for high throughput screening. First attempts of high throughput screening with various reporter cell-lines showed a high distribution of the response and consequent difficulties to reproduce the response, impairing an easy and clear identification of confirmed hits. In this study, we developed a robust and reproducible methodology to combine these two biological systems for high throughput application. We optimized experimental setups and completed them by appropriate statistical analysis tools allowing the use this innovative approach in a high throughput manner and on a broad range of reporter assays. We herewith present a methodology allowing a high throughput screening combining two biological systems. Therefore ideal conditions for homogeneity, sensitivity and reproducibility of both metagenomic clones as well as reporter cell lines have been identified and validated. We believe that this innovative method will allow the identification of new

  8. A High-Throughput, Multi-Cell Phenotype Assay for the Identification of Novel Inhibitors of Chemotaxis/Migration

    PubMed Central

    Liao, Xin-Hua; Meena, Netra Pal; Southall, Noel; Liu, Lunhua; Swaroop, Manju; Zhang, Arina Li; Xiang, Jan Jian; Parent, Carole A.; Zheng, Wei; Kimmel, Alan R.

    2016-01-01

    Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems. PMID:26956526

  9. A High-Throughput, Multi-Cell Phenotype Assay for the Identification of Novel Inhibitors of Chemotaxis/Migration.

    PubMed

    Liao, Xin-Hua; Meena, Netra Pal; Southall, Noel; Liu, Lunhua; Swaroop, Manju; Zhang, Arina Li; Xiang, Jan Jian; Parent, Carole A; Zheng, Wei; Kimmel, Alan R

    2016-01-01

    Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems. PMID:26956526

  10. Identification of Thyroid Hormone Receptor Active Compounds Using a Quantitative High-Throughput Screening Platform

    PubMed Central

    Freitas, Jaime; Miller, Nicole; Mengeling, Brenda J.; Xia, Menghang; Huang, Ruili; Houck, Keith; Rietjens, Ivonne M.C.M.; Furlow, J. David; Murk, Albertinka J.

    2014-01-01

    To adapt the use of GH3.TRE-Luc reporter gene cell line for a quantitative high-throughput screening (qHTS) platform, we miniaturized the reporter gene assay to a 1536-well plate format. 1280 chemicals from the Library of Pharmacologically Active Compounds (LOPAC) and the National Toxicology Program (NTP) 1408 compound collection were analyzed to identify potential thyroid hormone receptor (TR) agonists and antagonists. Of the 2688 compounds tested, eight scored as potential TR agonists when the positive hit cut-off was defined at ≥10% efficacy, relative to maximal triiodothyronine (T3) induction, and with only one of those compounds reaching ≥20% efficacy. One common class of compounds positive in the agonist assays were retinoids such as all-trans retinoic acid, which are likely acting via the retinoid-X receptor, the heterodimer partner with the TR. Five potential TR antagonists were identified, including the antiallergy drug tranilast and the anxiolytic drug SB 205384 but also some cytotoxic compounds like 5-fluorouracil. None of the inactive compounds were structurally related to T3, nor had been reported elsewhere to be thyroid hormone disruptors, so false negatives were not detected. None of the low potency (>100µM) TR agonists resembled T3 or T4, thus these may not bind directly in the ligand-binding pocket of the receptor. For TR agonists, in the qHTS, a hit cut-off of ≥20% efficacy at 100 µM may avoid identification of positives with low or no physiological relevance. The miniaturized GH3.TRE-Luc assay offers a promising addition to the in vitro test battery for endocrine disruption, and given the low percentage of compounds testing positive, its high-throughput nature is an important advantage for future toxicological screening. PMID:24772387

  11. High Throughput In Situ XAFS Screening of Catalysts

    SciTech Connect

    Tsapatsaris, Nikolaos; Beesley, Angela M.; Weiher, Norbert; Tatton, Helen; Schroeder, Sven L. M.; Dent, Andy J.; Mosselmans, Frederick J. W.; Tromp, Moniek; Russu, Sergio; Evans, John; Harvey, Ian; Hayama, Shu

    2007-02-02

    We outline and demonstrate the feasibility of high-throughput (HT) in situ XAFS for synchrotron radiation studies. An XAS data acquisition and control system for the analysis of dynamic materials libraries under control of temperature and gaseous environments has been developed. The system is compatible with the 96-well industry standard and coupled to multi-stream quadrupole mass spectrometry (QMS) analysis of reactor effluents. An automated analytical workflow generates data quickly compared to traditional individual spectrum acquisition and analyses them in quasi-real time using an HT data analysis tool based on IFFEFIT. The system was used for the automated characterization of a library of 91 catalyst precursors containing ternary combinations of Cu, Pt, and Au on {gamma}-Al2O3, and for the in situ characterization of Au catalysts supported on Al2O3 and TiO2.

  12. High Throughput In Situ XAFS Screening of Catalysts

    NASA Astrophysics Data System (ADS)

    Tsapatsaris, Nikolaos; Beesley, Angela M.; Weiher, Norbert; Tatton, Helen; Dent, Andy J.; Mosselmans, Frederick J. W.; Tromp, Moniek; Russu, Sergio; Evans, John; Harvey, Ian; Hayama, Shu; Schroeder, Sven L. M.

    2007-02-01

    We outline and demonstrate the feasibility of high-throughput (HT) in situ XAFS for synchrotron radiation studies. An XAS data acquisition and control system for the analysis of dynamic materials libraries under control of temperature and gaseous environments has been developed. The system is compatible with the 96-well industry standard and coupled to multi-stream quadrupole mass spectrometry (QMS) analysis of reactor effluents. An automated analytical workflow generates data quickly compared to traditional individual spectrum acquisition and analyses them in quasi-real time using an HT data analysis tool based on IFFEFIT. The system was used for the automated characterization of a library of 91 catalyst precursors containing ternary combinations of Cu, Pt, and Au on γ-Al2O3, and for the in situ characterization of Au catalysts supported on Al2O3 and TiO2.

  13. High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors

    PubMed Central

    Bageshwar, Umesh K.; VerPlank, Lynn; Baker, Dwight; Dong, Wen; Hamsanathan, Shruthi; Whitaker, Neal; Sacchettini, James C.; Musser, Siegfried M.

    2016-01-01

    The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors. PMID:26901445

  14. High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors.

    PubMed

    Bageshwar, Umesh K; VerPlank, Lynn; Baker, Dwight; Dong, Wen; Hamsanathan, Shruthi; Whitaker, Neal; Sacchettini, James C; Musser, Siegfried M

    2016-01-01

    The twin arginine translocation (Tat) pathway transports fully-folded and assembled proteins in bacteria, archaea and plant thylakoids. The Tat pathway contributes to the virulence of numerous bacterial pathogens that cause disease in humans, cattle and poultry. Thus, the Tat pathway has the potential to be a novel therapeutic target. Deciphering the Tat protein transport mechanism has been challenging since the active translocon only assembles transiently in the presence of substrate and a proton motive force. To identify inhibitors of Tat transport that could be used as biochemical tools and possibly as drug development leads, we developed a high throughput screen (HTS) to assay the effects of compounds in chemical libraries against protein export by the Escherichia coli Tat pathway. The primary screen is a live cell assay based on a fluorescent Tat substrate that becomes degraded in the cytoplasm when Tat transport is inhibited. Consequently, low fluorescence in the presence of a putative Tat inhibitor was scored as a hit. Two diverse chemical libraries were screened, yielding average Z'-factors of 0.74 and 0.44, and hit rates of ~0.5% and 0.04%, respectively. Hits were evaluated by a series of secondary screens. Electric field gradient (Δψ) measurements were particularly important since the bacterial Tat transport requires a Δψ. Seven low IC50 hits were eliminated by Δψ assays, suggesting ionophore activity. As Δψ collapse is generally toxic to animal cells and efficient membrane permeability is generally favored during the selection of library compounds, these results suggest that secondary screening of hits against electrochemical effects should be done early during hit validation. Though none of the short-listed compounds inhibited Tat transport directly, the screening and follow-up assays developed provide a roadmap to pursue Tat transport inhibitors. PMID:26901445

  15. Acoustic Droplet Ejection Technology and Its Application in High-Throughput RNA Interference Screening

    PubMed Central

    Nebane, N. Miranda; Coric, Tatjana; McKellip, Sara; Woods, LaKeisha; Sosa, Melinda; Rasmussen, Lynn; Bjornsti, Mary-Ann; White, E. Lucile

    2016-01-01

    The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte’s Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use. This includes the transfer of many biological reagents solubilized in aqueous buffers, including siRNAs. The Echo 555 is ideal for siRNA dispensing because it is accurate at low volumes and a step-down dilution is not necessary. The potential for liquid carryover and cross-contamination is eliminated, as no tips are needed. Herein, we describe the siRNA screening platform at Southern Research’s HTS Center using the ADE technology. With this technology, an siRNA library can be dispensed weeks or even months in advance of the assay itself. The protocol has been optimized to achieve assay parameters comparable to small-molecule screening parameters, and exceeding the norm reported for genomewide siRNA screens. PMID:26663785

  16. Microengineering Methods for Cell Based Microarrays and High-Throughput Drug Screening Applications

    PubMed Central

    Xu, Feng; Wu, JinHui; Wang, ShuQi; Durmus, Naside Gozde; Gurkan, Umut Atakan; Demirci, Utkan

    2011-01-01

    Screening for effective therapeutic agents from millions of drug candidates is costly, time-consuming and often face ethical concerns due to extensive use of animals. To improve cost-effectiveness, and to minimize animal testing in pharmaceutical research, in vitro monolayer cell microarrays with multiwell plate assays have been developed. Integration of cell microarrays with microfluidic systems have facilitated automated and controlled component loading, significantly reducing the consumption of the candidate compounds and the target cells. Even though these methods significantly increased the throughput compared to conventional in vitro testing systems and in vivo animal models, the cost associated with these platforms remains prohibitively high. Besides, there is a need for three-dimensional (3D) cell based drug-screening models, which can mimic the in vivo microenvironment and the functionality of the native tissues. Here, we present the state-of-the-art microengineering approaches that can be used to develop 3D cell based drug screening assays. We highlight the 3D in vitro cell culture systems with live cell-based arrays, microfluidic cell culture systems, and their application to high-throughput drug screening. We conclude that among the emerging microengineering approaches, bioprinting holds a great potential to provide repeatable 3D cell based constructs with high temporal, spatial control and versatility. PMID:21725152

  17. Lessons from high-throughput protein crystallization screening: 10 years of practical experience

    PubMed Central

    JR, Luft; EH, Snell; GT, DeTitta

    2011-01-01

    Introduction X-ray crystallography provides the majority of our structural biological knowledge at a molecular level and in terms of pharmaceutical design is a valuable tool to accelerate discovery. It is the premier technique in the field, but its usefulness is significantly limited by the need to grow well-diffracting crystals. It is for this reason that high-throughput crystallization has become a key technology that has matured over the past 10 years through the field of structural genomics. Areas covered The authors describe their experiences in high-throughput crystallization screening in the context of structural genomics and the general biomedical community. They focus on the lessons learnt from the operation of a high-throughput crystallization screening laboratory, which to date has screened over 12,500 biological macromolecules. They also describe the approaches taken to maximize the success while minimizing the effort. Through this, the authors hope that the reader will gain an insight into the efficient design of a laboratory and protocols to accomplish high-throughput crystallization on a single-, multiuser-laboratory or industrial scale. Expert Opinion High-throughput crystallization screening is readily available but, despite the power of the crystallographic technique, getting crystals is still not a solved problem. High-throughput approaches can help when used skillfully; however, they still require human input in the detailed analysis and interpretation of results to be more successful. PMID:22646073

  18. Hierarchical dose-response modeling for high-throughput toxicity screening of environmental chemicals.

    PubMed

    Wilson, Ander; Reif, David M; Reich, Brian J

    2014-03-01

    High-throughput screening (HTS) of environmental chemicals is used to identify chemicals with high potential for adverse human health and environmental effects from among the thousands of untested chemicals. Predicting physiologically relevant activity with HTS data requires estimating the response of a large number of chemicals across a battery of screening assays based on sparse dose-response data for each chemical-assay combination. Many standard dose-response methods are inadequate because they treat each curve separately and under-perform when there are as few as 6-10 observations per curve. We propose a semiparametric Bayesian model that borrows strength across chemicals and assays. Our method directly parametrizes the efficacy and potency of the chemicals as well as the probability of response. We use the ToxCast data from the U.S. Environmental Protection Agency (EPA) as motivation. We demonstrate that our hierarchical method provides more accurate estimates of the probability of response, efficacy, and potency than separate curve estimation in a simulation study. We use our semiparametric method to compare the efficacy of chemicals in the ToxCast data to well-characterized reference chemicals on estrogen receptor α (ERα) and peroxisome proliferator-activated receptor γ (PPARγ) assays, then estimate the probability that other chemicals are active at lower concentrations than the reference chemicals. PMID:24397816

  19. Noise Reduction in High-Throughput Gene Perturbation Screens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Motivation: Accurate interpretation of perturbation screens is essential for a successful functional investigation. However, the screened phenotypes are often distorted by noise, and their analysis requires specialized statistical analysis tools. The number and scope of statistical methods available...

  20. Better, Faster, Cheaper: Getting the Most Out of High-Throughput Screening with Zebrafish.

    PubMed

    Truong, Lisa; Simonich, Michael T; Tanguay, Robert L

    2016-01-01

    The field of toxicology is undergoing a vast change with high-throughput (HT) approaches that rapidly query huge swaths of chemico-structural space for bioactivity and hazard potential. Its practicality is due in large part to switching from high-cost, low-throughput mammalian models to faster and cheaper alternatives. We believe this is an improved approach because the immense breadth of the resulting data sets a foundation for predictive structure-activity-based toxicology. Moreover, rapidly uncovering structure-related bioactivity drives better decisions about where to commit resources to drill down to a mechanism, or pursue commercial leads. While hundreds of different in vitro toxicology assays can collectively serve as an alternative to mammalian animal model testing, far greater efficiency and ultimately more relevant data are obtained from the whole animal. The developmental zebrafish, with its well-documented advantages over many animal models, is now emerging as a true biosensor of chemical activity. Herein, we draw on nearly a decade of experience developing high-throughput toxicology screens in the developmental zebrafish to summarize the best practices in fulfilling the better, faster, cheaper goals. We include optimization and harmonization of dosing volume, exposure paradigms, chemical solubility, chorion status, experimental duration, endpoint definitions, and statistical analysis. PMID:27518627

  1. A Chromogenic Assay Suitable for High-Throughput Determination of Limit Dextrinase Activity in Barley Malt Extracts.

    PubMed

    Bøjstrup, Marie; Marri, Lucia; Lok, Finn; Hindsgaul, Ole

    2015-12-23

    Twenty-four malt samples were assayed for limit dextrinase activity using a chromogenic assay developed recently in our group. The assay utilizes a small soluble chromogenic substrate which is hydrolyzed selectively by limit dextrinase in a coupled assay to release the chromophore 2-chloro-4-nitrophenol. The release of the chromophore, corresponding to the activity of limit dextrinase, can be followed by measuring the UV absorption at 405 nm. The 24 malt samples represented a wide variation of limit dextrinase activities, and these activities could be clearly differentiated by the assay. The results obtained were comparable with the results obtained from a commercially available assay, Limit-Dextrizyme from Megazyme International Ireland. Furthermore, the improved assay uses a soluble substrate. That makes it well suited for high-throughput screening as it can be handled in a 96-well plate format. PMID:26615836

  2. Inhibitors of the Salicylate Synthase (MbtI) from Mycobacterium tuberculosis Discovered by High-Throughput Screening

    PubMed Central

    Vasan, Mahalakshmi; Neres, João; Williams, Jessica; Wilson, Daniel J.; Teitelbaum, Aaron M.; Remmel, Rory P.; Aldrich, Courtney C.

    2010-01-01

    A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole-2-thiones (class III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure–activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition. PMID:21053346

  3. A high-throughput method for assessing chemical toxicity using a Caenorhabditis elegans reproduction assay

    SciTech Connect

    Boyd, Windy A.; McBride, Sandra J.; Rice, Julie R.; Snyder, Daniel W.; Freedman, Jonathan H.

    2010-06-01

    The National Research Council has outlined the need for non-mammalian toxicological models to test the potential health effects of a large number of chemicals while also reducing the use of traditional animal models. The nematode Caenorhabditis elegans is an attractive alternative model because of its well-characterized and evolutionarily conserved biology, low cost, and ability to be used in high-throughput screening. A high-throughput method is described for quantifying the reproductive capacity of C. elegans exposed to chemicals for 48 h from the last larval stage (L4) to adulthood using a COPAS Biosort. Initially, the effects of exposure conditions that could influence reproduction were defined. Concentrations of DMSO vehicle {<=} 1% did not affect reproduction. Previous studies indicated that C. elegans may be influenced by exposure to low pH conditions. At pHs greater than 4.5, C. elegans reproduction was not affected; however below this pH there was a significant decrease in the number of offspring. Cadmium chloride was chosen as a model toxicant to verify that automated measurements were comparable to those of traditional observational studies. EC{sub 50} values for cadmium for automated measurements (176-192 {mu}M) were comparable to those previously reported for a 72-h exposure using manual counting (151 {mu}M). The toxicity of seven test toxicants on C. elegans reproduction was highly correlative with rodent lethality suggesting that this assay may be useful in predicting the potential toxicity of chemicals in other organisms.

  4. A HIGH-THROUGHPUT METHOD FOR ASSESSING CHEMICAL TOXICITY USING A CAENORHABDITIS ELEGANS REPRODUCTION ASSAY

    PubMed Central

    Boyd, Windy A.; McBride, Sandra J.; Rice, Julie R.; Snyder, Daniel W.; Freedman, Jonathan H.

    2010-01-01

    The National Research Council has outlined the need for non-mammalian toxicological models to test the potential health effects of a large number of chemicals while also reducing the use of traditional animal models. The nematode Caenorhabditis elegans is an attractive alternative model because of its well-characterized and evolutionarily-conserved biology, low cost, and ability to be used in high-throughput screening. A high-throughput method is described for quantifying the reproductive capacity of C. elegans exposed to chemicals for 48 h from the last larval stage (L4) to adulthood using a COPAS Biosort. Initially, the effects of exposure conditions that could influence reproduction were defined. Concentrations of DMSO vehicle ≤ 1% did not affect reproduction. Previous studies indicated that C. elegans may be influenced by exposure to low pH conditions. At pHs greater than 4.5, C. elegans reproduction was not affected, however below this pH there was a significant decrease in the number of offspring. Cadmium chloride was chosen as a model toxicant to verify that automated measurements were comparable to those of traditional observational studies. EC50 values for cadmium for automated measurements (176-192 μM) were comparable to those previously reported for a 72-h exposure using manual counting (151 μM). The toxicity of seven test toxicants on C. elegans reproduction was highly correlative with rodent lethality suggesting that this assay may be useful in predicting the potential toxicity of chemicals in other organisms. PMID:20206647

  5. Convenient, Sensitive and High-Throughput Method for Screening Botanic Origin

    PubMed Central

    Yuan, Yuan; Jiang, Chao; Liu, Libing; Yu, Shulin; Cui, Zhanhu; Chen, Min; Lin, Shufang; Wang, Shu; Huang, Luqi

    2014-01-01

    In this work, a rapid (within 4–5 h), sensitive and visible new method for assessing botanic origin is developed by combining loop-mediated isothermal amplification with cationic conjugated polymers. The two Chinese medicinal materials (Jin-Yin-Hua and Shan-Yin-Hua) with similar morphology and chemical composition were clearly distinguished by gene SNP genotyping assays. The identification of plant species in Patented Chinese drugs containing Lonicera buds is successfully performed using this detection system. The method is also robust enough to be used in high-throughput screening. This new method is very helpful to identify herbal materials, and is beneficial for detecting safety and quality of botanic products. PMID:24953704

  6. Resonant waveguide grating imagers for single cell analysis and high throughput screening

    NASA Astrophysics Data System (ADS)

    Fang, Ye

    2015-08-01

    Resonant waveguide grating (RWG) systems illuminate an array of diffractive nanograting waveguide structures in microtiter plate to establish evanescent wave for measuring tiny changes in local refractive index arising from the dynamic mass redistribution of living cells upon stimulation. Whole-plate RWG imager enables high-throughput profiling and screening of drugs. Microfluidics RWG imager not only manifests distinct receptor signaling waves, but also differentiates long-acting agonism and antagonism. Spatially resolved RWG imager allows for single cell analysis including receptor signaling heterogeneity and the invasion of cancer cells in a spheroidal structure through 3-dimensional extracellular matrix. High frequency RWG imager permits real-time detection of drug-induced cardiotoxicity. The wide coverage in target, pathway, assay, and cell phenotype has made RWG systems powerful tool in both basic research and early drug discovery process.

  7. Convenient, Sensitive and High-Throughput Method for Screening Botanic Origin

    NASA Astrophysics Data System (ADS)

    Yuan, Yuan; Jiang, Chao; Liu, Libing; Yu, Shulin; Cui, Zhanhu; Chen, Min; Lin, Shufang; Wang, Shu; Huang, Luqi

    2014-06-01

    In this work, a rapid (within 4-5 h), sensitive and visible new method for assessing botanic origin is developed by combining loop-mediated isothermal amplification with cationic conjugated polymers. The two Chinese medicinal materials (Jin-Yin-Hua and Shan-Yin-Hua) with similar morphology and chemical composition were clearly distinguished by gene SNP genotyping assays. The identification of plant species in Patented Chinese drugs containing Lonicera buds is successfully performed using this detection system. The method is also robust enough to be used in high-throughput screening. This new method is very helpful to identify herbal materials, and is beneficial for detecting safety and quality of botanic products.

  8. Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay.

    PubMed

    Wolter, Justin M; Kotagama, Kasuen; Babb, Cody S; Mangone, Marco

    2015-01-01

    Luminescent Identification of Functional Elements in 3'UTRs (3'LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3'UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3'LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3'LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3'UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3'LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets. PMID:26066857

  9. High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

    EPA Science Inventory

    Human ATAD5 is an excellent biomarker for identifying genotoxic compounds because ATADS protein levels increase post-transcriptionally following exposure to a variety of DNA damaging agents. Here we report a novel quantitative high-throughput ATAD5-Iuciferase assay that can moni...

  10. A high throughput fluorescence polarization assay for inhibitors of the GoLoco motif/G-alpha interaction.

    PubMed

    Kimple, Adam J; Yasgar, Adam; Hughes, Mark; Jadhav, Ajit; Willard, Francis S; Muller, Robin E; Austin, Christopher P; Inglese, James; Ibeanu, Gordon C; Siderovski, David P; Simeonov, Anton

    2008-06-01

    The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively. PMID:18537560

  11. Incorporating human dosimetry and exposure into high-throughput in vitro toxicity screening.

    PubMed

    Rotroff, Daniel M; Wetmore, Barbara A; Dix, David J; Ferguson, Stephen S; Clewell, Harvey J; Houck, Keith A; Lecluyse, Edward L; Andersen, Melvin E; Judson, Richard S; Smith, Cornelia M; Sochaski, Mark A; Kavlock, Robert J; Boellmann, Frank; Martin, Matthew T; Reif, David M; Wambaugh, John F; Thomas, Russell S

    2010-10-01

    Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multiple cellular pathways. In this study, metabolic clearance and plasma protein binding were experimentally measured for 35 ToxCast phase I chemicals. The experimental data were used to parameterize a population-based in vitro-to-in vivo extrapolation model for estimating the human oral equivalent dose necessary to produce a steady-state in vivo concentration equivalent to in vitro AC(50) (concentration at 50% of maximum activity) and LEC (lowest effective concentration) values from the ToxCast data. For 23 of the 35 chemicals, the range of oral equivalent doses for up to 398 ToxCast assays was compared with chronic aggregate human oral exposure estimates in order to assess whether significant in vitro bioactivity occurred within the range of maximum expected human oral exposure. Only 2 of the 35 chemicals, triclosan and pyrithiobac-sodium, had overlapping oral equivalent doses and estimated human oral exposures. Ranking by the potencies of the AC(50) and LEC values, these two chemicals would not have been at the top of a prioritization list. Integrating both dosimetry and human exposure information with the high-throughput toxicity screening efforts provides a better basis for making informed decisions on chemical testing priorities and regulatory attention. Importantly, these tools are necessary to move beyond hazard rankings to estimates of possible in vivo responses based on in vitro screens. PMID:20639261

  12. Substrate-Free High-Throughput Screening Identifies Selective Inhibitors for Uncharacterized Enzymes

    PubMed Central

    Bachovchin, Daniel A.; Brown, Steven J.; Rosen, Hugh; Cravatt, Benjamin F.

    2009-01-01

    Target-based high-throughput screening (HTS) is essential for the discovery of small-molecule modulators of proteins. Typical screening methods for enzymes rely on extensively tailored substrate assays, which are not available for targets of poorly characterized biochemical activity. Here, we report a general, substrate-free platform for HTS that overcomes this problem by monitoring the reaction of broad-spectrum, activity-based probes with enzymes using fluorescence polarization. We show that this platform is applicable to enzymes from multiple mechanistic classes, regardless of their degree of functional annotation, and can be coupled with secondary competitive activity-based proteomic assays to rapidly determine the specificity of screening hits. Using this platform, we identified the bioactive alkaloid emetine as a selective inhibitor of the uncharacterized cancer-associated hydrolase RBBP9. We furthermore show that the detoxification enzyme GSTO1, also implicated in cancer, is inhibited by several electrophilic compounds found in public libraries, some of which display high selectivity for this enzyme. PMID:19329999

  13. The essential roles of chemistry in high-throughput screening triage

    PubMed Central

    Dahlin, Jayme L; Walters, Michael A

    2015-01-01

    It is increasingly clear that academic high-throughput screening (HTS) and virtual HTS triage suffers from a lack of scientists trained in the art and science of early drug discovery chemistry. Many recent publications report the discovery of compounds by screening that are most likely artifacts or promiscuous bioactive compounds, and these results are not placed into the context of previous studies. For HTS to be most successful, it is our contention that there must exist an early partnership between biologists and medicinal chemists. Their combined skill sets are necessary to design robust assays and efficient workflows that will weed out assay artifacts, false positives, promiscuous bioactive compounds and intractable screening hits, efforts that ultimately give projects a better chance at identifying truly useful chemical matter. Expertise in medicinal chemistry, cheminformatics and purification sciences (analytical chemistry) can enhance the post-HTS triage process by quickly removing these problematic chemotypes from consideration, while simultaneously prioritizing the more promising chemical matter for follow-up testing. It is only when biologists and chemists collaborate effectively that HTS can manifest its full promise. PMID:25163000

  14. Learning from the data: mining of large high-throughput screening databases.

    PubMed

    Yan, S Frank; King, Frederick J; He, Yun; Caldwell, Jeremy S; Zhou, Yingyao

    2006-01-01

    High-throughput screening (HTS) campaigns in pharmaceutical companies have accumulated a large amount of data for several million compounds over a couple of hundred assays. Despite the general awareness that rich information is hidden inside the vast amount of data, little has been reported for a systematic data mining method that can reliably extract relevant knowledge of interest for chemists and biologists. We developed a data mining approach based on an algorithm called ontology-based pattern identification (OPI) and applied it to our in-house HTS database. We identified nearly 1500 scaffold families with statistically significant structure-HTS activity profile relationships. Among them, dozens of scaffolds were characterized as leading to artifactual results stemming from the screening technology employed, such as assay format and/or readout. Four types of compound scaffolds can be characterized based on this data mining effort: tumor cytotoxic, general toxic, potential reporter gene assay artifact, and target family specific. The OPI-based data mining approach can reliably identify compounds that are not only structurally similar but also share statistically significant biological activity profiles. Statistical tests such as Kruskal-Wallis test and analysis of variance (ANOVA) can then be applied to the discovered scaffolds for effective assignment of relevant biological information. The scaffolds identified by our HTS data mining efforts are an invaluable resource for designing SAR-robust diversity libraries, generating in silico biological annotations of compounds on a scaffold basis, and providing novel target family specific scaffolds for focused compound library design. PMID:17125181

  15. High-Throughput In Vitro Glycoside Hydrolase (HIGH) Screening for Enzyme Discovery

    SciTech Connect

    Kim, Tae-Wan; Chokhawala, Harshal A.; Hess, Matthias; Dana, Craig M.; Baer, Zachary; Sczyrba, Alexander; Rubin, Edward M.; Blanch, Harvey W.; Clark, Douglas S.

    2011-09-16

    A high-throughput protein-expression and screening method (HIGH method, see picture) provides a rapid approach to the discovery of active glycoside hydrolases in environmental samples. Finally, HIGH screening combines cloning, protein expression, and enzyme hydrolysis in one pot; thus, the entire process from gene expression to activity detection requires only three hours.

  16. Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.

    PubMed

    Hondowicz, Brian D; Schwedhelm, Katharine V; Kas, Arnold; Tasch, Michael A; Rawlings, Crystal; Ramchurren, Nirasha; McIntosh, Martin; D'Amico, Leonard A; Sanda, Srinath; Standifer, Nathan E; Shendure, Jay; Stone, Brad

    2012-01-01

    The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery. PMID:22253836

  17. High-throughput assay of levansucrase variants in search of feasible catalysts for the synthesis of fructooligosaccharides and levan.

    PubMed

    Mardo, Karin; Visnapuu, Triinu; Gromkova, Maria; Aasamets, Anneli; Viigand, Katrin; Vija, Heiki; Alamäe, Tiina

    2014-01-01

    Bacterial levansucrases polymerize fructose residues of sucrose to β-2,6 linked fructans-fructooligosaccharides (FOS) and levan. While β-2,1-linked FOS are widely recognized as prebiotics, the health-related effects of β-2,6 linked FOS are scarcely studied as they are not commercially available. Levansucrase Lsc3 (Lsc-3) of Pseudomonas syringae pv. tomato has very high catalytic activity and stability making it a promising biotechnological catalyst for FOS and levan synthesis. In this study we evaluate feasibility of several high-throughput methods for screening and preliminary characterization of levansucrases using 36 Lsc3 mutants as a test panel. Heterologously expressed and purified His-tagged levansucrase variants were studied for: (1) sucrose-splitting activity; (2) FOS production; (3) ability and kinetics of levan synthesis; (4) thermostability in a Thermofluor assay. Importantly, we show that sucrose-splitting activity as well as the ability to produce FOS can both be evaluated using permeabilized levansucrase-expressing E. coli transformants as catalysts. For the first time we demonstrate the key importance of Trp109, His113, Glu146 and Glu236 for the catalysis of Lsc3. Cost-effective and high-throughput methods presented here are applicable not only in the levansucrase assay, but have a potential to be adapted for high-throughput (automated) study of other enzymes. PMID:24955639

  18. Integration of dosimetry, exposure, and high-throughput screening data in chemical toxicity assessment.

    PubMed

    Wetmore, Barbara A; Wambaugh, John F; Ferguson, Stephen S; Sochaski, Mark A; Rotroff, Daniel M; Freeman, Kimberly; Clewell, Harvey J; Dix, David J; Andersen, Melvin E; Houck, Keith A; Allen, Brittany; Judson, Richard S; Singh, Reetu; Kavlock, Robert J; Richard, Ann M; Thomas, Russell S

    2012-01-01

    High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, clearance, and exposure. Hepatic metabolic clearance and plasma protein binding were experimentally measured for 239 ToxCast Phase I chemicals. The experimental data were used in a population-based in vitro-to-in vivo extrapolation model to estimate the daily human oral dose, called the oral equivalent dose, necessary to produce steady-state in vivo blood concentrations equivalent to in vitro AC(50) (concentration at 50% of maximum activity) or lowest effective concentration values across more than 500 in vitro assays. The estimated steady-state oral equivalent doses associated with the in vitro assays were compared with chronic aggregate human oral exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. A total of 18 (9.9%) chemicals for which human oral exposure estimates were available had oral equivalent doses at levels equal to or less than the highest estimated U.S. population exposures. Ranking the chemicals by nominal assay concentrations would have resulted in different chemicals being prioritized. The in vitro assay endpoints with oral equivalent doses lower than the human exposure estimates included cell growth kinetics, cytokine and cytochrome P450 expression, and cytochrome P450 inhibition. The incorporation of dosimetry and exposure provide necessary context for interpretation of in vitro toxicity screening data and are important considerations in determining chemical testing priorities. PMID:21948869

  19. Two Variants of a High-Throughput Fluorescent Microplate Assay of Polysaccharide Endotransglycosylases.

    PubMed

    Kováčová, Kristína; Farkaš, Vladimír

    2016-04-01

    Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated. PMID:26754421

  20. High throughput miniature drug-screening platform using bioprinting technology.

    PubMed

    Rodríguez-Dévora, Jorge I; Zhang, Bimeng; Reyna, Daniel; Shi, Zhi-dong; Xu, Tao

    2012-09-01

    In the pharmaceutical industry, new drugs are tested to find appropriate compounds for therapeutic purposes for contemporary diseases. Unfortunately, novel compounds emerge at expensive prices and current target evaluation processes have limited throughput, thus leading to an increase of cost and time for drug development. This work shows the development of the novel inkjet-based deposition method for assembling a miniature drug-screening platform, which can realistically and inexpensively evaluate biochemical reactions in a picoliter-scale volume at a high speed rate. As proof of concept, applying a modified Hewlett Packard model 5360 compact disc printer, green fluorescent protein expressing Escherichia coli cells along with alginate gel solution have been arrayed on a coverslip chip under a repeatable volume of 180% ± 26% picoliters per droplet; subsequently, different antibiotic droplets were patterned on the spots of cells to evaluate the inhibition of bacteria for antibiotic screening. The proposed platform was compared to the current screening process, validating its effectiveness. The viability and basic function of the printed cells were evaluated, resulting in cell viability above 98% and insignificant or no DNA damage to human kidney cells transfected. Based on the reduction of investment and compound volume used by this platform, this technique has the potential to improve the actual drug discovery process at its target evaluation stage. PMID:22728820

  1. High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1.

    PubMed

    Lokesh, G L; Rachamallu, Aparna; Kumar, G D Kishore; Natarajan, Amarnath

    2006-05-01

    The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening. PMID:16500609

  2. High-Throughput Screen of Natural Product Libraries for Hsp90 Inhibitors

    PubMed Central

    Davenport, Jason; Balch, Maurie; Galam, Lakshmi; Girgis, Antwan; Hall, Jessica; Blagg, Brian S. J.; Matts, Robert L.

    2014-01-01

    Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we have utilized a high-throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized as Hsp90 inhibitors. Four compounds, anthothecol, garcinol, piplartine, and rottlerin, were further characterized, and the ability of these compounds to inhibit the refolding of luciferase, and reduce the rate of growth of MCF7 breast cancer cells, correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase, and deplete cultured cells of Hsp90-dependent client proteins. Thus, this screen has identified an additional 44 compounds with known beneficial pharmacological properties, but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine. PMID:24833337

  3. MScreen: an integrated compound management and high-throughput screening data storage and analysis system.

    PubMed

    Jacob, Renju T; Larsen, Martha J; Larsen, Scott D; Kirchhoff, Paul D; Sherman, David H; Neubig, Richard R

    2012-09-01

    High-throughput screening (HTS) has historically been used by the pharmaceutical industry to rapidly test hundreds of thousands of compounds to identify potential drug candidates. More recently, academic groups have used HTS to identify new chemical probes or small interfering RNA (siRNA) that can serve as experimental tools to examine the biology or physiology of novel proteins, processes, or interactions. HTS presents a significant challenge with the vast and complex nature of data generated. This report describes MScreen, a Web-based, open-source cheminformatics application for chemical library and siRNA plate management, primary HTS and dose-response data handling, structure search, and administrative functions. Each project in MScreen can be secured with passwords or shared in an open-information environment that enables collaborators to easily compare data from many screens, providing a useful means to identify compounds with desired selectivity. Unique features include compound, substance, mixture, and siRNA plate creation and formatting; automated dose-response fitting and quality control (QC); and user, target, and assay method administration. MScreen provides an effective means to facilitate HTS information handling and analysis in the academic setting so that users can efficiently view their screening data and evaluate results for follow-up. PMID:22706349

  4. MScreen: An Integrated Compound Management and High Throughput Screening (HTS) Data Storage and Analysis System

    PubMed Central

    Jacob, Renju T.; Larsen, Martha J.; Larsen, Scott D.; Kirchhoff, Paul D.; Sherman, David H.; Neubig, Richard R.

    2013-01-01

    High-throughput screening (HTS) has historically been used by the pharmaceutical industry to rapidly test hundreds of thousands of compounds to identify potential drug candidates. More recently, academic groups have used HTS to identify new chemical probes or small interfering RNA (siRNA) that can serve as experimental tools to examine the biology or physiology of novel proteins, processes, or interactions. HTS presents a significant challenge with the vast and complex nature of data generated. This report describes MScreen, a web-based, open-source cheminformatics application for chemical library and siRNA plate management, primary HTS and dose-response data handling, structure search, and administrative functions. Each project in MScreen can be secured with passwords or shared in an open information environment which enables collaborators to easily compare data from many screens, providing a useful means to identify compounds with desired selectivity. Unique features include compound, substance, mixture, and siRNA plate creation and formatting; automated dose-response fitting and quality control (QC); and user, target, and assay method administration. MScreen provides an effective means to facilitate HTS information handling and analysis in the academic setting so that users can efficiently view their screening data and evaluate results for follow-up. PMID:22706349

  5. Optimized high-throughput screen for Hepatitis C virus translation inhibitors

    PubMed Central

    Berry, Katherine E.; Peng, Betty; Koditek, David; Beeman, Douglas; Pagratis, Nikos; Perry, Jason K.; Parrish, Jay; Zhong, Weidong; Doudna, Jennifer A.; Shih, I-hung

    2011-01-01

    Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. We developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5′ capped mRNA control. We screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1,700 initial hits. After secondary counter screening the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end we found no selective HCV IRES inhibitors but did discover a new scaffold of general translation inhibitor. The analysis of these molecules, and the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors. PMID:21297107

  6. Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors within the ToxCast Phase I and II Chemical Libraries

    EPA Science Inventory

    High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limi...

  7. Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

    PubMed Central

    Gardner, Thomas J.; Bolovan-Fritts, Cynthia; Teng, Melissa W.; Redmann, Veronika; Kraus, Thomas A.; Sperling, Rhoda; Moran, Thomas; Britt, William; Weinberger, Leor S.

    2013-01-01

    Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169IE2-YFP) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection. PMID:23389931

  8. High-Throughput parallel blind Virtual Screening using BINDSURF

    PubMed Central

    2012-01-01

    Background Virtual Screening (VS) methods can considerably aid clinical research, predicting how ligands interact with drug targets. Most VS methods suppose a unique binding site for the target, usually derived from the interpretation of the protein crystal structure. However, it has been demonstrated that in many cases, diverse ligands interact with unrelated parts of the target and many VS methods do not take into account this relevant fact. Results We present BINDSURF, a novel VS methodology that scans the whole protein surface in order to find new hotspots, where ligands might potentially interact with, and which is implemented in last generation massively parallel GPU hardware, allowing fast processing of large ligand databases. Conclusions BINDSURF is an efficient and fast blind methodology for the determination of protein binding sites depending on the ligand, that uses the massively parallel architecture of GPUs for fast pre-screening of large ligand databases. Its results can also guide posterior application of more detailed VS methods in concrete binding sites of proteins, and its utilization can aid in drug discovery, design, repurposing and therefore help considerably in clinical research. PMID:23095663

  9. Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni Redox Cascade

    PubMed Central

    Simeonov, Anton; Jadhav, Ajit; Sayed, Ahmed A.; Wang, Yuhong; Nelson, Michael E.; Thomas, Craig J.; Inglese, James; Williams, David L.; Austin, Christopher P.

    2008-01-01

    Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 µL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (∼25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel

  10. Quantitative High-Throughput Screening Identifies 8-Hydroxyquinolines as Cell-Active Histone Demethylase Inhibitors

    PubMed Central

    Kawamura, Akane; Rose, Nathan R.; Ng, Stanley S.; Quinn, Amy M.; Rai, Ganesha; Mott, Bryan T.; Beswick, Paul; Klose, Robert J.; Oppermann, Udo; Jadhav, Ajit; Heightman, Tom D.; Maloney, David J.; Schofield, Christopher J.; Simeonov, Anton

    2010-01-01

    Background Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. Nε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. Nε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. Principal Findings High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. Conclusions These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation. PMID:21124847

  11. A high throughput screen for biomining cellulase activity from metagenomic libraries.

    PubMed

    Mewis, Keith; Taupp, Marcus; Hallam, Steven J

    2011-01-01

    Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production (1). Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils (2), buffalo rumen (3) and the termite hind-gut (4) using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood (5)). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio (6). Other methods have been reported (7,8) but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate (9). Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction (10). The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system. PMID:21307835

  12. High-Throughput Screening Platform for Engineered Nanoparticle-Mediated Genotoxicity Using CometChip Technology

    PubMed Central

    2015-01-01

    The likelihood of intentional and unintentional engineered nanoparticle (ENP) exposure has dramatically increased due to the use of nanoenabled products. Indeed, ENPs have been incorporated in many useful products and have enhanced our way of life. However, there are many unanswered questions about the consequences of nanoparticle exposures, in particular, with regard to their potential to damage the genome and thus potentially promote cancer. In this study, we present a high-throughput screening assay based upon the recently developed CometChip technology, which enables evaluation of single-stranded DNA breaks, abasic sites, and alkali-sensitive sites in cells exposed to ENPs. The strategic microfabricated, 96-well design and automated processing improves efficiency, reduces processing time, and suppresses user bias in comparison to the standard comet assay. We evaluated the versatility of this assay by screening five industrially relevant ENP exposures (SiO2, ZnO, Fe2O3, Ag, and CeO2) on both suspension human lymphoblastoid (TK6) and adherent Chinese hamster ovary (H9T3) cell lines. MTT and CyQuant NF assays were employed to assess cellular viability and proliferation after ENP exposure. Exposure to ENPs at a dose range of 5, 10, and 20 μg/mL induced dose-dependent increases in DNA damage and cytotoxicity. Genotoxicity profiles of ZnO > Ag > Fe2O3 > CeO2 > SiO2 in TK6 cells at 4 h and Ag > Fe2O3 > ZnO > CeO2 > SiO2 in H9T3 cells at 24 h were observed. The presented CometChip platform enabled efficient and reliable measurement of ENP-mediated DNA damage, therefore demonstrating the efficacy of this powerful tool in nanogenotoxicity studies. PMID:24617523

  13. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening.

    PubMed

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R; Fry, Stephen C

    2015-09-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes' biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme-cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors - especially compounds that generate singlet oxygen ((1)O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting (1)O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (-)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (-)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  14. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening

    PubMed Central

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R.; Fry, Stephen C.

    2015-01-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  15. High-Throughput Plasmid cDNA Library Screening

    SciTech Connect

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  16. A Three-groups Model for High Throughput Survival Screens

    PubMed Central

    Shaby, Benjamin A.; Skibinski, Gaia; Ando, Michael; LaDow, Eva S.; Finkbeiner, Steven

    2016-01-01

    Summary Amyotrophic lateral sclerosis (ALS) is a neurodegenerative condition characterized by the progressive deterioration of motor neurons in the cortex and spinal cord. Using an automated robotic microscope platform that enables the longitudinal tracking of thousands of single neurons, we examine the effects a large library of compounds on modulating the survival of primary neurons expressing a mutation known to cause ALS. The goal of our analysis is to identify the few potentially beneficial compounds among the many assayed, the vast majority of which do not extend neuronal survival. This resembles the large-scale simultaneous inference scenario familiar from microarray analysis, but transferred to the survival analysis setting due to the novel experimental setup. We apply a three component mixture model to censored survival times of thousands of individual neurons subjected to hundreds of different compounds. The shrinkage induced by our model significantly improves performance in simulations relative to performing treatment-wise survival analysis and subsequent multiple testing adjustment. Our analysis identified compounds that provide insight into potential novel therapeutic strategies for ALS. PMID:26821783

  17. Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi

    PubMed Central

    Bilsland, Elizabeth; Bean, Daniel M.; Devaney, Eileen; Oliver, Stephen G.

    2016-01-01

    Background Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7–15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases. Methodology/Principal Findings We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts), and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds), we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi. Conclusions/Significance We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between

  18. What Is High-Throughput Virtual Screening? A Perspective from Organic Materials Discovery

    NASA Astrophysics Data System (ADS)

    Pyzer-Knapp, Edward O.; Suh, Changwon; Gómez-Bombarelli, Rafael; Aguilera-Iparraguirre, Jorge; Aspuru-Guzik, Alán

    2015-07-01

    A philosophy for defining what constitutes a virtual high-throughput screen is discussed, and the choices that influence decisions at each stage of the computational funnel are investigated, including an in-depth discussion of the generation of molecular libraries. Additionally, we provide advice on the storing, analysis, and visualization of data on the basis of extensive experience in our research group.

  19. Evaluation of food-relevant chemicals in the ToxCast high-throughput screening program

    EPA Science Inventory

    There are thousands of chemicals that are directly added to or come in contact with food, many of which have undergone little to no toxicological evaluation. The ToxCast high-throughput screening (HTS) program has evaluated over 1,800 chemicals in concentration-response across ~8...

  20. PLASMA PROTEIN PROFILING AS A HIGH THROUGHPUT TOOL FOR CHEMICAL SCREENING USING A SMALL FISH MODEL

    EPA Science Inventory

    Hudson, R. Tod, Michael J. Hemmer, Kimberly A. Salinas, Sherry S. Wilkinson, James Watts, James T. Winstead, Peggy S. Harris, Amy Kirkpatrick and Calvin C. Walker. In press. Plasma Protein Profiling as a High Throughput Tool for Chemical Screening Using a Small Fish Model (Abstra...

  1. AOPs and Biomarkers: Bridging High Throughput Screening and Regulatory Decision Making

    EPA Science Inventory

    As high throughput screening (HTS) plays a larger role in toxicity testing, camputational toxicology has emerged as a critical component in interpreting the large volume of data produced. Computational models designed to quantify potential adverse effects based on HTS data will b...

  2. Incorporating Human Dosimetry and Exposure into High-Throughput In Vitro Toxicity Screening

    EPA Science Inventory

    Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multip...

  3. Liquid marbles for high-throughput biological screening of anchorage-dependent cells.

    PubMed

    Oliveira, Nuno M; Correia, Clara R; Reis, Rui L; Mano, João F

    2015-01-28

    Stable liquid marbles (LM) are produced by coating liquid droplets with a hydrophobic powder. The used hydrophobic powder is produced by fluorosi-lanization of diatomaceous earth, used before to produce superhydrophobic structures. Here, the use of LM is proposed for high-throughput drug screening on anchorage-dependent cells. To provide the required cell adhesion sites inside the liquid environment of LM, surface-modified poly(l-lactic acid) microparticles are used. A simple method that takes advantage from LM appealing features is presented, such as the ability to inject liquid on LM without disrupting (self-healing ability), and to monitor color changes inside of LM. After promoting cell adhesion, a cytotoxic screening test is performed as a proof of concept. Fe(3+) is used as a model cytotoxic agent and is injected on LM. After incubation, AlamarBlue reagent is injected and used to assess the presence of viable cells, by monitoring color change from blue to red. Color intensity is measured by image processing and the analysis of pictures takes using an ordinary digital camera. The proposed method is fully validated in counterpoint to an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carbo​xymethoxyphenyl)-2-(4-sulfophenyl)-2H-te​trazolium) colorimetric assay, a well-known method used for the cytotoxicity assessment. PMID:25091700

  4. High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility.

    PubMed

    Pattenden, Samantha G; Simon, Jeremy M; Wali, Aminah; Jayakody, Chatura N; Troutman, Jacob; McFadden, Andrew W; Wooten, Joshua; Wood, Cameron C; Frye, Stephen V; Janzen, William P; Davis, Ian J

    2016-03-15

    Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways. PMID:26929321

  5. Intersection of toxicogenomics and high throughput screening in the Tox21 program: an NIEHS perspective

    PubMed Central

    Paules, Richard S.; Tice, Raymond R.

    2015-01-01

    Humans are exposed to thousands of chemicals with inadequate toxicological data. Advances in computational toxicology, robotic high throughput screening (HTS), and genome-wide expression have been integrated into the Tox21 program to better predict the toxicological effects of chemicals. Tox21 is a collaboration among US government agencies initiated in 2008 that aims to shift chemical hazard assessment from traditional animal toxicology to target-specific, mechanism-based, biological observations using in vitro assays and lower organism models. HTS uses biocomputational methods for probing thousands of chemicals in in vitro assays for gene-pathway response patterns predictive of adverse human health outcomes. In 1999, NIEHS began exploring the application of toxicogenomics to toxicology and recent advances in NextGen sequencing should greatly enhance the biological content obtained from HTS platforms. We foresee an intersection of new technologies in toxicogenomics and HTS as an innovative development in Tox21. Tox21 goals, priorities, progress, and challenges will be reviewed. PMID:27122658

  6. Environmental surveillance and monitoring the next frontier for pathway-based high throughput screening

    EPA Science Inventory

    In response to a proposed vision and strategy for toxicity testing in the 21st century nascent high throughput toxicology (HTT) programs have tested thousands of chemicals in hundreds of pathway-based biological assays. Although, to date, use of HTT data for safety assessment of ...

  7. Prioritizing Environmental Chemicals for Obesity and Diabetes Outcomes Research: A Screening Approach Using ToxCast™ High-Throughput Data

    PubMed Central

    Auerbach, Scott; Filer, Dayne; Reif, David; Walker, Vickie; Holloway, Alison C.; Schlezinger, Jennifer; Srinivasan, Supriya; Svoboda, Daniel; Judson, Richard; Bucher, John R.; Thayer, Kristina A.

    2016-01-01

    Background: Diabetes and obesity are major threats to public health in the United States and abroad. Understanding the role that chemicals in our environment play in the development of these conditions is an emerging issue in environmental health, although identifying and prioritizing chemicals for testing beyond those already implicated in the literature is challenging. This review is intended to help researchers generate hypotheses about chemicals that may contribute to diabetes and to obesity-related health outcomes by summarizing relevant findings from the U.S. Environmental Protection Agency (EPA) ToxCast™ high-throughput screening (HTS) program. Objectives: Our aim was to develop new hypotheses around environmental chemicals of potential interest for diabetes- or obesity-related outcomes using high-throughput screening data. Methods: We identified ToxCast™ assay targets relevant to several biological processes related to diabetes and obesity (insulin sensitivity in peripheral tissue, pancreatic islet and β cell function, adipocyte differentiation, and feeding behavior) and presented chemical screening data against those assay targets to identify chemicals of potential interest. Discussion: The results of this screening-level analysis suggest that the spectrum of environmental chemicals to consider in research related to diabetes and obesity is much broader than indicated by research papers and reviews published in the peer-reviewed literature. Testing hypotheses based on ToxCast™ data will also help assess the predictive utility of this HTS platform. Conclusions: More research is required to put these screening-level analyses into context, but the information presented in this review should facilitate the development of new hypotheses. Citation: Auerbach S, Filer D, Reif D, Walker V, Holloway AC, Schlezinger J, Srinivasan S, Svoboda D, Judson R, Bucher JR, Thayer KA. 2016. Prioritizing environmental chemicals for obesity and diabetes outcomes research

  8. Novel method for the high-throughput processing of slides for the comet assay.

    PubMed

    Karbaschi, Mahsa; Cooke, Marcus S

    2014-01-01

    Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. "Scoring", or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure. PMID:25425241

  9. High Throughput Screening in Duchenne Muscular Dystrophy: From Drug Discovery to Functional Genomics

    PubMed Central

    Gintjee, Thomas J.J.; Magh, Alvin S.H.; Bertoni, Carmen

    2014-01-01

    Centers for the screening of biologically active compounds and genomic libraries are becoming common in the academic setting and have enabled researchers devoted to developing strategies for the treatment of diseases or interested in studying a biological phenomenon to have unprecedented access to libraries that, until few years ago, were accessible only by pharmaceutical companies. As a result, new drugs and genetic targets have now been identified for the treatment of Duchenne muscular dystrophy (DMD), the most prominent of the neuromuscular disorders affecting children. Although the work is still at an early stage, the results obtained to date are encouraging and demonstrate the importance that these centers may have in advancing therapeutic strategies for DMD as well as other diseases. This review will provide a summary of the status and progress made toward the development of a cure for this disorder and implementing high-throughput screening (HTS) technologies as the main source of discovery. As more academic institutions are gaining access to HTS as a valuable discovery tool, the identification of new biologically active molecules is likely to grow larger. In addition, the presence in the academic setting of experts in different aspects of the disease will offer the opportunity to develop novel assays capable of identifying new targets to be pursued as potential therapeutic options. These assays will represent an excellent source to be used by pharmaceutical companies for the screening of larger libraries providing the opportunity to establish strong collaborations between the private and academic sectors and maximizing the chances of bringing into the clinic new drugs for the treatment of DMD. PMID:25405319

  10. A systematic study of mitochondrial toxicity of environmental chemicals using quantitative high throughput screening

    PubMed Central

    Attene-Ramos, Matias S.; Huang, Ruili; Sakamuru, Srilatha; Witt, Kristine L.; Beeson, Gyda C.; Shou, Louie; Schnellmann, Rick G.; Beeson, Craig C.; Tice, Raymond R.; Austin, Christopher P.; Xia, Menghang

    2014-01-01

    A goal of the Tox21 program is to transit toxicity testing from traditional in vivo models to in vitro assays that assess how chemicals affect cellular responses and toxicity pathways. A critical contribution of the NIH Chemical Genomics center (NCGC) to the Tox21 program is the implementation of a quantitative high throughput screening (qHTS) approach, using cell- and biochemical-based assays to generate toxicological profiles for thousands of environmental compounds. Here, we evaluated the effect of chemical compounds on mitochondrial membrane potential in HepG2 cells by screening a library of 1,408 compounds provided by the National Toxicology Program (NTP) in a qHTS platform. Compounds were screened over 14 concentrations, and results showed that 91 and 88 compounds disrupted mitochondrial membrane potential after treatment for one or five h, respectively. Seventy-six compounds active at both time points were clustered by structural similarity, producing 11 clusters and 23 singletons. Thirty-eight compounds covering most of the active chemical space were more extensively evaluated. Thirty-six of the 38 compounds were confirmed to disrupt mitochondrial membrane potential using a fluorescence plate reader and 35 were confirmed using a high content imaging approach. Among the 38 compounds, 4 and 6 induced LDH release, a measure of cytotoxicity, at 1 or 5 h, respectively. Compounds were further assessed for mechanism of action (MOA) by measuring changes in oxygen consumption rate, which enabled identification of 20 compounds as uncouplers. This comprehensive approach allows for evaluation of thousands of environmental chemicals for mitochondrial toxicity and identification of possible MOAs. PMID:23895456

  11. Identification of multiple salicylic acid-binding proteins using two high throughput screens

    PubMed Central

    Manohar, Murli; Tian, Miaoying; Moreau, Magali; Park, Sang-Wook; Choi, Hyong Woo; Fei, Zhangjun; Friso, Giulia; Asif, Muhammed; Manosalva, Patricia; von Dahl, Caroline C.; Shi, Kai; Ma, Shisong; Dinesh-Kumar, Savithramma P.; O'Doherty, Inish; Schroeder, Frank C.; van Wijk, Klass J.; Klessig, Daniel F.

    2014-01-01

    Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays. PMID:25628632

  12. A novel high-throughput activity assay for the Trypanosoma brucei editosome enzyme REL1 and other RNA ligases

    PubMed Central

    Zimmermann, Stephan; Hall, Laurence; Riley, Sean; Sørensen, Jesper; Amaro, Rommie E.; Schnaufer, Achim

    2016-01-01

    The protist parasite Trypanosoma brucei causes Human African trypanosomiasis (HAT), which threatens millions of people in sub-Saharan Africa. Without treatment the infection is almost always lethal. Current drugs for HAT are difficult to administer and have severe side effects. Together with increasing drug resistance this results in urgent need for new treatments. T. brucei and other trypanosomatid pathogens require a distinct form of post-transcriptional mRNA modification for mitochondrial gene expression. A multi-protein complex called the editosome cleaves mitochondrial mRNA, inserts or deletes uridine nucleotides at specific positions and re-ligates the mRNA. RNA editing ligase 1 (REL1) is essential for the re-ligation step and has no close homolog in the mammalian host, making it a promising target for drug discovery. However, traditional assays for RELs use radioactive substrates coupled with gel analysis and are not suitable for high-throughput screening of compound libraries. Here we describe a fluorescence-based REL activity assay. This assay is compatible with a 384-well microplate format and sensitive, satisfies statistical criteria for high-throughput methods and is readily adaptable for other polynucleotide ligases. We validated the assay by determining kinetic properties of REL1 and by identifying REL1 inhibitors in a library of small, pharmacologically active compounds. PMID:26400159

  13. Ultra High Throughput Screening of Natural Product Extracts to Identify Pro-apoptotic Inhibitors of Bcl-2 Family Proteins

    PubMed Central

    Hassig, Christian A.; Zeng, Fu-Yue; Kung, Paul; Kiankarimi, Mehrak; Kim, Sylvia; Diaz, Paul W.; Zhai, Dayong; Welsh, Kate; Morshedian, Shana; Su, Ying; O'Keefe, Barry; Newman, David J.; Rusman, Yudi; Kaur, Harneet; Salomon, Christine E.; Brown, Susan G.; Baire, Beeraiah; Michel, Andrew R.; Hoye, Thomas R.; Francis, Subhashree; Georg, Gunda I.; Walters, Michael A.; Divlianska, Daniela B.; Roth, Gregory P.; Wright, Amy E.; Reed, John C.

    2015-01-01

    Anti-apoptotic Bcl-2 family proteins are validated cancer targets comprised of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). While several isoform-selective inhibitors have been developed using structure-based design or high throughput screening (HTS) of synthetic chemical libraries, no large scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six anti-apoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally-relevant PPIs. The screens were conducted in 1,536-well format and displayed satisfactory overall HTS statistics, with Z’-factor values ranging from 0.72 to 0.83, and a hit confirmation rate between 16-64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra high throughput screening using natural product sources and highlight some of the challenges associated with this approach. PMID:24870016

  14. Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries

    PubMed Central

    Kalber, Tammy; Badar, Adam; Lythgoe, Mark; Pule, Martin

    2015-01-01

    The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins. PMID:26536118

  15. Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries.

    PubMed

    Stowe, Cassandra; Pizzey, Arnold; Kalber, Tammy; Badar, Adam; Lythgoe, Mark; Pule, Martin

    2015-01-01

    The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins. PMID:26536118

  16. A cell-based quantitative high-throughput image screening identified novel autophagy modulators.

    PubMed

    Li, Yuan; McGreal, Steven; Zhao, Jean; Huang, Ruili; Zhou, Yan; Zhong, Hua; Xia, Menghang; Ding, Wen-Xing

    2016-08-01

    Macroautophagy is a major cellular degradation pathway for long-lived proteins and cellular organelles to maintain cellular homeostasis. Reduced autophagy has been implicated in neurodegenerative diseases, metabolic syndrome, and tumorigenesis. In contrast, increased autophagy has been shown to protect against tissue injury and aging. Here we employed a cell-based quantitative high-throughput image screening (qHTS) for autophagy modulators using mouse embryonic fibroblasts (MEFs) that are stably expressing GFP-LC3. The library of pharmacologically active compounds (LOPAC) was used to screen for the autophagy modulators in compounds alone or in combination with the lysosome inhibitor chloroquine (CQ). The GFP-LC3 puncta were then quantified to measure autophagic flux. The primary screening revealed 173 compounds with efficacy more than 40%. These compounds were cherry-picked and re-tested at multiple different concentrations using the same assay. A number of novel autophagy inducers, inhibitors, and modulators with dual-effects on autophagy were identified from the cherry-pick screening. Interestingly, we found a group of compounds that induce autophagy are related to dopamine receptors and are commonly used as clinical psychiatric drugs. Among them, indatraline hydrochloride (IND), a dopamine inhibitor, and chlorpromazine hydrochloride (CPZ) and fluphenazine dihydrochloride (FPZ), two dopamine receptor antagonists, were further evaluated. We found that FPZ-induced autophagy through mTOR inhibition but IND and CPZ induced autophagy in an mTOR-independent manner. Our data suggest that image-based autophagic flux qHTS can efficiently identify autophagy inducers and inhibitors. PMID:27168224

  17. A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins.

    PubMed

    Izumi Willcoxon, Michi; Dennis, Jaclyn R; Lau, Sabina I; Xie, Weiping; You, You; Leng, Song; Fong, Ryan C; Yamamoto, Takashi

    2016-01-10

    A high-throughput, in-vitro assay for Bacillus thuringiensis (Bt) insecticidal proteins designated as Cry was developed and evaluated for screening a large number of Cry protein variants produced by DNA shuffling. This automation-amenable assay exploits an insect cell line expressing a single receptor of Bt Cry proteins. The Cry toxin used to develop this assay is a variant of the Cry1Ab protein called IP1-88, which was produced previously by DNA shuffling. Cell mortality caused by the activated Bt Cry toxin was determined by chemical cell viability assay in 96/384-well microtiter plates utilizing CellTiter 96(®) obtained from Promega. A widely-accepted mode-of-action theory of certain Bt Cry proteins suggests that the activated toxin binds to one or more receptors and forms a pore through the insect gut epithelial cell apical membrane. A number of insect proteins such as cadherin-like protein (Cad), aminopeptidase-N (APN), alkaline phosphatase (ALP) and ABC transporter (ABCC) have been identified as the receptors of Bt Cry toxins. In this study, Bt Cry toxin receptors Ostrinia nubilalis (European corn borer) cadherin-like protein (On-Cad) and aminopeptidase-N 1 and 3 (On-APN1, On-APN3) and Spodoptera frugiperda (fall armyworm) cadherin-like protein (Sf-Cad) were cloned in an insect cell line, Sf21, and a mammalian cell line, Expi293F. It was observed by ligand blotting and immunofluorescence microscopy that trypsin-activated IP1-88 bound to On-Cad and On-APN1, but not Sf-Cad or On-APN3. In contrast, IP1-88 bound only to APN1 in BBMV (Brush Border Membrane Vesicles) prepared from the third and fourth-instar O. nubilalis larval midgut. The sensitivity of the recombinant cells to the toxin was then tested. IP1-88 showed no toxicity to non-recombinant Sf21 and Expi293F. Toxicity was observed only when the On-Cad gene was cloned and expressed. Sf-Cad and On-APN1 were not able to make those cells sensitive to the toxin. Since the expression of On-Cad alone was

  18. Predictive models of prenatal developmental toxicity from ToxCast high-throughput screening data.

    PubMed

    Sipes, Nisha S; Martin, Matthew T; Reif, David M; Kleinstreuer, Nicole C; Judson, Richard S; Singh, Amar V; Chandler, Kelly J; Dix, David J; Kavlock, Robert J; Knudsen, Thomas B

    2011-11-01

    Environmental Protection Agency's ToxCast project is profiling the in vitro bioactivity of chemicals to assess pathway-level and cell-based signatures that correlate with observed in vivo toxicity. We hypothesized that developmental toxicity in guideline animal studies captured in the ToxRefDB database would correlate with cell-based and cell-free in vitro high-throughput screening (HTS) data to reveal meaningful mechanistic relationships and provide models identifying chemicals with the potential to cause developmental toxicity. To test this hypothesis, we built statistical associations based on HTS and in vivo developmental toxicity data from ToxRefDB. Univariate associations were used to filter HTS assays based on statistical correlation with distinct in vivo endpoint. This revealed 423 total associations with distinctly different patterns for rat (301 associations) and rabbit (122 associations) across multiple HTS assay platforms. From these associations, linear discriminant analysis with cross-validation was used to build the models. Species-specific models of predicted developmental toxicity revealed strong balanced accuracy (> 70%) and unique correlations between assay targets such as transforming growth factor beta, retinoic acid receptor, and G-protein-coupled receptor signaling in the rat and inflammatory signals, such as interleukins (IL) (IL1a and IL8) and chemokines (CCL2), in the rabbit. Species-specific toxicity endpoints were associated with one another through common Gene Ontology biological processes, such as cleft palate to urogenital defects through placenta and embryonic development. This work indicates the utility of HTS assays for developing pathway-level models predictive of developmental toxicity. PMID:21873373

  19. A fluorescence high throughput screening method for the detection of reactive electrophiles as potential skin sensitizers.

    PubMed

    Avonto, Cristina; Chittiboyina, Amar G; Rua, Diego; Khan, Ikhlas A

    2015-12-01

    Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, 'HTS-DCYA assay', is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time. PMID:26455772

  20. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Fenglei Li

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  1. Evaluation of high-throughput genotoxicity assays used in profiling the US EPA ToxCast chemicals.

    PubMed

    Knight, Andrew W; Little, Stephen; Houck, Keith; Dix, David; Judson, Richard; Richard, Ann; McCarroll, Nancy; Akerman, Gregory; Yang, Chihae; Birrell, Louise; Walmsley, Richard M

    2009-11-01

    Three high-throughput screening (HTS) genotoxicity assays-GreenScreen HC GADD45a-GFP (Gentronix Ltd.), CellCiphr p53 (Cellumen Inc.) and CellSensor p53RE-bla (Invitrogen Corp.)-were used to analyze the collection of 320 predominantly pesticide active compounds being tested in Phase I of US. Environmental Protection Agency's ToxCast research project. Between 9% and 12% of compounds were positive for genotoxicity in the assays. However, results of the varied tests only partially overlapped, suggesting a strategy of combining data from a battery of assays. The HTS results were compared to mutagenicity (Ames) and animal tumorigenicity data. Overall, the HTS assays demonstrated low sensitivity for rodent tumorigens, likely due to: screening at a low concentration, coverage of selected genotoxic mechanisms, lack of metabolic activation and difficulty detecting non-genotoxic carcinogens. Conversely, HTS results demonstrated high specificity, >88%. Overall concordance of the HTS assays with tumorigenicity data was low, around 50% for all tumorigens, but increased to 74-78% (vs. 60% for Ames) for those compounds producing tumors in rodents at multiple sites and, thus, more likely genotoxic carcinogens. The aim of the present study was to evaluate the utility of HTS assays to identify potential genotoxicity hazard in the larger context of the ToxCast project, to aid prioritization of environmentally relevant chemicals for further testing and assessment of carcinogenicity risk to humans. PMID:19591892

  2. Accelerating the Discovery of Biologically Active Small Molecules Using a High-Throughput Yeast Halo Assay#

    PubMed Central

    Gassner, Nadine C.; Tamble, Craig M.; Bock, Jonathan E.; Cotton, Naomi; White, Kimberly N.; Tenney, Karen; St. Onge, Robert P.; Proctor, Michael J.; Giaever, Guri; Davis, Ronald W.; Crews, Phillip; Holman, Theodore R.; Lokey, R. Scott

    2008-01-01

    The budding yeast Saccharomyces cerevisiae, a powerful model system for the study of basic eukaryotic cell biology, has been used increasingly as a screening tool for the identification of bioactive small molecules. We have developed a novel yeast toxicity screen that is easily automated and compatible with high-throughput screening robotics. The new screen is quantitative and allows inhibitory potencies to be determined, since the diffusion of the sample provides a concentration gradient and a corresponding toxicity halo. The efficacy of this new screen was illustrated by testing materials including 3,104 compounds from the NCI libraries, 167 marine sponge crude extracts, and 149 crude marine-derived fungal extracts. There were 46 active compounds among the NCI set. One very active extract was selected for bioactivity-guided fractionation resulting in the identification of crambescidin 800 as a potent antifungal agent. PMID:17291044

  3. [High-throughput functional screening using CRISPR/Cas9 system].

    PubMed

    Wang, Gancheng; Ming, Ma; Ye, Yanzhen; Xi, Jianzhong

    2016-05-01

    High-throughput screening, a powerful tool for the discovery of functionally important genes responsible for certain phenotypes, is performed according to loss-of-function or gain-of-function strategies. RNAi technology or knockout approaches have been widely used in high throughput screening due to their advantages of ease use, low cost and so on. However, imcomplete knockdown activity and off-target effect hindered their utility. More recently, CRISPR/Cas9 technology is becoming a robust tool for genome editing in diverse cells or animals, since it could generate a gene mutation in a target-specific manner. In this review, we first summarize the characterization of CRISPR/Cas9 and make comparison with traditional genetic tools, then describe recent achievements of genetic screen in several model organisms using CRISPR/Cas9, finally discuss on its future challenges and opportunities. PMID:27232487

  4. Establishing an Infrastructure for High-Throughput Short-Interfering RNA Screening.

    PubMed

    Yin, Hongwei; Sereduk, Chris; Tang, Nanyun

    2016-01-01

    RNA interference (RNAi) is a readily available research tool that can be used to accelerate the identification and functional validation of a multitude of new candidate drug targets by experimentally perturbing gene expression and function. High-throughput RNAi technology using libraries of short-interfering RNA (siRNA) makes it possible to rapidly identify genes and biomarkers associated with biological processes such as diseases or a cellular response to therapy. Thus, RNAi-based screening is an extremely powerful technology that can provide tremendous insights into the mechanisms of action and contexts of vulnerability of a particular drug treatment. This chapter describes the infrastructure requirements needed to successfully perform HT-RNAi screening. Information on the methodology, instrumentation, experimental design, and workflow aspects is provided, as well as insights on how to successfully implement a high-throughput RNAi screen. PMID:27581280

  5. Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity.

    PubMed

    Emert-Sedlak, Lori A; Narute, Purushottam; Shu, Sherry T; Poe, Jerrod A; Shi, Haibin; Yanamala, Naveena; Alvarado, John Jeff; Lazo, John S; Yeh, Joanne I; Johnston, Paul A; Smithgall, Thomas E

    2013-01-24

    HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of nonenzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents. PMID:23352142

  6. Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay.

    PubMed

    Kwon, Keehwan; Nagarajan, Rajesh; Stivers, James T

    2004-11-30

    Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover. PMID:15554707

  7. Bis-benzimidazole hits against Naegleria fowleri discovered with new high-throughput screens.

    PubMed

    Rice, Christopher A; Colon, Beatrice L; Alp, Mehmet; Göker, Hakan; Boykin, David W; Kyle, Dennis E

    2015-04-01

    Naegleria fowleri is a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two new in vitro assays suitable for medium- or high-throughput drug discovery and used these for N. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono- and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC50) of 177 nM. Similarly, we identified 10 additional analogues with IC50s of <1 μM, with many of these having reasonable selectivity indices. The most potent hits were >500 times more potent than pentamidine. In summary, the mono- and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections with N. fowleri. PMID:25605363

  8. Bis-Benzimidazole Hits against Naegleria fowleri Discovered with New High-Throughput Screens

    PubMed Central

    Rice, Christopher A.; Colon, Beatrice L.; Alp, Mehmet; Göker, Hakan; Boykin, David W.

    2015-01-01

    Naegleria fowleri is a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two new in vitro assays suitable for medium- or high-throughput drug discovery and used these for N. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono- and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC50) of 177 nM. Similarly, we identified 10 additional analogues with IC50s of <1 μM, with many of these having reasonable selectivity indices. The most potent hits were >500 times more potent than pentamidine. In summary, the mono- and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections with N. fowleri. PMID:25605363

  9. High-throughput metabolic genotoxicity screening with a fluidic microwell chip and electrochemiluminescence†

    PubMed Central

    Wasalathanthri, Dhanuka P.; Malla, Spundana; Bist, Itti; Tang, Chi K.; Faria, Ronaldo C.; Rusling, James F.

    2014-01-01

    A high throughput electrochemiluminescent (ECL) chip was fabricated and integrated into a fluidic system for screening toxicity-related chemistry of drug and pollutant metabolites. The chip base is conductive pyrolytic graphite onto which are printed 64 microwells capable of holding one-µL droplets. Films combining DNA, metabolic enzymes and an ECL-generating ruthenium metallopolymer (RuIIPVP) are fabricated in these microwells. The system runs metabolic enzyme reactions, and subsequently detects DNA damage caused by reactive metabolites. The performance of the chip was tested by measuring DNA damage caused by metabolites of the well-known procarcinogen benzo[a]pyrene (B[a]P). Liver microsomes and cytochrome P450 (cyt P450) enzymes were used with and without epoxide hydrolase (EH), a conjugative enzyme required for multi-enzyme bioactivation of B[a]P. DNA adduct formation was confirmed by determining specific DNA-metabolite adducts using similar films of DNA/enzyme on magnetic bead biocolloid reactors, hydrolyzing the DNA, and analyzing by capillary liquid chromatography-mass spectrometry (CapLC-MS/MS). The fluidic chip was also used to measure IC50-values of inhibitors of cyt P450s. All results show good correlation with reported enzyme activity and inhibition assays. PMID:24113555

  10. Monoclonal antibodies as surrogate receptors in a high throughput screen for compounds that enhance insulin sensitivity.

    PubMed

    Bright, S W; Gold, G; Sage, S W; Sportsman, J R; Tinsley, F C; Dominianni, S J; Schmiegel, K K; Kellam, M L; Fitch, L L; Yen, T T

    1997-01-01

    Monoclonal antibodies (MoAbs) were made to a known insulin sensitivity enhancer (ISE) compound, CS-045. The MoAbs were characterized with respect to binding other known thiazolidinedione ISE compounds using a CS-045 labeled with b-phycoerythrin in a competitive particle concentration fluorescence immunoassay (PCFIA). By comparing the rank order of IC50 values for each compound to its respective potency as an ISE, one MoAb (13E3) was selected for further characterization. This MoAb was also used as a surrogate receptor in a high throughput screen to identify novel compounds that compete for binding to CS-045. Some of the hits were found to have efficacy in reducing blood glucose. Subsequently, another group reported that several compounds with the core thiazolidinedione structure of the ISE compounds bound with high affinity to peroxisome proliferator-activating receptors (PPAR). Therefore, we used the MoAb assay to test these and other compounds that are known to bind to PPARgamma and noted crossreactivity with some of the compounds. PMID:9408053

  11. Discovery of bile salt hydrolase inhibitors using an efficient high-throughput screening system.

    PubMed

    Smith, Katie; Zeng, Ximin; Lin, Jun

    2014-01-01

    The global trend of restricting the use of antibiotic growth promoters (AGP) in animal production necessitates the need to develop valid alternatives to maintain productivity and sustainability of food animals. Previous studies suggest inhibition of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization, is a promising approach to promote animal growth performance. To achieve the long term goal of developing novel alternatives to AGPs, in this study, a rapid and convenient high-throughput screening (HTS) system was developed and successfully used for identification of BSH inhibitors. With the aid of a high-purity BSH from a chicken Lactobacillus salivarius strain, we optimized various screening conditions (e.g. BSH concentration, reaction buffer pH, incubation temperature and length, substrate type and concentration) and establish a precipitation-based screening approach to identify BSH inhibitors using 96-well or 384-well microplates. A pilot HTS was performed using a small compound library comprised of 2,240 biologically active and structurally diverse compounds. Among the 107 hits, several promising and potent BSH inhibitors (e.g. riboflavin and phenethyl caffeate) were selected and validated by standard BSH activity assay. Interestingly, the HTS also identified a panel of antibiotics as BSH inhibitor; in particular, various tetracycline antibiotics and roxarsone, the widely used AGP, have been demonstrated to display potent inhibitory effect on BSH. Together, this study developed an efficient HTS system and identified several BSH inhibitors with potential as alternatives to AGP. In addition, the findings from this study also suggest a new mode of action of AGP for promoting animal growth. PMID:24454844

  12. Discovery of Bile Salt Hydrolase Inhibitors Using an Efficient High-Throughput Screening System

    PubMed Central

    Smith, Katie; Zeng, Ximin; Lin, Jun

    2014-01-01

    The global trend of restricting the use of antibiotic growth promoters (AGP) in animal production necessitates the need to develop valid alternatives to maintain productivity and sustainability of food animals. Previous studies suggest inhibition of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization, is a promising approach to promote animal growth performance. To achieve the long term goal of developing novel alternatives to AGPs, in this study, a rapid and convenient high-throughput screening (HTS) system was developed and successfully used for identification of BSH inhibitors. With the aid of a high-purity BSH from a chicken Lactobacillus salivarius strain, we optimized various screening conditions (e.g. BSH concentration, reaction buffer pH, incubation temperature and length, substrate type and concentration) and establish a precipitation-based screening approach to identify BSH inhibitors using 96-well or 384-well microplates. A pilot HTS was performed using a small compound library comprised of 2,240 biologically active and structurally diverse compounds. Among the 107 hits, several promising and potent BSH inhibitors (e.g. riboflavin and phenethyl caffeate) were selected and validated by standard BSH activity assay. Interestingly, the HTS also identified a panel of antibiotics as BSH inhibitor; in particular, various tetracycline antibiotics and roxarsone, the widely used AGP, have been demonstrated to display potent inhibitory effect on BSH. Together, this study developed an efficient HTS system and identified several BSH inhibitors with potential as alternatives to AGP. In addition, the findings from this study also suggest a new mode of action of AGP for promoting animal growth. PMID:24454844

  13. Predictive model of rat reproductive toxicity from ToxCast high throughput screening.

    PubMed

    Martin, Matthew T; Knudsen, Thomas B; Reif, David M; Houck, Keith A; Judson, Richard S; Kavlock, Robert J; Dix, David J

    2011-08-01

    The U.S. Environmental Protection Agency's ToxCast research program uses high throughput screening (HTS) for profiling bioactivity and predicting the toxicity of large numbers of chemicals. ToxCast Phase I tested 309 well-characterized chemicals in more than 500 assays for a wide range of molecular targets and cellular responses. Of the 309 environmental chemicals in Phase I, 256 were linked to high-quality rat multigeneration reproductive toxicity studies in the relational Toxicity Reference Database. Reproductive toxicants were defined here as having achieved a reproductive lowest-observed-adverse-effect level of less than 500 mg kg(-1) day(-1). Eight-six chemicals were identified as reproductive toxicants in the rat, and 68 of those had sufficient in vitro bioactivity to model. Each assay was assessed for univariate association with the identified reproductive toxicants. Significantly associated assays were linked to gene sets and used for the subsequent predictive modeling. Using linear discriminant analysis and fivefold cross-validation, a robust and stable predictive model was produced capable of identifying rodent reproductive toxicants with 77% ± 2% and 74% ± 5% (mean ± SEM) training and test cross-validation balanced accuracies, respectively. With a 21-chemical external validation set, the model was 76% accurate, further indicating the model's potential for prioritizing the many thousands of environmental chemicals with little to no hazard information. The biological features of the model include steroidal and nonsteroidal nuclear receptors, cytochrome P450 enzyme inhibition, G protein-coupled receptors, and cell signaling pathway readouts-mechanistic information suggesting additional targeted, integrated testing strategies and potential applications of in vitro HTS to risk assessment. PMID:21565999

  14. A high-throughput neutralizing assay for antibodies and sera against hepatitis E virus

    PubMed Central

    Cai, Wei; Tang, Zi-Min; Wen, Gui-Ping; Wang, Si-Ling; Ji, Wen-Fang; Yang, Min; Ying, Dong; Zheng, Zi-Zheng; Xia, Ning-Shao

    2016-01-01

    Hepatitis E virus (HEV) is the aetiological agent of enterically transmitted hepatitis. The traditional methods for evaluating neutralizing antibody titres against HEV are real-time PCR and the immunofluorescence foci assay (IFA), which are poorly repeatable and operationally complicated, factors that limit their applicability to high-throughput assays. In this study, we developed a novel high-throughput neutralizing assay based on biotin-conjugated p239 (HEV recombinant capsid proteins, a.a. 368–606) and staining with allophycocyanin-conjugated streptavidin (streptavidin APC) to amplify the fluorescence signal. A linear regression analysis indicated that there was a high degree of correlation between IFA and the novel assay. Using this method, we quantitatively evaluated the neutralization of sera from HEV-infected and vaccinated macaques. The anti-HEV IgG level had good concordance with the neutralizing titres of macaque sera. However, the neutralization titres of the sera were also influenced by anti-HEV IgM responses. Further analysis also indicated that, although vaccination with HEV vaccine stimulated higher anti-HEV IgG and neutralization titres than infection with HEV in macaques, the proportions of neutralizing antibodies in the infected macaques’ sera were higher than in the vaccinated macaques with the same anti-HEV IgG levels. Thus, the infection more efficiently stimulated neutralizing antibody responses. PMID:27122081

  15. Novel diversity-oriented synthesis-derived respiratory syncytial virus inhibitors identified via a high throughput replicon-based screen.

    PubMed

    Duvall, Jeremy R; VerPlank, Lynn; Ludeke, Barbara; McLeod, Sarah M; Lee, Maurice D; Vishwanathan, Karthick; Mulrooney, Carol A; Le Quement, Sebastian; Yu, Qin; Palmer, Michelle A; Fleming, Paul; Fearns, Rachel; Foley, Michael A; Scherer, Christina A

    2016-07-01

    Respiratory syncytial virus (RSV) infections affect millions of children and adults every year. Despite the significant disease burden, there are currently no safe and effective vaccines or therapeutics. We employed a replicon-based high throughput screen combined with live-virus triaging assays to identify three novel diversity-oriented synthesis-derived scaffolds with activity against RSV. One of these small molecules is shown to target the RSV polymerase (L protein) to inhibit viral replication and transcription; the mechanisms of action of the other small molecules are currently unknown. The compounds described herein may provide attractive inhibitors for lead optimization campaigns. PMID:27059228

  16. A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting.

    PubMed

    Tseng, Hubert; Gage, Jacob A; Haisler, William L; Neeley, Shane K; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G; Wagoner, Matthew; Souza, Glauco R

    2016-01-01

    Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

  17. A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting

    PubMed Central

    Tseng, Hubert; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G.; Wagoner, Matthew; Souza, Glauco R.

    2016-01-01

    Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

  18. Probe molecules (PrM) approach in adverse outcome pathway (AOP) based high throughput screening (HTS): in vivo discovery for developing in vitro target methods

    EPA Science Inventory

    Efficient and accurate adverse outcome pathway (AOP) based high-throughput screening (HTS) methods use a systems biology based approach to computationally model in vitro cellular and molecular data for rapid chemical prioritization; however, not all HTS assays are grounded by rel...

  19. Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

    PubMed

    Zottig, Ximena; Meddeb-Mouelhi, Fatma; Beauregard, Marc

    2016-03-01

    A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility. PMID:26706798

  20. High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

    PubMed Central

    Han, Xiao; Guo, Jinhai; Deng, Weiwei; Zhang, Chenying; Du, Peige; Shi, Taiping; Ma, Dalong

    2008-01-01

    Background Estrogen receptor α (ERα) is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE) with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131) as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE). In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells. PMID:18847501

  1. The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond.

    PubMed

    Haslam, Carl; Hellicar, John; Dunn, Adrian; Fuetterer, Arne; Hardy, Neil; Marshall, Peter; Paape, Rainer; Pemberton, Michelle; Resemannand, Anja; Leveridge, Melanie

    2016-02-01

    Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent RapidFire system, have emerged that are capable of analyzing samples in <10 s. While dramatically faster than liquid chromatography-coupled MS, an analysis time of 8-10 s is still considered relatively slow for full-diversity high-throughput screening (HTS). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) offers an alternative for high-throughput MS detection. However, sample preparation and deposition onto the MALDI target, as well as interference from matrix ions, have been considered limitations for the use of MALDI for screening assays. Here we describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, we have generated robust data in a 1536 format and also increased sample deposition to 6144 samples per target. Using these methods, we demonstrate that this technology can deliver fast sample analysis time with low sample volume, and data comparable to that of current RapidFire assays. PMID:26428484

  2. High-Throughput Yeast-Based Reporter Assay to Identify Compounds with Anti-inflammatory Potential.

    PubMed

    Garcia, G; Santos, C Nunes do; Menezes, R

    2016-01-01

    The association between altered proteostasis and inflammatory responses has been increasingly recognized, therefore the identification and characterization of novel compounds with anti-inflammatory potential will certainly have a great impact in the therapeutics of protein-misfolding diseases such as degenerative disorders. Although cell-based screens are powerful approaches to identify potential therapeutic compounds, establishing robust inflammation models amenable to high-throughput screening remains a challenge. To bridge this gap, we have exploited the use of yeasts as a platform to identify lead compounds with anti-inflammatory properties. The yeast cell model described here relies on the high-degree homology between mammalian and yeast Ca(2+)/calcineurin pathways converging into the activation of NFAT and Crz1 orthologous proteins, respectively. It consists of a recombinant yeast strain encoding the lacZ gene under the control of Crz1-recongition elements to facilitate the identification of compounds interfering with Crz1 activation through the easy monitoring of β-galactosidase activity. Here, we describe in detail a protocol optimized for high-throughput screening of compounds with potential anti-inflammatory activity as well as a protocol to validate the positive hits using an alternative β-galactosidase substrate. PMID:27613055

  3. Label-Free Surface Enhanced Raman Scattering Approach for High-Throughput Screening of Biocatalysts.

    PubMed

    Westley, Chloe; Xu, Yun; Carnell, Andrew J; Turner, Nicholas J; Goodacre, Royston

    2016-06-01

    Biocatalyst discovery and directed evolution are central to many pharmaceutical research programs, yet the lack of robust high-throughput screening methods for large libraries of enzyme variants generated (typically 10(6)-10(8)) has hampered progress and slowed enzyme optimization. We have developed a label-free generally applicable approach based on Raman spectroscopy which results in significant reductions in acquisition times (>30-fold). Surface enhanced Raman scattering (SERS) is employed to monitor the enzyme-catalyzed conversion by xanthine oxidase of hypoxanthine to xanthine to uric acid. This approach measures the substrates and products directly and does not require chromogenic substrates or lengthy chromatography, was successfully benchmarked against HPLC, and shows high levels of accuracy and reproducibility. Furthermore, we demonstrate that this SERS approach has utility in monitoring enzyme inhibition illustrating additional medical significance to this high-throughput screening method. PMID:27132981

  4. Computational high-throughput screening of fluid permeability in heterogeneous fiber materials.

    PubMed

    Röding, Magnus; Schuster, Erich; Logg, Katarina; Lundman, Malin; Bergström, Per; Hanson, Charlotta; Gebäck, Tobias; Lorén, Niklas

    2016-07-20

    We explore computational high-throughput screening as a design strategy for heterogeneous, isotropic fiber materials. Fluid permeability, a key property in the design of soft porous materials, is systematically studied using a multi-scale lattice Boltzmann framework. After characterizing microscopic permeability as a function of solid volume fraction in the microstructure, we perform high-throughput computational screening of in excess of 35 000 macrostructures consisting of a continuous bulk interrupted by spherical/elliptical domains with either lower or higher microscopic permeability (hence with two distinct microscopic solid volume fractions and therefore two distinct microscopic permeabilities) to assess which parameters determine macroscopic permeability for a fixed average solid volume fraction. We conclude that the fractions of bulk and domains and the distribution of solid volume fraction between them are the primary determinants of macroscopic permeability, and that a substantial increase in permeability compared to the corresponding homogenous material is attainable. PMID:27367292

  5. Design and Application of a Novel High-throughput Screening Technique for 1-Deoxynojirimycin

    PubMed Central

    Jiang, Peixia; Mu, Shanshan; Li, Heng; Li, Youhai; Feng, Congmin; Jin, Jian-Ming; Tang, Shuang-Yan

    2015-01-01

    High-throughput screening techniques for small molecules can find intensive applications in the studies of biosynthesis of these molecules. A sensitive, rapid and cost-effective technique that allows high-throughput screening of endogenous production of the natural iminosugar 1-deoxynojirimycin (1-DNJ), an α-glucosidase inhibitor relevant to the pharmaceutical industry, was developed in this study, based on the inhibitory effects of 1-DNJ on the activity of the β-glycosidase LacS from Sulfolobus solfataricus. This technique has been demonstrated effective in engineering both the key enzyme and the expression levels of enzymes in the 1-DNJ biosynthetic pathway from Bacillus atrophaeus cloned in E. coli. Higher biosynthetic efficiency was achieved using directed evolution strategies. PMID:25708517

  6. Design and application of a novel high-throughput screening technique for 1-deoxynojirimycin.

    PubMed

    Jiang, Peixia; Mu, Shanshan; Li, Heng; Li, Youhai; Feng, Congmin; Jin, Jian-Ming; Tang, Shuang-Yan

    2015-01-01

    High-throughput screening techniques for small molecules can find intensive applications in the studies of biosynthesis of these molecules. A sensitive, rapid and cost-effective technique that allows high-throughput screening of endogenous production of the natural iminosugar 1-deoxynojirimycin (1-DNJ), an α-glucosidase inhibitor relevant to the pharmaceutical industry, was developed in this study, based on the inhibitory effects of 1-DNJ on the activity of the β-glycosidase LacS from Sulfolobus solfataricus. This technique has been demonstrated effective in engineering both the key enzyme and the expression levels of enzymes in the 1-DNJ biosynthetic pathway from Bacillus atrophaeus cloned in E. coli. Higher biosynthetic efficiency was achieved using directed evolution strategies. PMID:25708517

  7. High-Throughput Screening in Protein Engineering: Recent Advances and Future Perspectives

    PubMed Central

    Wójcik, Magdalena; Telzerow, Aline; Quax, Wim J.; Boersma, Ykelien L.

    2015-01-01

    Over the last three decades, protein engineering has established itself as an important tool for the development of enzymes and (therapeutic) proteins with improved characteristics. New mutagenesis techniques and computational design tools have greatly aided in the advancement of protein engineering. Yet, one of the pivotal components to further advance protein engineering strategies is the high-throughput screening of variants. Compartmentalization is one of the key features allowing miniaturization and acceleration of screening. This review focuses on novel screening technologies applied in protein engineering, highlighting flow cytometry- and microfluidics-based platforms. PMID:26492240

  8. High Throughput Screening for Inhibitors of Mycobacterium tuberculosis H37Rv

    PubMed Central

    ANANTHAN, SUBRAMANIAM; FAALEOLEA, ELLEN R.; GOLDMAN, ROBERT C.; HOBRATH, JUDITH V.; KWONG, CECIL D.; LAUGHON, BARBARA E.; MADDRY, JOSEPH A.; MEHTA, ALKA; RASMUSSEN, LYNN; REYNOLDS, ROBERT C.; SECRIST, JOHN A.; SHINDO, NICE; SHOWE, DUSTIN N.; SOSA, MELINDA I.; SULING, WILLIAM J.; WHITE, E. LUCILE

    2009-01-01

    SUMMARY There is an urgent need for the discovery and development of new antitubercular agents that target new biochemical pathways and treat drug resistant forms of the disease. One approach to addressing this need is through high throughput screening of medicinally relevant libraries against the whole bacterium in order to discover a variety of new, active scaffolds that will stimulate new biological research and drug discovery. Through the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (www.taacf.org), a large, medicinally relevant chemical library was screened against M. tuberculosis strain H37Rv. The screening methods and a medicinal chemistry analysis of the results are reported herein. PMID:19758845

  9. High-Throughput Screening of Tyrosine Kinase Inhibitor Resistant Genes in CML.

    PubMed

    Ma, Leyuan; Roderick, Justine; Kelliher, Michelle A; Green, Michael R

    2016-01-01

    Genome-wide RNA interference (RNAi) screening in mammalian cells has proven to be a powerful tool for identifying new genes and molecular pathways relevant to many cellular processes and diseases. For example, screening for genes that, when inactivated, lead to resistance to cancer therapeutic drugs can reveal new mechanisms for how resistance develops and identify potential targetable strategies to overcome drug resistance. Here, we describe a detailed procedure for performing a high-throughput RNAi screen using a genome-wide human short hairpin RNA (shRNA) library for identifying tyrosine kinase inhibitor (TKI)-resistance genes in a human CML cell line model. PMID:27581147

  10. High-throughput method for optimum solubility screening for homogeneity and crystallization of proteins

    DOEpatents

    Kim, Sung-Hou; Kim, Rosalind; Jancarik, Jamila

    2012-01-31

    An optimum solubility screen in which a panel of buffers and many additives are provided in order to obtain the most homogeneous and monodisperse protein condition for protein crystallization. The present methods are useful for proteins that aggregate and cannot be concentrated prior to setting up crystallization screens. A high-throughput method using the hanging-drop method and vapor diffusion equilibrium and a panel of twenty-four buffers is further provided. Using the present methods, 14 poorly behaving proteins have been screened, resulting in 11 of the proteins having highly improved dynamic light scattering results allowing concentration of the proteins, and 9 were crystallized.

  11. [Rapid and high throughput measurement of lipase thermo-stability through ANS fluorescence signal assay].

    PubMed

    Feng, Weizong; Lin, Junhan; Cai, Shaoli; Zou, Youtu; Chen, Guoren; Huang, Ping; Lin, Yajing; Wang, Bingbing; Lin, Lin

    2011-04-01

    We have developed a rapid and high throughput lipase-ANS (8-Anilino-l-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal's increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 degrees C to 65 degrees C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement. PMID:21847993

  12. Environmental Impact on Vascular Development Predicted by High-Throughput Screening

    PubMed Central

    Judson, Richard S.; Reif, David M.; Sipes, Nisha S.; Singh, Amar V.; Chandler, Kelly J.; DeWoskin, Rob; Dix, David J.; Kavlock, Robert J.; Knudsen, Thomas B.

    2011-01-01

    Background: Understanding health risks to embryonic development from exposure to environmental chemicals is a significant challenge given the diverse chemical landscape and paucity of data for most of these compounds. High-throughput screening (HTS) in the U.S. Environmental Protection Agency (EPA) ToxCast™ project provides vast data on an expanding chemical library currently consisting of > 1,000 unique compounds across > 500 in vitro assays in phase I (complete) and Phase II (under way). This public data set can be used to evaluate concentration-dependent effects on many diverse biological targets and build predictive models of prototypical toxicity pathways that can aid decision making for assessments of human developmental health and disease. Objective: We mined the ToxCast phase I data set to identify signatures for potential chemical disruption of blood vessel formation and remodeling. Methods: ToxCast phase I screened 309 chemicals using 467 HTS assays across nine assay technology platforms. The assays measured direct interactions between chemicals and molecular targets (receptors, enzymes), as well as downstream effects on reporter gene activity or cellular consequences. We ranked the chemicals according to individual vascular bioactivity score and visualized the ranking using ToxPi (Toxicological Priority Index) profiles. Results: Targets in inflammatory chemokine signaling, the vascular endothelial growth factor pathway, and the plasminogen-activating system were strongly perturbed by some chemicals, and we found positive correlations with developmental effects from the U.S. EPA ToxRefDB (Toxicological Reference Database) in vivo database containing prenatal rat and rabbit guideline studies. We observed distinctly different correlative patterns for chemicals with effects in rabbits versus rats, despite derivation of in vitro signatures based on human cells and cell-free biochemical targets, implying conservation but potentially differential

  13. Identification of new drug candidates against Borrelia burgdorferi using high-throughput screening

    PubMed Central

    Pothineni, Venkata Raveendra; Wagh, Dhananjay; Babar, Mustafeez Mujtaba; Inayathullah, Mohammed; Solow-Cordero, David; Kim, Kwang-Min; Samineni, Aneesh V; Parekh, Mansi B; Tayebi, Lobat; Rajadas, Jayakumar

    2016-01-01

    Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that >300,000 cases per annum are reported in USA alone. A total of 10%–20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved) that were screened are Library of Pharmacologically Active Compounds (LOPAC1280), the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150 unique compounds, which inhibited >90% of B. burgdorferi growth at a concentration of <25 µM. These 150 unique compounds comprise many safe antibiotics, chemical compounds, and also small molecules from plant sources. Of the 150 unique compounds, 101 compounds are FDA approved. We selected the top 20 FDA-approved molecules based on safety and potency and studied their minimum inhibitory concentration and minimum bactericidal concentration. The promising safe FDA-approved candidates that show low minimum inhibitory concentration and minimum bactericidal concentration values can be chosen as

  14. Integrated Protein Array Screening and High Throughput Validation of 70 Novel Neural Calmodulin-binding Proteins*

    PubMed Central

    O'Connell, David J.; Bauer, Mikael C.; O'Brien, John; Johnson, Winifred M.; Divizio, Catherine A.; O'Kane, Sara L.; Berggård, Tord; Merino, Alejandro; Åkerfeldt, Karin S.; Linse, Sara; Cahill, Dolores J.

    2010-01-01

    Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca2+ ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca2+. This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (KD ≤ 1 μm) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (koff ≤ 10−3 s−1) and high affinity (KD ≤ 1 μm), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We developed a microarray of the identified target proteins with which we can characterize the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage-gated channel Kv6.1 (residues 474–493), calmodulin kinase-like vesicle-associated protein (residues 302–316), EF-hand domain family member A2 (residues 202–216), and phosphatidylinositol-4-phosphate 5-kinase, type I, γ (residues 400–415). PMID:20068228

  15. Identification of new drug candidates against Borrelia burgdorferi using high-throughput screening.

    PubMed

    Pothineni, Venkata Raveendra; Wagh, Dhananjay; Babar, Mustafeez Mujtaba; Inayathullah, Mohammed; Solow-Cordero, David; Kim, Kwang-Min; Samineni, Aneesh V; Parekh, Mansi B; Tayebi, Lobat; Rajadas, Jayakumar

    2016-01-01

    Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that >300,000 cases per annum are reported in USA alone. A total of 10%-20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved) that were screened are Library of Pharmacologically Active Compounds (LOPAC1280), the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150 unique compounds, which inhibited >90% of B. burgdorferi growth at a concentration of <25 µM. These 150 unique compounds comprise many safe antibiotics, chemical compounds, and also small molecules from plant sources. Of the 150 unique compounds, 101 compounds are FDA approved. We selected the top 20 FDA-approved molecules based on safety and potency and studied their minimum inhibitory concentration and minimum bactericidal concentration. The promising safe FDA-approved candidates that show low minimum inhibitory concentration and minimum bactericidal concentration values can be chosen as lead

  16. Automated high-throughput in vitro screening of the acetylcholine esterase inhibiting potential of environmental samples, mixtures and single compounds.

    PubMed

    Froment, Jean; Thomas, Kevin V; Tollefsen, Knut Erik

    2016-08-01

    A high-throughput and automated assay for testing the presence of acetylcholine esterase (AChE) inhibiting compounds was developed, validated and applied to screen different types of environmental samples. Automation involved using the assay in 96-well plates and adapting it for the use with an automated workstation. Validation was performed by comparing the results of the automated assay with that of a previously validated and standardised assay for two known AChE inhibitors (paraoxon and dichlorvos). The results show that the assay provides similar concentration-response curves (CRCs) when run according to the manual and automated protocol. Automation of the assay resulted in a reduction in assay run time as well as in intra- and inter-assay variations. High-quality CRCs were obtained for both of the model AChE inhibitors (dichlorvos IC50=120µM and paraoxon IC50=0.56µM) when tested alone. The effect of co-exposure of an equipotent binary mixture of the two chemicals were consistent with predictions of additivity and best described by the concentration addition model for combined toxicity. Extracts of different environmental samples (landfill leachate, wastewater treatment plant effluent, and road tunnel construction run-off) were then screened for AChE inhibiting activity using the automated bioassay, with only landfill leachate shown to contain potential AChE inhibitors. Potential uses and limitations of the assay were discussed based on the present results. PMID:27085000

  17. Development of a highly sensitive, high-throughput assay for glycosyltransferases using enzyme-coupled fluorescence detection.

    PubMed

    Kumagai, Kazuo; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo

    2014-02-15

    Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z'-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators. PMID:24299989

  18. High-throughput screening and small animal models, where are we?

    PubMed Central

    Giacomotto, Jean; Ségalat, Laurent

    2010-01-01

    Current high-throughput screening methods for drug discovery rely on the existence of targets. Moreover, most of the hits generated during screenings turn out to be invalid after further testing in animal models. To by-pass these limitations, efforts are now being made to screen chemical libraries on whole animals. One of the most commonly used animal model in biology is the murine model Mus musculus. However, its cost limit its use in large-scale therapeutic screening. In contrast, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the fish Danio rerio are gaining momentum as screening tools. These organisms combine genetic amenability, low cost and culture conditions that are compatible with large-scale screens. Their main advantage is to allow high-throughput screening in a whole-animal context. Moreover, their use is not dependent on the prior identification of a target and permits the selection of compounds with an improved safety profile. This review surveys the versatility of these animal models for drug discovery and discuss the options available at this day. PMID:20423335

  19. Development of Cell-Based High-Throughput Chemical Screens for Protection Against Cisplatin-Induced Ototoxicity.

    PubMed

    Teitz, Tal; Goktug, Asli N; Chen, Taosheng; Zuo, Jian

    2016-01-01

    Various compounds have been tested in recent years for protection against cisplatin-induced hearing loss, but no compound has yet been FDA approved for clinical use in patients. Towards this goal, we developed an unbiased, high-throughput, mammalian cochlear cell-based chemical screen that allowed quantification of the protection ability of bioactive compounds and ranked them for future testing ex vivo in cochlear explant cultures and in vivo in animal models. In our primary screens, protection in the HEI-OC1 organ of Corti immortalized cell line was measured by the ability of each compound to inhibit caspase-3/7 activity triggered by cisplatin treatment (50 μM cisplatin for 22 h). A total of 4385 unique bioactive compounds were tested in a single dose of 8 μM and promising compounds were validated by dose response curves covering ten, 1:3 serial diluted concentrations. Primary hits were defined as having more than 60 % inhibition of the caspase-3/7 activity. Toxicity of the top compounds was measured by a CellTiter-Glo (CTG) assay that measured the viability of the cells in the presence of compound alone in similar dose responsive analysis. A combination of the caspase-3/7 inhibition activity assay (as measured by IC50) and the CTG viability assay (as determined by LD50) identified the top protective compounds in the HEI-OC1 cells. In the future, the top hits in our screens will be tested for their protective ability ex vivo in mouse cochlear explants and in vivo in animal models. Our mammalian cochlear cell-based, high-throughput chemical screening assays described here can be further modified and represent an initial successful step towards therapeutic intervention of hearing disorders, an unmet medical need of our society. PMID:27259939

  20. High-throughput fabrication and screening improves gold nanoparticle chemiresistor sensor performance.

    PubMed

    Hubble, Lee J; Cooper, James S; Sosa-Pintos, Andrea; Kiiveri, Harri; Chow, Edith; Webster, Melissa S; Wieczorek, Lech; Raguse, Burkhard

    2015-02-01

    Chemiresistor sensor arrays are a promising technology to replace current laboratory-based analysis instrumentation, with the advantage of facile integration into portable, low-cost devices for in-field use. To increase the performance of chemiresistor sensor arrays a high-throughput fabrication and screening methodology was developed to assess different organothiol-functionalized gold nanoparticle chemiresistors. This high-throughput fabrication and testing methodology was implemented to screen a library consisting of 132 different organothiol compounds as capping agents for functionalized gold nanoparticle chemiresistor sensors. The methodology utilized an automated liquid handling workstation for the in situ functionalization of gold nanoparticle films and subsequent automated analyte testing of sensor arrays using a flow-injection analysis system. To test the methodology we focused on the discrimination and quantitation of benzene, toluene, ethylbenzene, p-xylene, and naphthalene (BTEXN) mixtures in water at low microgram per liter concentration levels. The high-throughput methodology identified a sensor array configuration consisting of a subset of organothiol-functionalized chemiresistors which in combination with random forests analysis was able to predict individual analyte concentrations with overall root-mean-square errors ranging between 8-17 μg/L for mixtures of BTEXN in water at the 100 μg/L concentration. The ability to use a simple sensor array system to quantitate BTEXN mixtures in water at the low μg/L concentration range has direct and significant implications to future environmental monitoring and reporting strategies. In addition, these results demonstrate the advantages of high-throughput screening to improve the performance of gold nanoparticle based chemiresistors for both new and existing applications. PMID:25562398

  1. Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening

    PubMed Central

    Shukla, Sunita J.; Nguyen, Dac-Trung; MacArthur, Ryan; Simeonov, Anton; Frazee, William J.; Hallis, Tina M.; Marks, Bryan D.; Singh, Upinder; Eliason, Hildegard C.; Printen, John; Austin, Christopher P.; Inglese, James

    2009-01-01

    Abstract The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug–drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z′-factors of ≥0.5. Seven- to 15-point concentration–response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format. PMID:19505231

  2. High Throughput Screening for Small Molecule Therapy for Gaucher Disease Using Patient Tissue as the Source of Mutant Glucocerebrosidase

    PubMed Central

    Goldin, Ehud; Zheng, Wei; Motabar, Omid; Southall, Noel; Choi, Jae Hyuk; Marugan, Juan; Austin, Christopher P.; Sidransky, Ellen

    2012-01-01

    Gaucher disease (GD), the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase). Previously, wildtype GCase was used for high throughput screening (HTS) of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development. PMID:22272254

  3. Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow Cytometry.

    PubMed

    Fereshteh, Mark P; Li, Xin; Li, Sha; Fan, Yi; Zhang, Rosemary; Farr, Glen A; Kolodin, Garrett; Lippy, Jonathan; Naglich, Joseph G; Schieven, Gary; Schweizer, Liang; Zhang, Litao

    2016-09-01

    Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors. PMID:27142718

  4. Adaptation of High-Throughput Screening in Drug Discovery—Toxicological Screening Tests

    PubMed Central

    Szymański, Paweł; Markowicz, Magdalena; Mikiciuk-Olasik, Elżbieta

    2012-01-01

    High-throughput screening (HTS) is one of the newest techniques used in drug design and may be applied in biological and chemical sciences. This method, due to utilization of robots, detectors and software that regulate the whole process, enables a series of analyses of chemical compounds to be conducted in a short time and the affinity of biological structures which is often related to toxicity to be defined. Since 2008 we have implemented the automation of this technique and as a consequence, the possibility to examine 100,000 compounds per day. The HTS method is more frequently utilized in conjunction with analytical techniques such as NMR or coupled methods e.g., LC-MS/MS. Series of studies enable the establishment of the rate of affinity for targets or the level of toxicity. Moreover, researches are conducted concerning conjugation of nanoparticles with drugs and the determination of the toxicity of such structures. For these purposes there are frequently used cell lines. Due to the miniaturization of all systems, it is possible to examine the compound’s toxicity having only 1–3 mg of this compound. Determination of cytotoxicity in this way leads to a significant decrease in the expenditure and to a reduction in the length of the study. PMID:22312262

  5. A strategy for high-throughput screening of ligands suitable for molecular imprinting of proteins.

    PubMed

    Eppler, Stefan; Schröder, Tim; Friedle, Jürgen; Michl, Simone; Dangel, Werner; Mizaikoff, Boris

    2012-05-15

    For facilitating the identification of appropriate functionalities that may serve as a binding motif of functional monomers, a selection strategy based on high-throughput screening of the binding properties of readily available sorbent materials has been developed. Thereby, the affinity of such ligands to the protein of interest may be rapidly determined. From these studies, it is anticipated that ligand functionalities will be derived, which may lead to advanced selection and design of dedicated functional monomers suitable for decorating the surface of a scavenger material. Thus, specific binding of the target protein of interest should be enabled even in complex solutions such as e.g., biotechnologically relevant cell lysates. In the present contribution, an automated screening method for studying ligand interactions of selected sorbent materials with pepsin - a protein of the protease family - was developed. Aqueous buffer solutions containing pepsin at known constant concentration were pipetted through an array of miniaturized chromatographic solid phase extraction (SPE) columns containing a variety of sorbent materials, and the eluted solutions were analyzed by UV/vis spectroscopy. The established screening protocol was validated against resin materials of known interaction with pepsin. Finally, the developed screening strategy was adapted for a robot system enabling high-throughput screening for a wide variety of sorbent materials and ligand functionalities in a fully automated approach. The obtained results clearly indicate that the established screening routine provides valuable data for characterizing resin-immobilized ligands, and their affinity toward pepsin. PMID:22472529

  6. Micro-colony array based high throughput platform for enzyme library screening.

    PubMed

    Pohn, Brigitte; Gerlach, Jochen; Scheideler, Marcel; Katz, Hermann; Uray, Martina; Bischof, Horst; Klimant, Ingo; Schwab, Helmut

    2007-03-30

    Enzymes are becoming increasingly important tools for synthesizing and modifying fine and bulk chemicals. The availability of biocatalysts which fulfil the requirements of industrial processes is often limited. Recruiting suited enzymes from natural (e.g. metagenomes) and artificial (e.g. directed evolution) biodiversity is based on screening libraries of microbial clones expressing enzyme variants. However, exploring the complex diversity of such libraries needs efficient screening methods. Overcoming the "screening bottleneck" requires rapid high throughput technology allowing the analysis of a large diversity of different enzymes and applying different screening conditions. Facing these facts an efficient and cost effective method for high throughput screening of large enzyme libraries at the colony level was developed. Therefore, ordered high density micro-colony arrays were combined with optical sensor technology and automated image analysis. The system generally allows the simultaneous monitoring of enzyme activities reflected by up to 7000 micro-colonies spotted on a filter in the size of a micro-titer plate. A developed replica option also allows the analysis of clones under varying external conditions. The method was verified by a model screening using esterases and was proved to provide reliable enzyme activity measurements within single micro-colonies allowing the discrimination of activity differences in the range of 10-20%. PMID:17174002

  7. Liquid gradient in two-dimensional matrix for high throughput screening

    PubMed Central

    Hu, Shan-Wen; Xu, Bi-Yi; Xu, Jing-Juan; Chen, Hong-Yuan

    2013-01-01

    Based on the ingenious combination of two different gradient generation mechanisms, this work reports a novel approach for a high throughput linear liquid gradient in a two-dimensional (2D) matrix. Specifically, a typical Christmas Tree structure with two inlets was designed as the first mixture gradient generator, upon which the second diffusion gradient generator was coupled to produce the desired concentration series on the basis of the distance difference. Rather than a simple 1D line, the integration of the two generators would result in an innovative 2D matrix of reservoirs, which was then characterized both theoretically and experimentally. Theoretically, calculation of fluid field demonstrated the formation of a concentration gradient, which was then confirmed by the dye solution visualization analysis. For high throughput screening application, doxorubicin (Dox) was then selected as model medicine to treat the acute myeloblastic leukemia (HL-60) cells. Cell viability displayed that cell death rate enhanced with the increase of drug concentration, and this result was higher than that on a 96-well plate, and the corresponding mechanism was properly discussed. Subsequently, Dox and quercetin were employed simultaneously to generate an overlapping gradient and its effect on HL-60 cells was investigated. Due to the automatic formation of concentration gradient that could improve the work efficiency, this work provides a promising tool for future high throughput drug screening. PMID:24396550

  8. Transfection microarrays for high-throughput phenotypic screening of genes involved in cell migration.

    PubMed

    Onuki-Nagasaki, Reiko; Nagasaki, Akira; Hakamada, Kazumi; Uyeda, Taro Q P; Fujita, Satoshi; Miyake, Masato; Miyake, Jun

    2010-01-01

    Cell migration is important in several biological phenomena, such as cancer metastasis. Therefore, the identification of genes involved in cell migration might facilitate the discovery of antimetastatic drugs. However, screening of genes by the current methods can be complicated by factors related to cell stimulation, for example, abolition of contact inhibition and the release inflammatory cytokines from wounded cells during examinations of wound healing in vitro. To overcome these problems and identify genes involved in cell migration, in this chapter we describe the use of transfection microarrays for high-throughput phenotypic screening. PMID:20387151

  9. A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities

    SciTech Connect

    McNally, N.; Liu, Xiang Yang; Choudary, P.V.

    1997-01-01

    The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also be used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.

  10. Phenotype MicroArrays for High-Throughput Phenotypic Testing and Assay of Gene Function

    PubMed Central

    Bochner, Barry R.; Gadzinski, Peter; Panomitros, Eugenia

    2001-01-01

    The bacterium Escherichia coli is used as a model cellular system to test and validate a new technology called Phenotype MicroArrays (PMs). PM technology is a high-throughput technology for simultaneous testing of a large number of cellular phenotypes. It consists of preconfigured well arrays in which each well tests a different cellular phenotype and an automated instrument that continuously monitors and records the response of the cells in all wells of the arrays. For example, nearly 700 phenotypes of E. coli can be assayed by merely pipetting a cell suspension into seven microplate arrays. PMs can be used to directly assay the effects of genetic changes on cells, especially gene knock-outs. Here, we provide data on phenotypic analysis of six strains and show that we can detect expected phenotypes as well as, in some cases, unexpected phenotypes. PMID:11435407

  11. Decoding the Chemical Language of Motile Bacteria by Using High-Throughput Microfluidic Assays

    PubMed Central

    Crooks, John A.; Stilwell, Matthew D.; Oliver, Piercen M.; Zhong, Zhou

    2015-01-01

    Motile bacteria navigate chemical environments by using chemoreceptors. The output of these protein sensors is linked to motility machinery and enables bacteria to follow chemical gradients. Understanding the chemical specificity of different families of chemoreceptors is essential for predicting and controlling bacterial behavior in ecological niches, including symbiotic and pathogenic interactions with plants and mammals. The identification of chemical(s) recognized by specific families of receptors is limited by the low throughput and complexity of chemotaxis assays. To address this challenge, we developed a microfluidic-based chemotaxis assay that is quantitative, simple, and enables high-throughput measurements of bacterial response to different chemicals. Using the model bacterium Escherichia coli, we demonstrated a strategy for identifying molecules that activate chemoreceptors from a diverse compound library and for determining how global behavioral strategies are tuned to chemical environments. PMID:26285783

  12. High-throughput single-molecule screen for small-molecule perturbation of splicing and transcription kinetics.

    PubMed

    Day, Christopher R; Chen, Huimin; Coulon, Antoine; Meier, Jordan L; Larson, Daniel R

    2016-03-01

    In eukaryotes, mRNA synthesis is catalyzed by RNA polymerase II and involves several distinct steps, including transcript initiation, elongation, cleavage, and transcript release. Splicing of RNA can occur during (co-transcriptional) or after (post-transcriptional) RNA synthesis. Thus, RNA synthesis and processing occurs through the concerted activity of dozens of enzymes, each of which is potentially susceptible to perturbation by small molecules. However, there are few, if any, high-throughput screening strategies for identifying drugs which perturb a specific step in RNA synthesis and processing. Here we have developed a high-throughput fluorescence microscopy approach in single cells to screen for inhibitors of specific enzymatic steps in RNA synthesis and processing. By utilizing the high affinity interaction between bacteriophage capsid proteins (MS2, PP7) and RNA stem loops, we are able to fluorescently label the intron and exon of a β-globin reporter gene in human cells. This approach allows one to measure the kinetics of transcription, splicing and release in both fixed and living cells using a tractable, genetically encoded assay in a stable cell line. We tested this reagent in a targeted screen of molecules that target chromatin readers and writers and identified three compounds that slow transcription elongation without changing transcription initiation. PMID:26655523

  13. Hi-Plex for high-throughput mutation screening: application to the breast cancer susceptibility gene PALB2

    PubMed Central

    2013-01-01

    Background Massively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour. Methods Here, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run. Results We observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing. Conclusions These results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings. PMID:24206657

  14. High-Throughput Thin Film Approach for Screening of Temperature-Pressure-Composition Phase Space

    SciTech Connect

    Zakutayev, A.; Subramaniyan, A.; Caskey, C. M.; Ndione, P. F.; Richards, R. M.; O'Hayre, R.; Ginley, D. S.

    2013-01-01

    Many solar energy technologies, for example CIGS and CdTe photovoltaics, utilize materials in thin film form. The equilibrium phase diagrams for these and other more novel solar energy materials are not known or are irrelevant because of the non-equilibrium character of the thin film growth processes. We demonstrate a high-throughput thin film approach for screening of temperature-pressure-composition phase diagrams and phase spaces. The examples in focus are novel solar absorbers Cu-N, Cu-O and p-type transparent conductors in the Cr2O3-MnO system. The composition axis of the Cr2O3-MnO phase diagram was screened using a composition spread method. The temperature axis of the Mn-O phase diagram was screened using a temperature spread method. The pressure axes of the Cu-N and Cu-O phase diagrams were screened using rate spread method with the aid of non-equilibrium growth phenomena. Overall these three methods constitute an approach to high-throughput screening of inorganic thin film phase diagrams. This research is supported by U.S. Department of Energy as a part of two NextGen Sunshot projects and an Energy Frontier Research Center.

  15. Complementary cell-based high-throughput screens identify novel modulators of the unfolded protein response.

    PubMed

    Fribley, Andrew M; Cruz, Patricia G; Miller, Justin R; Callaghan, Michael U; Cai, Peter; Narula, Neha; Neubig, Richard R; Showalter, Hollis D; Larsen, Scott D; Kirchhoff, Paul D; Larsen, Martha J; Burr, Douglas A; Schultz, Pamela J; Jacobs, Renju R; Tamayo-Castillo, Giselle; Ron, David; Sherman, David H; Kaufman, Randal J

    2011-09-01

    Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis

  16. A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity.

    PubMed

    Bachovchin, Daniel A; Koblan, Luke W; Wu, Wengen; Liu, Yuxin; Li, Youhua; Zhao, Peng; Woznica, Iwona; Shu, Ying; Lai, Jack H; Poplawski, Sarah E; Kiritsy, Christopher P; Healey, Sarah E; DiMare, Matthew; Sanford, David G; Munford, Robert S; Bachovchin, William W; Golub, Todd R

    2014-08-01

    The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling and suggest that such profiling can be incorporated into the earliest stages of drug discovery. PMID:24997602

  17. An exposure:activity profiling method for interpreting high-throughput screening data for estrogenic activity--proof of concept.

    PubMed

    Becker, Richard A; Friedman, Katie Paul; Simon, Ted W; Marty, M Sue; Patlewicz, Grace; Rowlands, J Craig

    2015-04-01

    Rapid high throughput in vitro screening (HTS) assays are now available for characterizing dose-responses in assays that have been selected for their sensitivity in detecting estrogen-related endpoints. For example, EPA's ToxCast™ program recently released endocrine assay results for more than 1800 substances and the interagency Tox21 consortium is in the process of releasing data for approximately 10,000 chemicals. But such activity measurements alone fall short for the purposes of priority setting or screening because the relevant exposure context is not considered. Here, we extend the method of exposure:activity profiling by calculating the exposure:activity ratios (EARs) using human exposure estimates and AC50 values for a range of chemicals tested in a suite of seven estrogenic assays in ToxCast™ and Tox21. To provide additional context, relative estrogenic exposure:activity quotients (REEAQ) were derived by comparing chemical-specific EARs to the EAR of the ubiquitous dietary phytoestrogen, genistein (GEN). Although the activity of a substance in HTS-endocrine assays is not a measure of health hazard or risk, understanding how such a dose compares to human exposures provides a valuable additional metric that can be used in decision-making; substances with small EARs and REEAQs would indicate low priority for further endocrine screening or testing. PMID:25656492

  18. Development and Validation of a Universal High-Throughput UDP-Glycosyltransferase Assay with a Time-Resolved FRET Signal.

    PubMed

    Zielinski, Thomas; Reichman, Melvin; Donover, Preston S; Lowery, Robert G

    2016-05-01

    Glycosyltransferase enzymes play diverse metabolic and regulatory roles by catalyzing the transfer of sugar molecules to protein, lipid, and carbohydrate acceptors, and they are increasingly of interest as therapeutic targets in a number of diseases, including metabolic disorders, cancer, and infectious diseases. The glycosyltransferases are a challenging target class from an assay development perspective because of the diversity of both donor and acceptor substrates and the lack of suitable glycan detection methods. However, many glycosyltransferases use uridine 5'-diphosphate (UDP) sugars as donor substrates, and detection of the free UDP reaction product provides a generic approach for measuring the activity of those enzymes. To exploit this approach for a broadly applicable high-throughput screening (HTS) assay for discovery of glycosyltransferase inhibitors, we developed a Transcreener(®) assay for immunodetection of UDP with a time-resolved Förster resonance energy transfer (TR-FRET) signal. We optimized the assay for detection of glycosyltransferase activity with nucleotide diphosphate (NDP) sugars at concentrations from 10 μM to 1 mM, achieving Z' values of 0.6 or higher. The assay was validated by orthogonal pooled screening with 8,000 compounds using polypeptide N-acetylgalactosaminyltransferase T3 as the target, and the hits were confirmed using an orthogonal readout. The reagents and signal were both stable for more than 8 h at room temperature, insuring robust performance in automated HTS environments. The TR-FRET-based UDP detection assay provides a broadly applicable approach for screening glycosyltransferases that use a UDP-sugar donor. PMID:27136323

  19. A Novel High-Throughput Assay for Islet Respiration Reveals Uncoupling of Rodent and Human Islets

    PubMed Central

    Wikstrom, Jakob D.; Sereda, Samuel B.; Stiles, Linsey; Elorza, Alvaro; Allister, Emma M.; Neilson, Andy; Ferrick, David A.; Wheeler, Michael B.; Shirihai, Orian S.

    2012-01-01

    Background The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. Methodology/Principal Findings The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. Conclusions/Significance The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells. PMID:22606219

  20. Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases.

    PubMed

    Vardakou, Maria; Salmon, Melissa; Faraldos, Juan A; O'Maille, Paul E

    2014-01-01

    Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography-mass spectrometry (GC-MS). In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity.•We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs.•We validate the method by direct comparison with established assays. The agreement of k cat/K M among methods makes this adaptation optimal for rapid evaluation of TPSs.•We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries. PMID:26150952

  1. Development and optimization of a high-throughput assay to measure neutralizing antibodies against Clostridium difficile binary toxin.

    PubMed

    Xie, Jinfu; Horton, Melanie; Zorman, Julie; Antonello, Joseph M; Zhang, Yuhua; Arnold, Beth A; Secore, Susan; Xoconostle, Rachel; Miezeiewski, Matthew; Wang, Su; Price, Colleen E; Thiriot, David; Goerke, Aaron; Gentile, Marie-Pierre; Skinner, Julie M; Heinrichs, Jon H

    2014-05-01

    Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates. PMID:24623624

  2. Development and Optimization of a High-Throughput Assay To Measure Neutralizing Antibodies against Clostridium difficile Binary Toxin

    PubMed Central

    Xie, Jinfu; Horton, Melanie; Zorman, Julie; Antonello, Joseph M.; Zhang, Yuhua; Arnold, Beth A.; Secore, Susan; Xoconostle, Rachel; Miezeiewski, Matthew; Wang, Su; Price, Colleen E.; Thiriot, David; Goerke, Aaron; Gentile, Marie-Pierre; Skinner, Julie M.

    2014-01-01

    Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates. PMID:24623624

  3. High-throughput radiometric CYP2C19 inhibition assay using tritiated (S)-mephenytoin.

    PubMed

    Di Marco, Annalise; Cellucci, Antonella; Chaudhary, Ashok; Fonsi, Massimiliano; Laufer, Ralph

    2007-10-01

    A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high-performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4'-hydroxylation of (S)-mephenytoin labeled with tritium in the 4' position. Because this reaction is subject to an NIH shift, tritium was also introduced into the 3'- and 5'-positions of the tracer to enhance formation of a tritiated water product. Tritiated water was separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC(50) values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4'-hydroxy-(S)-mephenytoin product, using HPLC coupled to mass spectrometric detection. All of the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in a 96-well format and can be automated. This assay represents a non-HPLC, high-throughput version of the classic (S)-mephenytoin 4'-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 inhibition of new chemical entities. PMID:17600081

  4. Optical tools for high-throughput screening of abrasion resistance of combinatorial libraries of organic coatings

    NASA Astrophysics Data System (ADS)

    Potyrailo, Radislav A.; Chisholm, Bret J.; Olson, Daniel R.; Brennan, Michael J.; Molaison, Chris A.

    2002-02-01

    Design, validation, and implementation of an optical spectroscopic system for high-throughput analysis of combinatorially developed protective organic coatings are reported. Our approach replaces labor-intensive coating evaluation steps with an automated system that rapidly analyzes 8x6 arrays of coating elements that are deposited on a plastic substrate. Each coating element of the library is 10 mm in diameter and 2 to 5 micrometers thick. Performance of coatings is evaluated with respect to their resistance to wear abrasion because this parameter is one of the primary considerations in end-use applications. Upon testing, the organic coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Coatings are abraded using industry-accepted abrasion test methods at single-or multiple-abrasion conditions, followed by high- throughput analysis of abrasion-induced light scatter. The developed automated system is optimized for the analysis of diffusively scattered light that corresponds to 0 to 30% haze. System precision of 0.1 to 2.5% relative standard deviation provides capability for the reliable ranking of coatings performance. While the system was implemented for high-throughput screening of combinatorially developed organic protective coatings for automotive applications, it can be applied to a variety of other applications where materials ranking can be achieved using optical spectroscopic tools.

  5. High-Throughput Synthesis and Screening of Titania-Based Photocatalysts.

    PubMed

    Nursam, Natalita M; Wang, Xingdong; Caruso, Rachel A

    2015-10-12

    Titanium dioxide is widely known as a prominent photocatalyst material and research in this area has increased substantially over the last decades. However, the photoactivity of TiO2 is hindered by several factors, such as a relatively high photogenerated electron-hole recombination rate and a wide bandgap of ∼ 3.2 eV, rendering it inactive under visible light. Approaches to optimize the TiO2 photocatalyst, either by altering its morphological or chemical properties, have been conducted for many years, yet further modification of this semiconductor has the potential to yield photocatalysts with excellent properties and higher photocatalytic activity. This could be effectively explored using combinatorial synthesis coupled with high-throughput characterization approaches. Such an approach has been widely applied for the discovery of new functional materials, including photocatalysts. By using high-throughput synthesis and characterization technology, preparation and screening of materials on small sample scales can be accelerated; hence, new TiO2-based photocatalysts with enhanced photocatalytic activity can be acquired more rapidly. Additionally, the large database of materials being systematically examined will greatly build our fundamental understanding of the relation between materials structure/composition and photocatalytic activity. This review details various high-throughput syntheses and characterization techniques applied to improve the photocatalytic properties of TiO2 materials and discuss several challenges that have been raised or may be encountered in the future when using this approach. PMID:26371558

  6. High-throughput screening to identify selective inhibitors of microbial sulfate reduction (and beyond)

    NASA Astrophysics Data System (ADS)

    Carlson, H. K.; Coates, J. D.; Deutschbauer, A. M.

    2015-12-01

    The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive and corrosive. Current strategies to selectively inhibit sulfidogenesis are based on non-specific biocide treatments, bio-competitive exclusion by alternative electron acceptors or sulfate-analogs which are competitive inhibitors or futile/alternative substrates of the sulfate reduction pathway. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to target SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Once inhibitor selectivity is defined, high-throughput characterization of microbial community structure across compound gradients and identification of fitness determinants using isolate bar-coded transposon mutant libraries can give insights into the genetic mechanisms whereby compounds structure microbial communities. The high-throughput (HT) approach we present can be readily applied to target SRM in diverse environments and more broadly, could be used to identify and quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant for engineering environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

  7. Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis

    PubMed Central

    Shen, Yun-An A; Niu, Jianqin; Zhao, Chao; Presley, Bryan; Miao, Edna; Lee, Seonok; Mayoral, Sonia R; Redmond, Stephanie A; Etxeberria, Ainhoa; Xiao, Lan; Franklin, Robin J M; Green, Ari; Hauser, Stephen L; Chan, Jonah R

    2016-01-01

    Functional screening for compounds that promote remyelination represents a major hurdle in the development of rational therapeutics for multiple sclerosis. Screening for remyelination is problematic, as myelination requires the presence of axons. Standard methods do not resolve cell-autonomous effects and are not suited for high-throughput formats. Here we describe a binary indicant for myelination using micropillar arrays (BIMA). Engineered with conical dimensions, micropillars permit resolution of the extent and length of membrane wrapping from a single two-dimensional image. Confocal imaging acquired from the base to the tip of the pillars allows for detection of concentric wrapping observed as ‘rings’ of myelin. The platform is formatted in 96-well plates, amenable to semiautomated random acquisition and automated detection and quantification. Upon screening 1,000 bioactive molecules, we identified a cluster of antimuscarinic compounds that enhance oligodendrocyte differentiation and remyelination. Our findings demonstrate a new high-throughput screening platform for potential regenerative therapeutics in multiple sclerosis. PMID:24997607

  8. The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

    PubMed Central

    Smith, Clyde A.; Cohen, Aina E.

    2008-01-01

    The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening and data collection. At the Stanford Synchrotron Radiation Laboratory (SSRL), a National User Facility which provides extremely brilliant X-ray photon beams for use in materials science, environmental science and structural biology research, the incorporation of advanced robotics has enabled crystals to be screened in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Automated Mounter, or SAM) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter’s home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines. PMID:19956359

  9. The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

    SciTech Connect

    Smith, C.A.; Cohen, A.E.

    2009-05-26

    The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be screened in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Auto-Mounting System) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter's home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.

  10. Integration of High-Throughput Screening Data with Dosimetry and Human Exposure in the Toxicity Assessment of Environmental Chemicals

    EPA Science Inventory

    High-throughput in vitro screening and computational tools provide government an efficient way to identify those chemicals that warrant further testing while conserving limited testing resources. Incorporation of kinetic and exposure information should provide a more meaningful i...

  11. High-throughput drug library screening identifies colchicine as a thyroid cancer inhibitor

    PubMed Central

    Zhang, Le; Yang, Zhaoying; Granieri, Letizia; Pasculescu, Adrian; Datti, Alessandro; Asa, Sylvia L.; Xu, Zheli; Ezzat, Shereen

    2016-01-01

    We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer. PMID:26942566

  12. High-throughput drug library screening identifies colchicine as a thyroid cancer inhibitor.

    PubMed

    Zhang, Le; Yang, Zhaoying; Granieri, Letizia; Pasculescu, Adrian; Datti, Alessandro; Asa, Sylvia L; Xu, Zheli; Ezzat, Shereen

    2016-04-12

    We employed a high-throughput drug library screening platform to identify novel agents affecting thyroid cancer cells. We used human thyroid cancer cell lines to screen a collection of approximately 5200 small molecules with biological and/or pharmacologial properties. Parallel primary screens yielded a number of hits differentially active between thyroid and melanoma cells. Amongst compounds specifically targeting thyroid cancer cells, colchicine emerged as an effective candidate. Colchicine inhibited cell growth which correlated with G2 cell cycle arrest and apoptosis. These effects were hampered through inhibition of MEK1/2 and JNK. In contrast, inhibition of p38-MAPK had little effect, and AKT had no impact on colchicine action. Systemic colchicine inhibited thyroid cancer progression in xenografted mice. These findings demonstrate that our screening platform is an effective vehicle for drug reposition and show that colchicine warrants further attention in well-defined clinical niches such as thyroid cancer. PMID:26942566

  13. Novel inhibitors for PRMT1 discovered by high-throughput screening using activity-based fluorescence polarization.

    PubMed

    Dillon, Myles B C; Bachovchin, Daniel A; Brown, Steven J; Finn, M G; Rosen, Hugh; Cravatt, Benjamin F; Mowen, Kerri A

    2012-07-20

    Protein arginine methyltransferases (PRMTs) catalyze the posttranslational methylation of arginine using S-adenosylmethionine (SAM) as a methyl-donor. The PRMT family is widely expressed and has been implicated in biological functions such as RNA splicing, transcriptional control, signal transduction, and DNA repair. Therefore, specific inhibitors of individual PRMTs have potentially significant research and therapeutic value. In particular, PRMT1 is responsible for >85% of arginine methyltransferase activity, but currently available inhibitors of PRMT1 lack specificity, efficacy, and bioavailability. To address this limitation, we developed a high-throughput screening assay for PRMT1 that utilizes a hyper-reactive cysteine within the active site, which is lacking in almost all other PRMTs. This assay, which monitors the kinetics of the fluorescence polarization signal increase upon PRMT1 labeling by a rhodamine-containing cysteine-reactive probe, successfully identified two novel inhibitors selective for PRMT1 over other SAM-dependent methyltransferases. PMID:22506763

  14. A sensitive high-throughput HPLC assay for simultaneous determination of everolimus and clobetasol propionate.

    PubMed

    Kamberi, Marika; Fu, Katherine; Lu, Jianmin; Chemaly, G Mike; Feder, Debra

    2008-01-01

    A novel sensitive high-throughput high-performance liquid chromatography assay is developed and validated for the simultaneous determination of everolimus and clobetasol propionate in pharmaceutical formulations. The chromatographic separation is achieved on a Zorbax Eclipse XDB-C18 reversed-phase column using a gradient elution, with solvent A: ammonium acetate (pH 6.8; 0.01 M) and solvent B: acetonitrile. The mean recovery ranges from 95.1% to 100.0% for clobetasol propionate and from 97.9% to 103.7% for everolimus. The limit of quantitation for each analyte is 0.02 microg/mL. The percent relative standard deviations are less than 3% for intra- and inter-day analyses. The proposed method can be used for the routine quality control of everolimus and clobetasol propionate in complex pharmaceutical formulations, especially the drug-delivery systems with a low total drug-load. PMID:18218184

  15. Lipid-sensing high-throughput ApoA-I assays.

    PubMed

    Niedziela-Majka, Anita; Lad, Latesh; Chisholm, Jeffrey W; Lagpacan, Leanna; Schwartz, Karen; Hung, Magdeleine; Jin, Debi; Fung, Wanchi; Brendza, Katherine M; Liu, Xiaohong; Pagratis, Nikos; Sakowicz, Roman

    2012-09-01

    Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647-labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation. PMID:22811478

  16. High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells.

    PubMed

    Karmaus, Agnes L; Toole, Colleen M; Filer, Dayne L; Lewis, Kenneth C; Martin, Matthew T

    2016-04-01

    Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis. PMID:26781511

  17. High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells

    PubMed Central

    Toole, Colleen M.; Filer, Dayne L.; Lewis, Kenneth C.; Martin, Matthew T.

    2016-01-01

    Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis. PMID:26781511

  18. High throughput identification of promiscuous inhibitors from screening libraries with the use of a thiol-containing fluorescent probe

    PubMed Central

    McCallum, Megan M.; Nandhikonda, Premchendar; Temmer, Jonathan J.; Eyermann, Charles; Simeonov, Anton; Jadhav, Ajit; Yasgar, Adam; Maloney, David; Arnold, Leggy A.

    2013-01-01

    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 in 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 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 in respect to thiol-reactive compounds. PMID:23446699

  19. Screensaver: an open source lab information management system (LIMS) for high throughput screening facilities

    PubMed Central

    2010-01-01

    Background Shared-usage high throughput screening (HTS) facilities are becoming more common in academe as large-scale small molecule and genome-scale RNAi screening strategies are adopted for basic research purposes. These shared facilities require a unique informatics infrastructure that must not only provide access to and analysis of screening data, but must also manage the administrative and technical challenges associated with conducting numerous, interleaved screening efforts run by multiple independent research groups. Results We have developed Screensaver, a free, open source, web-based lab information management system (LIMS), to address the informatics needs of our small molecule and RNAi screening facility. Screensaver supports the storage and comparison of screening data sets, as well as the management of information about screens, screeners, libraries, and laboratory work requests. To our knowledge, Screensaver is one of the first applications to support the storage and analysis of data from both genome-scale RNAi screening projects and small molecule screening projects. Conclusions The informatics and administrative needs of an HTS facility may be best managed by a single, integrated, web-accessible application such as Screensaver. Screensaver has proven useful in meeting the requirements of the ICCB-Longwood/NSRB Screening Facility at Harvard Medical School, and has provided similar benefits to other HTS facilities. PMID:20482787

  20. High-throughput assay for measuring monoclonal antibody self-association and aggregation in serum.

    PubMed

    Li, Xiaoning; Geng, Steven B; Chiu, Mark L; Saro, Dorina; Tessier, Peter M

    2015-03-18

    Subcutaneous delivery is one of the preferred administration routes for therapeutic monoclonal antibodies (mAbs). High antibody dosing requirements and small injection volumes necessitate formulation and delivery of highly concentrated mAb solutions. Such elevated antibody concentrations can lead to undesirable solution behaviors such as mAb self-association and aggregation, which are relatively straightforward to detect using various biophysical methods because of the high purity and concentration of antibody formulations. However, the biophysical properties of mAbs in serum can also impact antibody activity, but these properties are less well understood because of the difficulty characterizing mAbs in such a complex environment. Here we report a high-throughput assay for directly evaluating mAb self-association and aggregation in serum. Our approach involves immobilizing polyclonal antibodies specific for human mAbs on gold nanoparticles, and then using these conjugates to capture human antibodies at a range of subsaturating to saturating mAb concentrations in serum. Antibody aggregation is detected at subsaturating mAb concentrations via blue-shifted plasmon wavelengths due to the reduced efficiency of capturing mAb aggregates relative to monomers, which reduces affinity cross-capture of mAbs by multiple conjugates. In contrast, antibody self-association is detected at saturating mAb concentrations via red-shifted plasmon wavelengths due to attractive interparticle interactions between immobilized mAbs. The high-throughput nature of this assay along with its compatibility with unusually dilute mAb solutions (0.1-10 μg per mL) should make it useful for identifying antibody candidates with high serum stability during early antibody discovery. PMID:25714504

  1. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    PubMed Central

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2015-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

  2. High-throughput screening of tissue-nonspecific alkaline phosphatase for identification of effectors with diverse modes of action.

    PubMed

    Sergienko, Eduard A; Millán, José Luis

    2010-08-01

    Here we describe a protocol for the identification of effectors of tissue-nonspecific alkaline phosphatase (TNAP). It is based on a highly sensitive method for detecting TNAP activity. After dephosphorylation by TNAP, a dioxetane-based substrate undergoes a series of chemical transformations resulting in light production. Light intensity serves as a quantitative measure of the velocity of the TNAP-catalyzed reaction in the steady state. This protocol includes guidelines for optimizing the assay and for high-throughput screening in multiwell plates. The assay is sensitive to the influence of diverse effectors of TNAP as long as the assay optimization steps are repeated for each new batch of the enzyme; full optimization is accomplished in under 2 d. Depending on the available equipment, 10,000-100,000 compounds can be screened in an 8-h period. This protocol provides a method of screening TNAP that is 1,000-fold more sensitive and 10-fold faster than a conventional colorimetric assay with p-nitrophenyl phosphate. PMID:20671726

  3. High-Throughput Screening of the Asymmetric Decarboxylative Alkylation Reaction of Enolate-Stabilized Enol Carbonates.

    PubMed

    McDougal, Nolan T; Virgil, Scott C; Stoltz, Brian M

    2010-01-01

    The use of high-throughput screening allowed for the optimization of reaction conditions for the palladium-catalyzed asymmetric decarboxylative alkylation reaction of enolate-stabilized enol carbonates. Changing to a non-polar reaction solvent and to an electron-deficient PHOX derivative as ligand from our standard reaction conditions improved the enantioselectivity for the alkylation of a ketal-protected,1,3-diketone-derived enol carbonate from 28% ee to 84% ee. Similar improvements in enantioselectivity were seen for a β-keto-ester derived- and an α-phenyl cyclohexanone-derived enol carbonate. PMID:21072327

  4. A High-Throughput Screen Reveals New Small-Molecule Activators and Inhibitors of Pantothenate Kinases

    PubMed Central

    2016-01-01

    Pantothenate kinase (PanK) is a regulatory enzyme that controls coenzyme A (CoA) biosynthesis. The association of PanK with neurodegeneration and diabetes suggests that chemical modifiers of PanK activity may be useful therapeutics. We performed a high throughput screen of >520000 compounds from the St. Jude compound library and identified new potent PanK inhibitors and activators with chemically tractable scaffolds. The HTS identified PanK inhibitors exemplified by the detailed characterization of a tricyclic compound (7) and a preliminary SAR. Biophysical studies reveal that the PanK inhibitor acts by binding to the ATP–enzyme complex. PMID:25569308

  5. Screening and Crystallization Plates for Manual and High-throughput Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    Thorne, Robert E. (Inventor); Berejnov, Viatcheslav (Inventor); Kalinin, Yevgeniy (Inventor)

    2010-01-01

    In one embodiment, a crystallization and screening plate comprises a plurality of cells open at a top and a bottom, a frame that defines the cells in the plate, and at least two films. The first film seals a top of the plate and the second film seals a bottom of the plate. At least one of the films is patterned to strongly pin the contact lines of drops dispensed onto it, fixing their position and shape. The present invention also includes methods and other devices for manual and high-throughput protein crystal growth.

  6. A High-Throughput Biophotonics Instrument to Screen for Novel Ocular Photosensitizing Therapeutic Agents

    PubMed Central

    Butler, Mark C.; Itotia, Patrick N.

    2010-01-01

    Purpose. High-throughput techniques are needed to identify and optimize novel photodynamic therapy (PDT) agents with greater efficacy and to lower toxicity. Novel agents with the capacity to completely ablate pathologic angiogenesis could be of substantial utility in diseases such as wet age-related macular degeneration (AMD). Methods. An instrument and approach was developed based on light-emitting diode (LED) technology for high-throughput screening (HTS) of libraries of potential chemical and biological photosensitizing agents. Ninety-six-well LED arrays were generated at multiple wavelengths and under rigorous intensity control. Cell toxicity was measured in 96-well culture arrays with the nuclear dye SYTOX Green (Invitrogen-Molecular Probes, Eugene, OR). Results. Rapid screening of photoactivatable chemicals or biological molecules has been realized in 96-well arrays of cultured human cells. This instrument can be used to identify new PDT agents that exert cell toxicity on presentation of light of the appropriate energy. The system is further demonstrated through determination of the dose dependence of model compounds having or lacking cellular phototoxicity. Killer Red (KR), a genetically encoded red fluorescent protein expressed from transfected plasmids, is examined as a potential cellular photosensitizing agent and offers unique opportunities as a cell-type–specific phototoxic protein. Conclusions. This instrument has the capacity to screen large chemical or biological libraries for rapid identification and optimization of potential novel phototoxic lead candidates. KR and its derivatives have unique potential in ocular gene therapy for pathologic angiogenesis or tumors. PMID:19834043

  7. High-throughput transformation method for Yarrowia lipolytica mutant library screening.

    PubMed

    Leplat, Christophe; Nicaud, Jean-Marc; Rossignol, Tristan

    2015-09-01

    As a microorganism of major biotechnological importance, the oleaginous yeast Yarrowia lipolytica is subjected to intensive genetic engineering and functional genomic analysis. Future advancements in this area, however, require a system that will generate a large collection of mutants for high-throughput screening. Here, we report a rapid and efficient method for high-throughput transformation of Y. lipolytica in 96-well plates. We developed plasmids and strains for the large-scale screening of overexpression mutant strains, using Gateway® vectors that were adapted for specific locus integration in Y. lipolytica. As an example, a collection of mutants that overexpressed the alkaline extracellular protease (AEP) was obtained in a single transformation experiment. The platform strain that we developed to receive the overexpression cassette was designed to constitutively express a fluorescent protein as a convenient growth reporter for screening in non-translucid media. An example of growth comparison in skim milk-based medium between AEP overexpression and deletion mutants is provided. PMID:26100263

  8. A BSL-4 high-throughput screen identifies sulfonamide inhibitors of Nipah virus.

    PubMed

    Tigabu, Bersabeh; Rasmussen, Lynn; White, E Lucile; Tower, Nichole; Saeed, Mohammad; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W; Noah, James W

    2014-04-01

    Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses. PMID:24735442

  9. An electrode probe for high-throughput screening of electrochemical libraries

    NASA Astrophysics Data System (ADS)

    Jiang, Rongzhong; Chu, Deryn

    2005-06-01

    A pen-shaped O2 electrode probe is designed for high-throughput screening of electrochemical libraries. The electrode probe consists of a large-area O2 electrode and a cylindrical electrolyte sponge with a short cone tip for screening. This type of design can easily minimize the probe resistance contributed by the electrolyte. A zinc electrode library is generated using a nonautomated method to deposit metal zinc on a graphite plate. The zinc electrode library and the O2-electrode probe form an electrochemical library containing 128 micro zinc/air batteries. High-throughput screening of the zinc/air batteries are carried out by moving the tip of the electrode probe under constant potential (1.0V) and measuring the current. A Gaussian distribution is used for statistical analysis of the experimental data. These data obtained with the combinatorial method have a relative standard deviation of 8.9% based on a nonautomated coating procedure. The O2 electrode probe is used to study the effect of addition of Cu in the anode on the performance of the zinc/air battery.

  10. A BSL-4 High-Throughput Screen Identifies Sulfonamide Inhibitors of Nipah Virus

    PubMed Central

    Tigabu, Bersabeh; Rasmussen, Lynn; White, E. Lucile; Tower, Nichole; Saeed, Mohammad; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W.

    2014-01-01

    Abstract Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses. PMID:24735442

  11. Detecting and overcoming systematic bias in high-throughput screening technologies: a comprehensive review of practical issues and methodological solutions.

    PubMed

    Caraus, Iurie; Alsuwailem, Abdulaziz A; Nadon, Robert; Makarenkov, Vladimir

    2015-11-01

    Significant efforts have been made recently to improve data throughput and data quality in screening technologies related to drug design. The modern pharmaceutical industry relies heavily on high-throughput screening (HTS) and high-content screening (HCS) technologies, which include small molecule, complementary DNA (cDNA) and RNA interference (RNAi) types of screening. Data generated by these screening technologies are subject to several environmental and procedural systematic biases, which introduce errors into the hit identification process. We first review systematic biases typical of HTS and HCS screens. We highlight that study design issues and the way in which data are generated are crucial for providing unbiased screening results. Considering various data sets, including the publicly available ChemBank data, we assess the rates of systematic bias in experimental HTS by using plate-specific and assay-specific error detection tests. We describe main data normalization and correction techniques and introduce a general data preprocessing protocol. This protocol can be recommended for academic and industrial researchers involved in the analysis of current or next-generation HTS data. PMID:25750417

  12. Comprehensive analysis of high-throughput screens with HiTSeekR.

    PubMed

    List, Markus; Schmidt, Steffen; Christiansen, Helle; Rehmsmeier, Marc; Tan, Qihua; Mollenhauer, Jan; Baumbach, Jan

    2016-08-19

    High-throughput screening (HTS) is an indispensable tool for drug (target) discovery that currently lacks user-friendly software tools for the robust identification of putative hits from HTS experiments and for the interpretation of these findings in the context of systems biology. We developed HiTSeekR as a one-stop solution for chemical compound screens, siRNA knock-down and CRISPR/Cas9 knock-out screens, as well as microRNA inhibitor and -mimics screens. We chose three use cases that demonstrate the potential of HiTSeekR to fully exploit HTS screening data in quite heterogeneous contexts to generate novel hypotheses for follow-up experiments: (i) a genome-wide RNAi screen to uncover modulators of TNFα, (ii) a combined siRNA and miRNA mimics screen on vorinostat resistance and (iii) a small compound screen on KRAS synthetic lethality. HiTSeekR is publicly available at http://hitseekr.compbio.sdu.dk It is the first approach to close the gap between raw data processing, network enrichment and wet lab target generation for various HTS screen types. PMID:27330136

  13. High-Throughput, Liquid-Based Genome-Wide RNAi Screening in C. elegans.

    PubMed

    O'Reilly, Linda P; Knoerdel, Ryan R; Silverman, Gary A; Pak, Stephen C

    2016-01-01

    RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) molecules mediate the inhibition of gene expression. RNAi in C. elegans can be achieved by simply feeding animals with bacteria expressing dsRNA against the gene of interest. This "feeding" method has made it possible to conduct genome-wide RNAi experiments for the systematic knockdown and subsequent investigation of almost every single gene in the genome. Historically, these genome-scale RNAi screens have been labor and time intensive. However, recent advances in automated, high-throughput methodologies have allowed the development of more rapid and efficient screening protocols. In this report, we describe a fast and efficient, liquid-based method for genome-wide RNAi screening. PMID:27581291

  14. Discovery of novel TAOK2 inhibitor scaffolds from high-throughput screening.

    PubMed

    Piala, Alexander T; Akella, Radha; Potts, Malia B; Dudics-Giagnocavo, Stephanie A; He, Haixia; Wei, Shuguang; White, Michael A; Posner, Bruce A; Goldsmith, Elizabeth J

    2016-08-15

    The MAP3K (Mitogen Activated Protein Kinase Kinase Kinase) TAOK2 (Thousand-And-One Kinase 2) is an activator of p38 MAP kinase cascade that is up-regulated in response to environmental stresses. A synthetic lethal screen performed using a NSCLC (non-small cell lung cancer) cell line, and a second screen identifying potential modulators of autophagy have implicated TAOK2 as a potential cancer therapeutic target. Using a 200,000 compound high throughput screen, we identified three specific small molecule compounds that inhibit the kinase activity of TAOK2. These compounds also showed inhibition of autophagy. Based on SAR (structure-activity relationship) studies, we have predicted the modifications on the reactive groups for the three compounds. PMID:27426302

  15. Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays.

    PubMed

    Le Goff, Gaelle C; Desmet, Cloé; Brès, Jean-Charles; Rigal, Dominique; Blum, Loïc J; Marquette, Christophe A

    2010-12-15

    We are reporting here a low cost colorimetric device for high-throughput multiplexed blood group genotyping and allergy diagnosis, displayed as an automated 96-well microtiter plate format. A porous polymeric membrane sealed at the bottom of each well accounts for the sensor support. For each sensing unit, a 6×6 matrix of specific probes is spotted on the external surface of the membrane resulting in 5 mm(2) microarrays. Thanks to the membrane porosity, reagents dispensed into the well can be eliminated through vacuum soaking. This unusual design drastically reduces the assay background signal. The system was first validated on robust models composed of either two complementary oligonucleotide sequences or one allergen/specific rabbit IgG pair. The quality of both oligonucleotide and protein immobilisation on the membrane substrate was then demonstrated together with the capacity to use the arrayed biomolecules as probes for the quantitative detection of specific targets (respectively complementary oligonucleotide and specific antibody). On the basis of these good results, two multiplex assays were developed for crude biological samples testing, focussing on two human in vitro diagnosis applications: a hybridisation assay for multiplex blood group genotyping and a multiparametric immunoassay for allergy diagnosis. In both cases, the transfer to crude biological samples testing was successful i.e. high signal to noise ratio of the stained membranes, reproducibility and good correlation with results obtained using routine testing procedures. PMID:20663657

  16. High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission

    PubMed Central

    Plouffe, David M.; Wree, Melanie; Du, Alan Y.; Meister, Stephan; Li, Fengwu; Patra, Kailash; Lubar, Aristea; Okitsu, Shinji L.; Flannery, Erika L.; Kato, Nobutaka; Tanaseichuk, Olga; Comer, Eamon; Zhou, Bin; Kuhen, Kelli; Zhou, Yingyao; Leroy, Didier; Schreiber, Stuart L.; Scherer, Christina A.; Vinetz, Joseph; Winzeler, Elizabeth A.

    2016-01-01

    Summary Preventing transmission is an important element of malaria control. However, most of the current available methods to assay for malaria transmission blocking are relatively low throughput and cannot be applied to large chemical libraries. We have developed a high-throughput and cost-effective assay, the Saponin-lysis Sexual Stage Assay (SaLSSA), for identifying small molecules with transmission-blocking capacity. SaLSSA analysis of 13,983 unique compounds uncovered that >90% of well-characterized antimalarials, including endoperoxides and 4-aminoquinolines, as well as compounds active against asexual blood stages, lost most of their killing activity when parasites developed into metabolically quiescent stage V gametocytes. On the other hand, we identified compounds with consistent low nanomolar transmission-blocking activity, some of which showed cross-reactivity against asexual blood and liver stages. The data clearly emphasize substantial physiological differences between sexual and asexual parasites and provide a tool and starting points for the discovery and development of transmission-blocking drugs. PMID:26749441

  17. Formulation, High Throughput In Vitro Screening and In Vivo Functional Characterization of Nanoemulsion-Based Intranasal Vaccine Adjuvants

    PubMed Central

    Wong, Pamela T.; Leroueil, Pascale R.; Smith, Douglas M.; Ciotti, Susan; Bielinska, Anna U.; Janczak, Katarzyna W.; Mullen, Catherine H.; Groom, Jeffrey V.; Taylor, Erin M.; Passmore, Crystal; Makidon, Paul E.; O’Konek, Jessica J.; Myc, Andrzej; Hamouda, Tarek; Baker, James R.

    2015-01-01

    Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (TH1, TH2, TH17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications. PMID:25962136

  18. Identification of small molecule inhibitors of ricin and shiga toxin using a cell-based high-throughput screen

    PubMed Central

    Wahome, Paul G.; Bai, Yan; Neal, Lori M.; Robertus, Jon D.; Mantis, Nicholas J.

    2010-01-01

    The Category B agents, ricin and shiga toxin (Stx), are RNA N-glycosidases that target a highly conserved adenine residue within the sarcin-ricin loop of eukaryotic 28S ribosomal RNA. In an effort to identify small-molecule inhibitors of these toxins that could serve as lead compounds for potential therapeutics, we have developed a simple Vero cell-based high-throughput cytotoxicity assay and have used it to screen ∼81,300 compounds in 17 commercially available chemical libraries. This initial screen identified ∼300 compounds with weak (≥30-<50%), moderate (≥50-<80%), or strong (≥80%) ricin inhibitory activity. Secondary analysis of 244 of these original “hits” was performed, and 20 compounds that were capable of reducing ricin cytotoxicity by >50% were chosen for further study. Four compounds demonstrated significant dose-dependent ricin inhibitory activity in the Vero cell-based assay, with 50% effective inhibitory concentration (EC50) values ranging from 25 to 60 μM. The same 20 compounds were tested in parallel for the ability to inhibit ricin's and Stx1's enzymatic activities in an in vitro translation reaction. Three of the 20 compounds, including the most effective compound in the cell-based assay, had discernible anti-toxin activity. One compound in particular, 4-fluorophenyl methyl 2-(furan-2-yl)quinoline-4-carboxylate (“compound 8”), had 50% inhibitory concentration (IC50) of 30 μM, a value indicating > 10-fold higher potency than is the case for previously described ricin-Stx1 inhibitors. Computer modeling predicted that compound 8 is capable of docking within the ricin active site. In conclusion, we have used a simple high-throughput cell-based method to identify several new small-molecule inhibitors of ricin and Stx. PMID:20350563

  19. Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams.

    PubMed

    Georgantzoglou, Antonios; Merchant, Michael J; Jeynes, Jonathan C G; Mayhead, Natalie; Punia, Natasha; Butler, Rachel E; Jena, Rajesh

    2015-01-01

    Charged particle therapy is increasingly becoming a valuable tool in cancer treatment, mainly due to the favorable interaction of particle radiation with matter. Its application is still limited due, in part, to lack of data regarding the radiosensitivity of certain cell lines to this radiation type, especially to high-linear energy transfer (LET) particles. From the earliest days of radiation biology, the clonogenic survival assay has been used to provide radiation response data. This method produces reliable data but it is not optimized for high-throughput microbeam studies with high-LET radiation where high levels of cell killing lead to a very low probability of maintaining cells' clonogenic potential. A new method, therefore, is proposed in this paper, which could potentially allow these experiments to be conducted in a high-throughput fashion. Cells are seeded in special polypropylene dishes and bright-field illumination provides cell visualization. Digital images are obtained and cell detection is applied based on corner detection, generating individual cell targets as x-y points. These points in the dish are then irradiated individually by a micron field size high-LET microbeam. Post-irradiation, time-lapse imaging follows cells' response. All irradiated cells are tracked by linking trajectories in all time-frames, based on finding their nearest position. Cell divisions are detected based on cell appearance and individual cell temporary corner density. The number of divisions anticipated is low due to the high probability of cell killing from high-LET irradiation. Survival curves are produced based on cell's capacity to divide at least four to five times. The process is repeated for a range of doses of radiation. Validation shows the efficiency of the proposed cell detection and tracking method in finding cell divisions. PMID:26835414

  20. Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams

    PubMed Central

    Georgantzoglou, Antonios; Merchant, Michael J.; Jeynes, Jonathan C. G.; Mayhead, Natalie; Punia, Natasha; Butler, Rachel E.; Jena, Rajesh

    2016-01-01

    Charged particle therapy is increasingly becoming a valuable tool in cancer treatment, mainly due to the favorable interaction of particle radiation with matter. Its application is still limited due, in part, to lack of data regarding the radiosensitivity of certain cell lines to this radiation type, especially to high-linear energy transfer (LET) particles. From the earliest days of radiation biology, the clonogenic survival assay has been used to provide radiation response data. This method produces reliable data but it is not optimized for high-throughput microbeam studies with high-LET radiation where high levels of cell killing lead to a very low probability of maintaining cells’ clonogenic potential. A new method, therefore, is proposed in this paper, which could potentially allow these experiments to be conducted in a high-throughput fashion. Cells are seeded in special polypropylene dishes and bright-field illumination provides cell visualization. Digital images are obtained and cell detection is applied based on corner detection, generating individual cell targets as x–y points. These points in the dish are then irradiated individually by a micron field size high-LET microbeam. Post-irradiation, time-lapse imaging follows cells’ response. All irradiated cells are tracked by linking trajectories in all time-frames, based on finding their nearest position. Cell divisions are detected based on cell appearance and individual cell temporary corner density. The number of divisions anticipated is low due to the high probability of cell killing from high-LET irradiation. Survival curves are produced based on cell’s capacity to divide at least four to five times. The process is repeated for a range of doses of radiation. Validation shows the efficiency of the proposed cell detection and tracking method in finding cell divisions. PMID:26835414

  1. Protocol: High-throughput and quantitative assays of auxin and auxin precursors from minute tissue samples

    PubMed Central

    2012-01-01

    Background The plant hormone auxin, indole-3-acetic acid (IAA), plays important roles in plant growth and development. The signaling response to IAA is largely dependent on the local concentration of IAA, and this concentration is regulated by multiple mechanisms in plants. Therefore, the precise quantification of local IAA concentration provides insights into the regulation of IAA and its biological roles. Meanwhile, pathways and genes involved in IAA biosynthesis are not fully understood, so it is necessary to analyze the production of IAA at the metabolite level for unbiased studies of IAA biosynthesis. Results We have developed high-throughput methods to quantify plant endogenous IAA and its biosynthetic precursors including indole, tryptophan, indole-3-pyruvic acid (IPyA), and indole-3-butyric acid (IBA). The protocol starts with homogenizing plant tissues with stable-labeled internal standards added, followed by analyte purification using solid phase extraction (SPE) tips and analyte derivatization. The derivatized analytes are finally analyzed by selected reaction monitoring on a gas chromatograph-mass spectrometer (GC-MS/MS) to determine the precise abundance of analytes. The amount of plant tissue required for the assay is small (typically 2–10 mg fresh weight), and the use of SPE tips is simple and convenient, which allows preparation of large sets of samples within reasonable time periods. Conclusions The SPE tips and GC-MS/MS based method enables high-throughput and accurate quantification of IAA and its biosynthetic precursors from minute plant tissue samples. The protocol can be used for measurement of these endogenous compounds using isotope dilution, and it can also be applied to analyze IAA biosynthesis and biosynthetic pathways using stable isotope labeling. The method will potentially advance knowledge of the role and regulation of IAA. PMID:22883136

  2. A Cell-Based High-Throughput Screening for Inducers of Myeloid Differentiation

    PubMed Central

    Radomska, Hanna S.; Jernigan, Finith; Nakayama, Sohei; Jorge, Susan E.; Sun, Lijun; Tenen, Daniel G.; Kobayashi, Susumu S.

    2015-01-01

    Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory and development of novel therapeutics is required. CCAAT/Enhancer Binding Protein α (C/EBPα) is one of crucial transcription factors that induce granulocytic differentiation and its activity is perturbed in human myeloid leukemias. As its re-expression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human AML patients treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly impact care of AML patients with unfavorable prognosis in the near future. PMID:26109609

  3. High-throughput screening of antibiotic-resistant bacteria in picodroplets.

    PubMed

    Liu, X; Painter, R E; Enesa, K; Holmes, D; Whyte, G; Garlisi, C G; Monsma, F J; Rehak, M; Craig, F F; Smith, C A

    2016-04-26

    The prevalence of clinically-relevant bacterial strains resistant to current antibiotic therapies is increasing and has been recognized as a major health threat. For example, multidrug-resistant tuberculosis and methicillin-resistant Staphylococcus aureus are of global concern. Novel methodologies are needed to identify new targets or novel compounds unaffected by pre-existing resistance mechanisms. Recently, water-in-oil picodroplets have been used as an alternative to conventional high-throughput methods, especially for phenotypic screening. Here we demonstrate a novel microfluidic-based picodroplet platform which enables high-throughput assessment and isolation of antibiotic-resistant bacteria in a label-free manner. As a proof-of-concept, the system was used to isolate fusidic acid-resistant mutants and estimate the frequency of resistance among a population of Escherichia coli (strain HS151). This approach can be used for rapid screening of rare antibiotic-resistant mutants to help identify novel compound/target pairs. PMID:27033300

  4. High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics

    PubMed Central

    Beneyton, Thomas; Wijaya, I. Putu Mahendra; Postros, Prexilia; Najah, Majdi; Leblond, Pascal; Couvent, Angélique; Mayot, Estelle; Griffiths, Andrew D.; Drevelle, Antoine

    2016-01-01

    Filamentous fungi are an extremely important source of industrial enzymes because of their capacity to secrete large quantities of proteins. Currently, functional screening of fungi is associated with low throughput and high costs, which severely limits the discovery of novel enzymatic activities and better production strains. Here, we describe a nanoliter-range droplet-based microfluidic system specially adapted for the high-throughput sceening (HTS) of large filamentous fungi libraries for secreted enzyme activities. The platform allowed (i) compartmentalization of single spores in ~10 nl droplets, (ii) germination and mycelium growth and (iii) high-throughput sorting of fungi based on enzymatic activity. A 104 clone UV-mutated library of Aspergillus niger was screened based on α-amylase activity in just 90 minutes. Active clones were enriched 196-fold after a single round of microfluidic HTS. The platform is a powerful tool for the development of new production strains with low cost, space and time footprint and should bring enormous benefit for improving the viability of biotechnological processes. PMID:27270141

  5. A Cell-Based High-Throughput Screening for Inducers of Myeloid Differentiation.

    PubMed

    Radomska, Hanna S; Jernigan, Finith; Nakayama, Sohei; Jorge, Susan E; Sun, Lijun; Tenen, Daniel G; Kobayashi, Susumu S

    2015-10-01

    Recent progress of genetic studies has dramatically unveiled pathogenesis of acute myeloid leukemia (AML). However, overall survival of AML still remains unsatisfactory, and development of novel therapeutics is required. CCAAT/enhancer binding protein α (C/EBPα) is one of the crucial transcription factors that induce granulocytic differentiation, and its activity is perturbed in human myeloid leukemias. As its reexpression can induce differentiation and subsequent apoptosis of leukemic cells in vitro, we hypothesized that chemical compounds that restore C/EBPα expression and/or activity would lead to myeloid differentiation of leukemic cells. Using a cell-based high-throughput screening, we identified 2-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-3-(2-methoxyphenyl)-4(3H)-quinazolinone as a potent inducer of C/EBPα and myeloid differentiation. Leukemia cell lines and primary blast cells isolated from human patients with AML treated with ICCB280 demonstrated evidence of morphological and functional differentiation, as well as massive apoptosis. We performed conformational analyses of the high-throughput screening hit compounds to postulate the spatial requirements for high potency. Our results warrant a development of novel differentiation therapies and significantly affect care of patients with AML with unfavorabl