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Sample records for drug screening assay

  1. Primary and secondary drug screening assays for Friedreich ataxia.

    PubMed

    Cotticelli, M Grazia; Rasmussen, Lynn; Kushner, Nicole L; McKellip, Sara; Sosa, Melinda Ingrum; Manouvakhova, Anna; Feng, Shuang; White, E Lucile; Maddry, Joseph A; Heemskerk, Jill; Oldt, Robert J; Surrey, Lea F; Ochs, Rachel; Wilson, Robert B

    2012-03-01

    Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardiodegenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix for the assembly of iron-sulfur clusters (ISCs), which are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain (ETC). Decreased expression of frataxin or the yeast frataxin orthologue, Yfh1p, is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. Using yeast depleted of Yfh1p, a high-throughput screening (HTS) assay was developed in which mitochondrial function was monitored by reduction of the tetrazolium dye WST-1 in a growth medium with a respiration-only carbon source. Of 101 200 compounds screened, 302 were identified that effectively rescue mitochondrial function. To confirm activities in mammalian cells and begin understanding mechanisms of action, secondary screening assays were developed using murine C2C12 cells and yeast mutants lacking specific complexes of the ETC, respectively. The compounds identified in this study have potential relevance for other neurodegenerative disorders associated with mitochondrial dysfunction, such as Parkinson disease. PMID:22086726

  2. Development of an in vitro drug screening assay using Schistosoma haematobium schistosomula

    PubMed Central

    2012-01-01

    Background The development of novel antischistosomal drugs is crucial, as currently no vaccine and only a single drug is available for the treatment of schistosomiasis. Fast and accurate in vitro assays are urgently needed to identify new drug candidates and research efforts should include Schistosoma haematobium. The aim of the present study was to develop a S. haematobium drug sensitivity assay based on newly transformed schistosomula (NTS). Methods We first undertook comparative studies on the cercarial emergence rhythms of the intermediate host snails Biomphalaria glabrata (S. mansoni) and Bulinus truncatus (S. haematobium). Two transformation methods as well as three purification methods were studied on S. haematobium cercariae in order to produce a large number of viable and clean NTS. Known antischistosomal drugs were tested in the established NTS assay in vitro. Drug effects were evaluated either microscopically or fluorometrically, using a resazurin based viability marker. Microscopically obtained IC50 values were compared with results obtained for S. mansoni. Results A circadian rhythm existed in both snail species. Infected B. truncatus snails shed less cercariae than B. glabrata during the testing period. The highest transformation rate (69%) of S. haematobium cercariae into NTS was obtained with the vortex transformation (mechanical input) and the highest purification factor was observed using Percoll®. The fluorimetric readout based on resazurin was very precise in detecting dead or/and severely damaged schistosomula. Conclusions With the use of viability markers such as resazurin, drug screening assays using S. haematobium NTS can be efficiently performed. However, drugs acting on the morphology and motility of S. haematobium NTS, such as metrifonate are missed. Drug sensitivity assays with NTS of both species, S. haematobium and S. mansoni, showed very similar results using known antischistosomal drugs. The S. mansoni NTS assay might be more

  3. Application of a human tumor colony-forming assay to new drug screening.

    PubMed

    Shoemaker, R H; Wolpert-DeFilippes, M K; Kern, D H; Lieber, M M; Makuch, R W; Melnick, N R; Miller, W T; Salmon, S E; Simon, R M; Venditti, J M

    1985-05-01

    The applicability of a human tumor colony-forming assay to drug screening was investigated in terms of feasibility, validity, and potential for discovering new antitumor drugs. Feasibility was addressed in a pilot study during which basic methods, appropriate assay quality controls, and a standardized protocol for screening were developed. Considerable variability was noted in the availability and colony growth of different tumor types. The majority of the evaluable experiments utilized breast, colorectal, kidney, lung, melanoma, or ovarian tumors. For many tumor types, little evidence of growth was observed, or only rare specimens formed colonies. Colony-forming efficiencies ranged from 0.05 to 0.11% for the six most useful tumors listed above. A set of quality control measures was developed to address technical problems inherent in the assay. Testing of standard agents in the pilot study established that most of these agents could be detected as active. However, it also identified three assay limitations: compounds requiring systemic metabolic activation are inactive; medium constituents may block the activity of certain antimetabolites; and compounds without therapeutic efficacy may be positive in the assay. The assay categorized nontoxic clinically ineffective agents as true negatives with 97% accuracy. Of 79 compounds which were negative in the current National Cancer Institute prescreen (leukemia P388), 14 were active in the assay. Several demonstrated outstanding in vitro activity and are structurally unrelated to compounds already in development or in clinical trials. A subset of these active compounds were found to lack activity in a P388 in vitro colony-forming assay. This indication of differential cytotoxicity to human tumor cells makes this subset of compounds particularly interesting as antitumor drug leads. The demonstrated sensitivity to most standard agents, discrimination of nontoxic compounds, reproducibility of survival values within assays and

  4. 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

  5. Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs.

    PubMed

    Escribano, Alexandra Ibáñez; Marcel, Alfredo Meneses; Tugores, Yanetsy Machado; Ruiz, Juan José Nogal; Redó, Vicente J Arán; García-Trevijano, José Antonio Escario; Barrio, Alicia Gómez

    2012-08-01

    A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis. PMID:22850954

  6. An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience

    PubMed Central

    Kaja, Simon; Payne, Andrew J.; Singh, Tulsi; Ghuman, Jasleen K.; Sieck, Erin G.; Koulen, Peter

    2015-01-01

    Background Quantification of lactate dehydrogenase (LDH) release is a widely accepted assay for the quantitative determination of cell viability and late-stage apoptosis. Major disadvantages of commercially available LDH assay kits include proprietary formulations, limited options for optimization and high cost, all resulting in limited reproducibility in research applications. New Method Here, we describe a novel, custom LDH assay suitable in the context of plate reader-based screening of drug candidates for glioprotection, but with wide applicability to other cell types and experimental paradigms. Results We developed a novel and highly reproducible LDH release assay that is more cost-effective than commercially available assays with comparable performance. The assay was validated by assessing 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid antioxidant protection against tert-butylhydroperoxide-induced oxidative stress in C6 astroglioma cells. Assay performance was validated by direct comparison and compatible with other methods of measuring cellular viability, namely 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 6-carboxy-2′, 7′ dichlorodihydrofluorescein diacetate assays. Comparison with Existing Method(s) There was no statistically significant difference between results obtained with the novel custom assay and a commercially available assay CytoTox96® (Promega, Madison, WI). Conclusions The novel custom LDH release assay allows the reproducible quantification of cell viability and is highly cost-effective when compared to commercially available assays (approximately 25 times cheaper). In addition and in contrast to commercially available assays, the identification and detailed description of all assay components and procedures provide greater control over experimental conditions and design. We provide a detailed standard operating procedure permitting our novel assay to be readily adapted depending on experimental requirements

  7. 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

  8. 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

  9. 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.

  10. Cell-Based Assay Design for High-Content Screening of Drug Candidates.

    PubMed

    Nierode, Gregory; Kwon, Paul S; Dordick, Jonathan S; Kwon, Seok-Joon

    2016-02-01

    To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as highcontent screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner. PMID:26428732

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

    PubMed

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

    2013-10-01

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

  12. Development of specific dengue virus 2'-O- and N7-methyltransferase assays for antiviral drug screening.

    PubMed

    Barral, K; Sallamand, C; Petzold, C; Coutard, B; Collet, A; Thillier, Y; Zimmermann, J; Vasseur, J-J; Canard, B; Rohayem, J; Debart, F; Decroly, E

    2013-09-01

    Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA(2'OMe)-RNA, at the 5'-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA(2'OMe)-RNA₇₄, by ligation of two RNA fragments. Then, we use GpppA(2'OMe)-RNA₇₄ as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate (7Me)GpppA-RNA₇₄ in order to characterize the DENV 2'-O-MTase activity specifically on long capped RNA. We next compared the N7- and 2'-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development. PMID:23769894

  13. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites

    PubMed Central

    Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses. PMID:25915529

  14. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    PubMed

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo

    2015-01-01

    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses. PMID:25915529

  15. A Systematic Comparison Identifies an ATP-Based Viability Assay as Most Suitable Read-Out for Drug Screening in Glioma Stem-Like Cells

    PubMed Central

    Kleijn, A.; Kloezeman, J. J.; Balvers, R. K.; van der Kaaij, M.; Dirven, C. M. F.; Leenstra, S.; Lamfers, M. L. M.

    2016-01-01

    Serum-free culture methods for patient-derived primary glioma cultures, selecting for glioma stem-like cells (GSCs), are becoming the gold standard in neurooncology research. These GSCs can be implemented in drug screens to detect patient-specific responses, potentially bridging the translational gap to personalized medicine. Since numerous compounds are available, a rapid and reliable readout for drug efficacies is required. This can be done using approaches that measure viability, confluency, cytotoxicity, or apoptosis. To determine which assay is best suitable for drug screening, 10 different assays were systematically tested on established glioma cell lines and validated on a panel of GSCs. General applicability was assessed using distinct treatment modalities, being temozolomide, radiation, rapamycin, and the oncolytic adenovirus Delta24-RGD. The apoptosis and cytotoxicity assays did not unequivocally detect responses and were excluded from further testing. The NADH- and ATP-based viability assays revealed comparable readout for all treatments; however, the latter had smaller standard deviations and direct readout. Importantly, drugs that interfere with cell metabolism require alternative techniques such as confluency monitoring to accurately measure treatment effects. Taken together, our data suggest that the combination of ATP luminescence assays with confluency monitoring provides the most specific and reproducible readout for drug screening on primary GSCs. PMID:27274737

  16. Urine drug screen

    MedlinePlus

    Drug screen -- urine ... detect the presence of illegal and some prescription drugs in your urine. Their presence indicates that you recently used these drugs. Some drugs may remain in your system for ...

  17. 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

  18. Establishment of a novel experimental protocol for drug-induced seizure liability screening based on a locomotor activity assay in zebrafish.

    PubMed

    Koseki, Naoteru; Deguchi, Jiro; Yamashita, Akihito; Miyawaki, Izuru; Funabashi, Hitoshi

    2014-08-01

    As drug-induced seizures have severe impact on drug development, evaluating seizure induction potential of candidate drugs at the early stages of drug discovery is important. A novel assay system using zebrafish has attracted interest as a high throughput toxicological in vivo assay system, and we tried to establish an experimental method for drug-induced seizure liability on the basis of locomotor activity in zebrafish. We monitored locomotor activity at high-speed movement (> 20 mm/sec) for 60 min immediately after exposure, and assessed seizure liability potential in some drugs using locomotor activity. However this experimental procedure was not sufficient for predicting seizures because the potential of several drugs with demonstrated seizure potential in mammals was not detected. We, therefore, added other parameters for locomotor activity such as extending exposure time or conducting flashlight stimulation (10 Hz) which is a known seizure induction stimulus, and these additional parameters improved seizure potential detection in some drugs. The validation study using the improved methodology was used to assess 52 commercially available drugs, and the prediction rate was approximately 70%. The experimental protocol established in this present study is considered useful for seizure potential screening during early stages of drug discovery. PMID:25056783

  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. 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

  1. Cross-reactivity of the CEDIA buprenorphine assay in drugs-of-abuse screening: influence of dose and metabolites of opioids

    PubMed Central

    Berg, Jon Andsnes; Schjøtt, Jan; Fossan, Kjell O; Riedel, Bettina

    2015-01-01

    Purpose The cloned enzyme donor immunoassay (CEDIA) for buprenorphine is applied for both urine drugs-of-abuse screening and compliance monitoring. Sensitivity, specificity, and optimal cutoff of this assay have differed between studies. This may indicate that cross-reactivity has to be taken into account during assay evaluation. We therefore investigated the performance of the CEDIA buprenorphine assay for use in our patient population and explored the impact of cross-reactivity on assay accuracy. Methods The CEDIA buprenorphine assay and high-performance liquid chromatography–tandem mass spectrometry were employed to analyze drugs-of-abuse in urine samples from a healthy drug-naïve male volunteer after intake of two tablets of a prescription drug containing 400 mg paracetamol +30 mg codeine phosphate, and in urine samples (n=2,272) from drug-addicted patients. Receiver operating characteristic analyses were performed to express the diagnostic accuracy of the CEDIA buprenorphine assay. Results CEDIA buprenorphine was positive in one urine sample from the drug-naïve person after intake of the prescription drug. Twenty-five (1.1%) of the patient urine samples were positive for buprenorphine by CEDIA, but negative by high-performance liquid chromatography–tandem mass spectrometry. Codeine, morphine, and their respective metabolites were prevalent in samples that were false positive for buprenorphine. The specificity of the CEDIA buprenorphine assay increased to 99.7% when the cutoff was increased from 5 ng/mL to 10 ng/mL. Conclusion Intake of a therapeutic dose of codeine can yield a false-positive CEDIA buprenorphine result. Additive effects from metabolites of codeine contribute to cross-reactivity in concentrations much lower than listed in the manufacturer’s cross-reactivity guide. Raising the cutoff from 5 ng/mL to 10 ng/mL increased the diagnostic accuracy. Clinicians should be informed about the risk of false-positive results with the CEDIA

  2. In vitro bioassays for anticancer drug screening: effects of cell concentration and other assay parameters on growth inhibitory activity.

    PubMed

    Lieberman, M M; Patterson, G M; Moore, R E

    2001-11-01

    In vitro growth inhibition assays were performed using human cancer cell lines at various concentrations with experimental anticancer drugs such as the cryptophycins and other cytotoxins. The effect of variations in assay parameters on the observed growth inhibition of these anticancer therapeutic agents was determined. The results demonstrated that the observed inhibitory activity of these compounds varied inversely with the cell concentrations used. The observed differences in activity between different cytotoxins were not necessarily proportionate. Thus, the relative activities of two toxins also varied with cell concentration. Furthermore, the sensitivity of these cell lines to the cytostatic purine analog, 6-mercaptopurine (used as a control), varied with cell concentration as well. The activity of this compound was dependent on the medium used for cell growth, yielding good activity in Eagle's minimum essential medium, but not in Ham's F-12 (Kaigin) medium. Moreover, growth inhibition by cryptophycin as well as 6-mercaptopurine was also dependent on the serum concentration in the medium. Finally, the sensitivity of the cancer cell lines to various organic solvents commonly used as drug vehicles for in vitro testing, such as ethanol, dimethylformamide, and dimethylsulfoxide, was likewise found to vary inversely with cell concentration. PMID:11578805

  3. An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.

    PubMed

    Loo, Jacky F C; Lau, P M; Ho, H P; Kong, S K

    2013-10-15

    Based on a recently reported ultra-sensitive bio-barcode (BBC) assay, we have developed an aptamer-based bio-barcode (ABC) alternative to detect a cell death marker cytochrome-c (Cyto-c) and its subsequent application to screen anti-cancer drugs. Aptamer is a short single-stranded DNA selected from a synthetic DNA library by virtue of its high binding affinity and specificity to its target based on its unique 3D structure from the nucleotide sequence after folding. In the BBC assay, an antigen (Ag) in analytes is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Abs). Gold nanoparticles (NPs) with another recognition Ab against the same target and hundreds of identical DNA molecules of known sequence are subsequently added to allow the formation of sandwich structures ([MMP-Ab1]-Ag-[Ab2-NP-DNA]). After isolating the sandwiches by a magnetic field, the DNAs hybridized to their complementary DNAs covalently bound on the NPs are released from the sandwiches after heating. Acting as an Ag identification tag, these bio-barcode DNAs with known DNA sequence are then amplified by polymerase chain reaction (PCR) and detected by fluorescence. In our ABC assay, we employed a Cyto-c-specific aptamer to substitute both the recognition Ab and barcode DNAs on the NPs in the BBC assay; and a novel isothermal recombinase polymerase amplification for the time-consuming PCR. The detection limit of our ABC assay for the Cyto-c was found to be 10 ng/mL and this new assay can be completed within 3h. Several potential anti-cancer drugs have been tested in vitro for their efficacy to kill liver cancer with or without multi-drug resistance. PMID:24054573

  4. Developing a microbiological growth inhibition screening assay for the detection of 27 veterinary drugs from 13 different classes in animal feedingstuffs.

    PubMed

    Bohn, Torsten; Pellet, Terence; Boscher, Aurore; Hoffmann, Lucien

    2013-01-01

    Many regulations prohibit using veterinary drugs in feedingstuffs to protect consumers and animals alike. Within this investigation we developed a simple, cost-efficient primary screening method for detecting antibiotics and coccidiostats in animal feeds. Thirty-two veterinary drugs were originally considered. Following matrix-free testing to optimise detection, an assay based on matrix extraction with methanol/acetonitrile/phosphate buffer followed by inoculation and diffusion in agar plates was developed. Final validation was performed with 14 representative drugs (one per drug class) and four bacteria (Escherichia coli ATCC11303 and ATCC27166, Staphylococcus aureus ATCC6538P, Micrococcus luteus ATCC9341) in bovine, lamb and swine fodder, measuring growth inhibition zones. Of the original drugs tested, 27 remained detectable in feed matrices at or below 20 mg kg(-1). Of the 14 validated representatives, two had estimated minimum detectable concentrations of 10-11 mg kg(-1), others of 5 mg kg(-1) or lower, an earlier minimum European Union inclusion rate for many veterinary drugs. No significant matrix effect on inhibition zones was detected. Per cent wrong negative deviations ranged from 0% (nine of 14 compounds) to 20-27% (two of 14), while inter-day precision based on inhibition zones had relative standard deviations (RSDs) of 6-109% (mean of 40%). When setting a 1 mm inhibition zone, the maximum observed for negative controls, as a cut-off level, no false-positives were found. While not all targeted antibiotics were detectable in complex matrices, the majority of veterinary drugs were detected with reasonable sensitivity, indicating that this method could be suitable for screening feedingstuffs prior to further confirmatory investigation of positive findings such as by LC-MS/MS. PMID:24053648

  5. Evaluation of a range of anti-proliferative assays for the preclinical screening of anti-psoriatic drugs: a comparison of colorimetric and fluorimetric assays with the thymidine incorporation assay.

    PubMed

    George, Suja Elizabeth; Anderson, Rosaleen J; Cunningham, Anne; Donaldson, Michael; Groundwater, Paul W

    2010-06-01

    Established treatments for psoriasis are generally based on antiproliferative, anti-inflammatory, or differentiation-modifying activity, or a combination of these effects. New agents for the treatment of psoriasis could be identified by high-throughput screening (HTS) of large compound libraries using keratinocyte proliferation models. Although several new proliferation assays have been developed, the radioactive [(3)H]-thymidine incorporation assay is still considered to be the gold standard for the evaluation of keratinocyte proliferation in vitro. In this study, we compare a number of simple, and reliable, colorimetric (MTT, NRU, SRB, and CVS), and fluorimetric (CAM and AB) methods with the [(3)H]-thymidine incorporation assay for the measurement of keratinocyte proliferation in the exponential growth phase in 96-well formats. The concentrations that induced 50% growth inhibition (GI(50)) were determined by each assay for the established antipsoriatics, dithranol, and methotrexate. Strong correlations were observed between the percentage growth inhibitions determined by the radioactive and the colorimetric assays, with no significant differences (P > 0.05) between their GI(50) values. The colorimetric assays are thus suitable alternatives to the radioactive assay for quantifying keratinocyte growth inhibition. We have also validated the use of the HaCaT cell line as a representative of the hyperproliferative psoriatic epidermis, in the preclinical screening of experimental anti-psoriatic agents. PMID:20482335

  6. Fluorescence Polarization Assays in Small Molecule Screening

    PubMed Central

    Lea, Wendy A.; Simeonov, Anton

    2011-01-01

    Importance of the field Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely utilized in clinical and biomedical settings, including the diagnosis of certain diseases and monitoring therapeutic drug levels in body fluids. Recent developments in the field has been symbolized by the facile adoption of FP in high-throughput screening (HTS) and small molecule drug discovery of an increasing range of target classes. Areas covered in this review The article provides a brief overview on the theoretical foundation of FP, followed by updates on recent advancements in its application for various drug target classes, including G-protein coupled receptors (GPCRs), enzymes and protein-protein interactions (PPIs). The strengths and weaknesses of this method, practical considerations in assay design, novel applications, and future directions are also discussed. What the reader will gain The reader will be informed of the most recent advancements and future directions of FP application to small molecule screening. Take home message In addition to its continued utilization in high-throughput screening, FP has expanded into new disease and target areas and has been marked by increased use of labeled small molecule ligands for receptor binding studies. PMID:22328899

  7. A new microperfusion system for the cultivation of tumor-cells invitro - approach to integrate pharmacokinetic parameters in screening assays for cytostatic drugs.

    PubMed

    Gimmel, S; Kinawi, A; Maurer, H

    1993-01-01

    By a newly introduced microperfusion system absorption and elimination rates can be simulated in vitro. This article describes the optimization of culture conditions (medium composition, membrane filters, pumping rates, and stirring speeds) of tumor cell lines (L1210, KB) maintained in suspension in an ultrafiltration-flat chamber. Viability and colony-forming ability are measured. Our results indicate that tumor cells can be cultured under serum-free conditions over a five hour incubation period with only minimal decrease in colony-forming ability. Survival of cells is independent from the pumping rate in the tested range, but is dependent of the stirring speed. Each cell line requires its own stirring speed. Ultrafiltration membranes with minimal nonspecific adsorption properties proved to be the best in terms of cell adsorption and toxicity to retain cells in the chamber. This system might improve the tumor cell colony assay for cytostatic drug screening. PMID:21573513

  8. LC-MS vs. GC-MS, online extraction systems, advantages of technology for drug screening assays.

    PubMed

    Marquet, Pierre

    2012-01-01

    This chapter reviews recent applications of mass spectrometry to systematic toxicological analysis (STA), where extended lists of compounds of toxicological interest are screened, as well as to the general unknown screening (GUS), where all exogenous compounds present in a sample are tentatively detected and identified, without any preselection. Many recent improvements in sample preparation, chromatographic separation, gas chromatography-mass spectrometry, and above all liquid chromatography-mass spectrometry techniques are described, which are applicable or have been applied to STA and/or GUS, generally with promising results. These improvements come from miniaturization and automation of solid-phase extraction, turbulent-flow or ultrahigh-pressure liquid chromatography, linear ion traps, accurate (e.g., time of flight or orbital trap) mass spectrometry, as well as software refinements to alternate between different ionization modes or automatically interpret the results. It also shows that robust LC-MS/MS techniques already exist for STA or GUS, which are at least as efficient as the traditional techniques used in most toxicology laboratories, such as GC-MS or high-performance liquid chromatography with diode-array detection, as shown by three comparative studies. However, the major drawback of LC-MS/MS in the full-scan mode for STA or GUS is that it still lacks universal reference libraries due to insufficient reproducibility of LC-MS(/MS) mass spectra obtained with different instrument types. PMID:22767104

  9. TOXICITY SCREENING WITH ZEBRAFISH ASSAY

    EPA Science Inventory

    The proposed toxicity screening will help EPA to prioritize chemicals for further testing, and it may also alert chemical manufacturers that some of their commercial products may be toxic. The proposed toxicity pathway studies will improve the research community’s abi...

  10. Solid-Phase Biological Assays for Drug Discovery

    NASA Astrophysics Data System (ADS)

    Forsberg, Erica M.; Sicard, Clémence; Brennan, John D.

    2014-06-01

    In the past 30 years, there has been a significant growth in the use of solid-phase assays in the area of drug discovery, with a range of new assays being used for both soluble and membrane-bound targets. In this review, we provide some basic background to typical drug targets and immobilization protocols used in solid-phase biological assays (SPBAs) for drug discovery, with emphasis on particularly labile biomolecular targets such as kinases and membrane-bound receptors, and highlight some of the more recent approaches for producing protein microarrays, bioaffinity columns, and other devices that are central to small molecule screening by SPBA. We then discuss key applications of such assays to identify drug leads, with an emphasis on the screening of mixtures. We conclude by highlighting specific advantages and potential disadvantages of SPBAs, particularly as they relate to particular assay formats.

  11. In vitro screening for drug repositioning.

    PubMed

    Wilkinson, Graeme F; Pritchard, Kevin

    2015-02-01

    Drug repositioning or repurposing has received much coverage in the scientific literature in recent years and has been responsible for the generation of both new intellectual property and investigational new drug submissions. The literature indicates a significant trend toward the use of computational- or informatics-based methods for generating initial repositioning hypotheses, followed by focused assessment of biological activity in phenotypic assays. Another viable method for drug repositioning is in vitro screening of known drugs or drug-like molecules, initially in disease-relevant phenotypic assays, to identify and validate candidates for repositioning. This approach can use large compound libraries or can focus on subsets of known drugs or drug-like molecules. In this short review, we focus on ways to generate and validate repositioning candidates in disease-related in vitro and phenotypic assays, and we discuss specific examples of this approach as applied to a variety of disease areas. We propose that in vitro screens offer several advantages over biochemical or in vivo methods as a starting point for drug repositioning. PMID:25527136

  12. 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

  13. Urine drug screen

    MedlinePlus

    Pincus MR, Abraham NZ Jr. Toxicology and therapeutic drug monitoring. In: McPherson RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods . 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:chap 23.

  14. High-content screening for biofilm assays.

    PubMed

    Peng, Fubing; Hoek, Eric M V; Damoiseaux, Robert

    2010-08-01

    The authors describe a novel high-throughput screening platform that provides rapid, reliable, quantitative assessment of biofilm formation and removal on engineered surfaces. Unlike traditional biofilm assays based on plate readers, this assay platform is based on high-content screening, which allows for multiplexing to simultaneously quantify the number of bacterial adhesions per unit area and the viability of adhered cells using fluorescent dye combinations. This platform is fully automated and has a throughput of more than 10,000 wells per day. The authors used this platform to examine the influence of different assay buffer systems on bacterial adhesion, viability, and removal on cross-linked polyvinyl alcohol coating films synthesized directly onto the bottoms of 384-well plates. The results indicated that water chemistry, bacteria cell type, and film chemistry combine to govern biofilm formation. In general, both reversible and irreversible bacterial adhesion increased with the extent of cross-linking in coating films, which correlates strongly with coating film cross-linking degree and hydrophobicity, which is closely related. The high-throughput platform offers a powerful tool for rapid evaluation of fouling-resistant coating films in addition to elucidation of fundamental mechanisms governing bacterial adhesion. PMID:20639506

  15. 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

  16. 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

  17. 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

  18. Transporter assays and assay ontologies: useful tools for drug discovery.

    PubMed

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P

    2014-06-01

    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays. PMID:25027375

  19. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies.

    PubMed

    Partridge, Michael A; Purushothama, Shobha; Elango, Chinnasamy; Lu, Yanmei

    2016-01-01

    Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline. PMID:27556048

  20. Emerging Technologies and Generic Assays for the Detection of Anti-Drug Antibodies

    PubMed Central

    Elango, Chinnasamy

    2016-01-01

    Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline. PMID:27556048

  1. Reporting biological assay screening results for maximum impact.

    PubMed

    Bolton, Evan

    2015-07-01

    A very large corpus of biological assay screening results exist in the public domain. The ability to compare and analyze this data is hampered due to missing details and lack of a commonly used terminology to describe assay protocols and assay endpoints. Minimum reporting guidelines exist that, if followed, would greatly enhance the utility of biological assay screening data so it may be independently reproduced, readily integrated, effectively compared, and rapidly analyzed. PMID:26194585

  2. Miniaturized INtrinsic DISsolution Screening (MINDISS) assay for preformulation.

    PubMed

    Alsenz, Jochem; Haenel, Elisabeth; Anedda, Aline; Du Castel, Pauline; Cirelli, Giorgio

    2016-05-25

    This study describes a novel Miniaturized INtrinsic DISsolution Screening (MINDISS) assay for measuring disk intrinsic dissolution rates (DIDR). In MINDISS, compacted mini disks of drugs (2-5mg/disk) are prepared in custom made holders with a surface area of 3mm(2). Disks are immersed, pellet side down, into 0.35ml of appropriate dissolution media per well in 96-well microtiter plates, media are stirred and disk-holders are transferred to new wells after defined periods of time. After filtration, drug concentration in dissolution media is quantified by Ultra Performance Liquid Chromatography (UPLC) and solid state property of the disk is characterized by Raman spectroscopy. MINDISS was identified as an easy-to-use tool for rapid, parallel determination of DIDR of compounds that requires only small amounts of compound and of dissolution medium. Results obtained with marketed drugs in MINDISS correlate well with large scale DIDR methods and indicate that MINDISS can be used for (1) rank-ordering of compounds by intrinsic dissolution in late phase discovery and early development, (2) comparison of polymorphic forms and salts, (3) screening and selection of appropriate dissolution media, and (4) characterization of the intestinal release behavior of compounds along the gastro intestinal tract by changing biorelevant media during experiments. PMID:26360839

  3. 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

  4. Microfluidic assay without blocking for rapid HIV screening and confirmation.

    PubMed

    Song, Lusheng; Zhang, Yi; Wang, Wenjun; Ma, Liying; Liu, Yong; Hao, Yanlin; Shao, Yiming; Zhang, Wei; Jiang, Xingyu

    2012-08-01

    The essential step for HIV spreading limitation is the screening tests. However, there are multiple disadvantages in current screening assays which need further confirmation test. Herein we developed a rapid HIV assay combining screening and confirmation test by using the microfluidic network assay. Meanwhile, the assay is accelerated by bypassing the step of blocking. We call this method as microfluidic assay without blocking (MAWB). Both the limit of detection and reagent incubation time of MAWB are determined by screening of one model protein pair: ovalbumin and its antibody. The assay time is accelerated about 25% while the limit of detection (LOD) is well kept. Formatting the method in for both HIV screening (testing 8 HIV-related samples) and confirmation (assaying 6 kinds of HIV antibodies of each sample) within 30 min was successful. Fast HIV screening and confirmation of 20 plasma samples were also demonstrated by this method. MAWB improved the assay speed while keeping the LOD of conventional ELISA. Meanwhile, both the accuracy and throughput of MAWB were well improved, which made it an excellent candidate for a quick HIV test for both screening and confirmation. Methods like this one will find wide applications in clinical diagnosis and biochemical analysis based on the interactions between pairs of molecules. PMID:22374476

  5. 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

  6. 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

  7. Zebrafish developmental toxicity assay: A fishy solution to reproductive toxicity screening, or just a red herring?

    PubMed

    Van den Bulck, Kathleen; Hill, Adrian; Mesens, Natalie; Diekman, Heike; De Schaepdrijver, Luc; Lammens, Lieve

    2011-09-01

    The zebrafish embryotoxicity/teratogenicity assay is described as a useful alternative screening model to evaluate the effect of drugs on embryofoetal development. Fertilized eggs were exposed to different concentrations of 15 compounds with teratogenic (8) and non-teratogenic (7) potential until 96h post-fertilization when 28 morphological endpoints and the level of compound uptake was assessed. The majority of drugs testing positive in mammals was also positive in zebrafish (75% sensitivity), while a relative high number of false positives were noted (43% specificity). Compound uptake determination appears useful for clarifying classifications as teratogenic or potential overdose although assay sensitivity could be improved to 71% if the exposure threshold, previously suggested as ∼50ng/larvae, is reconsidered. The zebrafish assay shows some potential, though limited in its current form, as a screening tool for developmental toxicity within Janssen drug development. Further assay refinement with respect to endpoints and body burden threshold is required. PMID:21704152

  8. Addressing drug effects on cut point determination for an anti-drug antibody assay.

    PubMed

    Barbosa, Maria D F S; Gleason, Carol R; Phillips, Kelli R; Berisha, Flora; Stouffer, Bruce; Warrack, Bethanne M; Chen, Guodong

    2012-10-31

    The effect of trough levels of a monoclonal antibody drug (drugB) on screening cut point (CP) determination for an anti-drug antibody (ADA) assay was scrutinized and the conclusions substantiated by data from a phase 3 cancer clinical study. The ADA assay utilized an acid dissociation step and either 0 or 100 μg/ml drugB was added to the samples prior to obtaining the signals used for CP calculations. Serum samples from three different drug-naive populations were tested (healthy individuals, cancer patients enrolled in the drugB clinical trial and cancer patients whose serum samples were available commercially). For the same disease state samples, both the screening CP and confirmation CP were different when calculated during validation or from study sample analysis. It is reasonable to assume that variability was due to the patient heterogeneity, as they could have been at distinct stages of disease progression, and/or taking different medications, amongst other differences. The patients enrolled in the clinical trial were stratified as per protocol and hence represented a more homogeneous population. Drug effects on CP may be population dependent and also assay dependent. PMID:22750627

  9. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India

    PubMed Central

    2016-01-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  10. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India.

    PubMed

    Rajput, Manoj Kumar

    2016-02-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  11. Developing predictive assays: the phenotypic screening "rule of 3".

    PubMed

    Vincent, Fabien; Loria, Paula; Pregel, Marko; Stanton, Robert; Kitching, Linda; Nocka, Karl; Doyonnas, Regis; Steppan, Claire; Gilbert, Adam; Schroeter, Thomas; Peakman, Marie-Claire

    2015-06-24

    Phenotypic drug discovery approaches can positively affect the translation of preclinical findings to patients. However, not all phenotypic assays are created equal. A critical question then follows: What are the characteristics of the optimal assays? We analyze this question and propose three specific criteria related to the disease relevance of the assay-system, stimulus, and end point-to help design the most predictive phenotypic assays. PMID:26109101

  12. Screening for Drugs Against the Plasmodium falciparum Digestive Vacuole by Imaging Flow Cytometry.

    PubMed

    Lee, Yan Quan; Hall, Brian E; Tan, Kevin S W

    2016-01-01

    Phenotypic assays are increasingly employed to provide clues about drug mechanisms. In antimalarial drug screening, however, the majority of assays are designed to only measure parasite-killing activity. We describe here a high-content assay to detect drug-mediated perturbation of the digestive vacuole integrity in the trophozoite stage of Plasmodium falciparum, using the ImageStream imaging flow cytometer. PMID:27460247

  13. Rapid screening assay for calcium bioavailability studies

    SciTech Connect

    Luhrsen, K.R.; Hudepohl, G.R.; Smith, K.T.

    1986-03-01

    Calcium bioavailability has been studied by numerous techniques. The authors report here the use of the gamma emitting isotope of calcium (/sup 47/Ca) in a whole body retention assay system. In this system, calcium sources are administered by oral gavage and subsequent counts are determined and corrected for isotopic decay. Unlike iron and zinc retention curves, which exhibit a 2-3 day equilibration period, calcium reaches equilibration after 24 hours. Autoradiographic analysis of the femurs indicate that the newly absorbed calcium is rapidly distributed to the skeletal system. Moreover, the isotope is distributed along the entire bone. Comparisons of calcium bioavailability were made using intrinsic/extrinsic labeled milk from two species i.e. rat and goat as well as CaCO/sub 3/. In addition, extrinsic labeled cow milk was examined. In the rat, the extrinsic labeled calcium from milk was better absorbed than the intrinsic calcium. This was not the case in goat milk or the calcium carbonate which exhibited no significant differences. Chromatographic analysis of the labeled milk indicates a difference in distribution of the /sup 47/Ca. From these data, the authors recommend the use of this assay system in calcium bioavailability studies. The labeling studies and comparisons indicate caution should be used, however, in labeling techniques and species milk comparison.

  14. An axenic amastigote system for drug screening.

    PubMed Central

    Callahan, H L; Portal, A C; Devereaux, R; Grogl, M

    1997-01-01

    Currently available primary screens for selection of candidate antileishmanial compounds are not ideal. The choices include screens that are designed to closely reflect the situation in vivo but are labor-intensive and expensive (intracellular amastigotes and animal models) and screens that are designed to facilitate rapid testing of a large number of drugs but do not use the clinically relevant parasite stage (promastigote model). The advent of successful in vitro culture of axenic amastigotes permits the development of a primary screen which is quick and easy like the promastigote screen but still representative of the situation in vivo, since it uses the relevant parasite stage. We have established an axenic amastigote drug screening system using a Leishmania mexicana strain (strain M379). A comparison of the 50% inhibitory concentration (IC50) drug sensitivity profiles of M379 promastigotes, intracellular amastigotes, and axenic amastigotes for six clinically relevant antileishmanial drugs (sodium stibogluconate, meglumine antimoniate, pentamidine, paromomycin, amphotericin B, WR6026) showed that M379 axenic amastigotes are a good model for a primary drug screen. Promastigote and intracellular amastigote IC50s differed for four of the six drugs tested by threefold or more; axenic amastigote and intracellular amastigote IC50s differed by twofold for only one drug. This shows that the axenic amastigote susceptibility to clinically used reference drugs is comparable to the susceptibility of amastigotes in macrophages. These data also suggest that for the compounds tested, susceptibility is intrinsic to the parasite stage. This contradicts previous hypotheses that suggested that the activities of antimonial agents against intracellular amastigotes were solely a function of the macrophage. PMID:9087496

  15. Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

    PubMed Central

    Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

    2012-01-01

    The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

  16. A rapid screening assay for identifying mycobacteria targeted nanoparticle antibiotics.

    PubMed

    Donnellan, Samantha; Tran, Lang; Johnston, Helinor; McLuckie, Joyce; Stevenson, Karen; Stone, Vicki

    2016-08-01

    Antibiotic resistance is a serious problem. Nanotechnology offers enormous potential in medicine, yet there is limited knowledge regarding the toxicity of nanoparticles (NP) for mycobacterial species that cause serious human diseases (e.g. tuberculosis (TB) and leprosy). Mycobacterial diseases are a major global health problem; TB caused by Mycobacterium tuberculosis (Mtb) kills up to 2 million people annually and there are over 200 000 leprosy cases each year caused by Mycobacterium leprae (M. leprae). Few drugs are effective against these mycobacteria and increasing antibiotic resistance exacerbates the problem. As such, alternative therapies are urgently needed but most current assays used to assess the effectiveness of therapeutics against mycobacteria are slow and expensive. This study aimed to develop a rapid, low-cost assay which can be used for screening the antimicrobial properties of compounds against pathogenic mycobacteria and to assess the toxicity of three NP (silver [Ag], copper oxide [Cu(II)O], and zinc oxide [ZnO]) against a green fluorescent protein reporter strain of Mycobacterium avium subspecies paratuberculosis, a slow growing, pathogenic mycobacterial species causing paratuberculosis in ruminants. Fluorescence was used to monitor mycobacterial growth over time, with NP concentrations of 6.25-100 μg/mL tested for up to 7 days, and a method of data analysis was designed to permit comparison between results. Mycobacterial sensitivity to the NP was found to be NP composition specific and toxicity could be ranked in the following order: Ag > Cu(II)O > ZnO. PMID:26618564

  17. The virtual heart as a platform for screening drug cardiotoxicity.

    PubMed

    Yuan, Yongfeng; Bai, Xiangyun; Luo, Cunjin; Wang, Kuanquan; Zhang, Henggui

    2015-12-01

    To predict the safety of a drug at an early stage in its development is a major challenge as there is a lack of in vitro heart models that correlate data from preclinical toxicity screening assays with clinical results. A biophysically detailed computer model of the heart, the virtual heart, provides a powerful tool for simulating drug-ion channel interactions and cardiac functions during normal and disease conditions and, therefore, provides a powerful platform for drug cardiotoxicity screening. In this article, we first review recent progress in the development of theory on drug-ion channel interactions and mathematical modelling. Then we propose a family of biomarkers that can quantitatively characterize the actions of a drug on the electrical activity of the heart at multi-physical scales including cellular and tissue levels. We also conducted some simulations to demonstrate the application of the virtual heart to assess the pro-arrhythmic effects of cisapride and amiodarone. Using the model we investigated the mechanisms responsible for the differences between the two drugs on pro-arrhythmogenesis, even though both prolong the QT interval of ECGs. Several challenges for further development of a virtual heart as a platform for screening drug cardiotoxicity are discussed. PMID:25363597

  18. [Comparison of four drug interaction screening programs].

    PubMed

    Ing Lorenzini, K; Reutemann, B; Samer, C F; Guignard, B; Bonnabry, P; Dayer, P; Perrier, A; Desmeules, J

    2012-10-17

    Adverse drug events (ADE) are a major public health issue, with drug-drug interactions (DDI) being one of well-recognized causes of ADE that could be preventable by the use of DDI screening software. We compared the ability of four programs to detect clinically important DDI. We tested 62 drug pairs with and 12 drug pairs without clinically important DDI. Lexi-Interact and Epocrates were the most sensitive (95%) compared to the Compendium and Theriaque (80 and 73%, respectively). The Compendium and Theriaque also showed the lowest negative predictive value. All programs showed high specificity and positive predictive value. The qualitative assessment showed the best performances for Compendium and Lexi-Interact. The last one seems to be the best screening program, but the Compendium is in French and is freely available. PMID:23198652

  19. Sulfonylureas and Glinides as New PPARγ Agonists:. Virtual Screening and Biological Assays

    NASA Astrophysics Data System (ADS)

    Scarsi, Marco; Podvinec, Michael; Roth, Adrian; Hug, Hubert; Kersten, Sander; Albrecht, Hugo; Schwede, Torsten; Meyer, Urs A.; Rücker, Christoph

    2007-12-01

    This work combines the predictive power of computational drug discovery with experimental validation by means of biological assays. In this way, a new mode of action for type 2 diabetes drugs has been unvealed. Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target PPARγ improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides bind to PPARγ and exhibit PPARγ agonistic activity. This result was predicted in silico by virtual screening and confirmed in vitro by three biological assays. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, since it provides evidence that drugs can be designed which target both the sulfonylurea receptor and PPARγ. Targeting both receptors could in principle allow to increase pancreatic insulin secretion, as well as to improve insulin resistance.

  20. Synthetic Tumor Networks for Screening Drug Delivery Systems

    PubMed Central

    Prabhakarpandian, Balabhaskar; Shen, Ming-Che; Nichols, Joseph B.; Garson, Charles J.; Mills, Ivy R.; Matar, Majed M.; Fewell, Jason G.; Pant, Kapil

    2015-01-01

    Tumor drug delivery is a complex phenomenon affected by several elements in addition to drug or delivery vehicle’s physico-chemical properties. A key factor is tumor microvasculature with complex effects including convective transport, high interstitial pressure and enhanced vascular permeability due to the presence of “leaky vessels”. Current in vitro models of the tumor microenvironment for evaluating drug delivery are oversimplified and, as a result, show poor correlation with in vivo performance. In this study, we report on the development of a novel microfluidic platform that models the tumor microenvironment more accurately, with physiologically and morphologically realistic microvasculature including endothelial cell lined leaky capillary vessels along with 3D solid tumors. Endothelial cells and 3D spheroids of cervical tumor cells were co-cultured in the networks. Drug vehicle screening was demonstrated using GFP gene delivery by different formulations of nanopolymers. The synthetic tumor network was successful in predicting in vivo delivery efficiencies of the drug vehicles. The developed assay will have critical applications both in basic research, where it can be used to develop next generation delivery vehicles, and in drug discovery where it can be used to study drug transport and delivery efficacy in realistic tumor microenvironment, thereby enabling drug compound and/or delivery vehicle screening. PMID:25599856

  1. Synthetic tumor networks for screening drug delivery systems.

    PubMed

    Prabhakarpandian, Balabhaskar; Shen, Ming-Che; Nichols, Joseph B; Garson, Charles J; Mills, Ivy R; Matar, Majed M; Fewell, Jason G; Pant, Kapil

    2015-03-10

    Tumor drug delivery is a complex phenomenon affected by several elements in addition to drug or delivery vehicle's physico-chemical properties. A key factor is tumor microvasculature with complex effects including convective transport, high interstitial pressure and enhanced vascular permeability due to the presence of "leaky vessels". Current in vitro models of the tumor microenvironment for evaluating drug delivery are oversimplified and, as a result, show poor correlation with in vivo performance. In this study, we report on the development of a novel microfluidic platform that models the tumor microenvironment more accurately, with physiologically and morphologically realistic microvasculature including endothelial cell lined leaky capillary vessels along with 3D solid tumors. Endothelial cells and 3D spheroids of cervical tumor cells were co-cultured in the networks. Drug vehicle screening was demonstrated using GFP gene delivery by different formulations of nanopolymers. The synthetic tumor network was successful in predicting in vivo delivery efficiencies of the drug vehicles. The developed assay will have critical applications both in basic research, where it can be used to develop next generation delivery vehicles, and in drug discovery where it can be used to study drug transport and delivery efficacy in realistic tumor microenvironment, thereby enabling drug compound and/or delivery vehicle screening. PMID:25599856

  2. Rapid fluorescence screening assay for tetracyclines in chicken muscle.

    PubMed

    Schneider, Marilyn J; Lehotay, Steven J

    2004-01-01

    A simple, rapid fluorescence assay was developed for screening tetracyclines in chicken muscle at the U.S. tolerance level (2 mg/kg). The method requires only a homogenization of the tissue in acetonitrile-ammonium hydroxide, centrifugation, addition of Mg+2, and another centrifugation before fluorescence of the supernatant is measured at 505 nm (excitation at 385 nm). Comparison of the fluorescence of control chicken muscle extracts with extracts from muscle fortified with either 2 mg/kg tetracycline, oxytetracycline, or chlortetracycline showed no overlap. A threshold level set at the average fluorescence for a series of fortified 2 mg/kg samples minus 3sigma minimized false-negative responses to provide a successful screening method. The method was tested with blinded samples as controls or samples fortified with tetracycline, oxytetracycline, or chlortetracycline in order to demonstrate its utility. This approach can provide an alternative to microbial screening assays. PMID:15287655

  3. 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

  4. 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

  5. An assay for screening microbial cultures for chalkophore production.

    PubMed

    Yoon, Sukhwan; Kraemer, Stephan M; Dispirito, Alan A; Semrau, Jeremy D

    2010-04-01

    Methanotrophs, bacteria that utilize methane as their sole carbon and energy source, are known to have high requirements for copper. These bacteria have recently been found to synthesize a copper-chelating agent, or chalkophore, termed methanobactin. To aid in screening methanobactin production by methanotrophs, a plate assay developed from the chrome azurol S (CAS) assay for siderophore production, was modified. In the typical CAS assay, a colour change from blue to orange in iron-CAS plates is observed as iron (III) ion weakly bound to CAS is sequestered by siderophores with higher affinities. In our modified assay, iron (III) chloride of the original CAS solution was substituted with copper (II) chloride, and removal of copper from CAS caused a colour change from blue to yellow. Assay results indicated that of the four tested methanotrophs (Methylosinus trichosporium OB3b, Methylococcus capsulatus Bath, Methylomicrobium album BG8 and Methylocystis parvus OBBP), only M. trichosporium OB3b, M. capsulatus Bath and M. album BG8 produced chalkophores capable of competing with CAS for copper, while M. parvus OBBP did not or did not export sufficient concentrations of methanobactin for detection by this assay. It was also found using Fe-CAS plates that at least M. trichosporium OB3b and M. album BG8 produce siderophores. These results may be expanded for the detection of chalkophores in other microorganisms as well as for screening of putative mutants of chalkophore synthesis. PMID:23766081

  6. Biomimetic three-dimensional tissue models for advanced high-throughput drug screening

    PubMed Central

    Nam, Ki-Hwan; Smith, Alec S.T.; Lone, Saifullah; Kwon, Sunghoon; Kim, Deok-Ho

    2015-01-01

    Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately recreate the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when utilizing such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models which accurately mimic the physiological properties of native tissue samples, and highlight the advantages of using such 3D micro-tissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based-on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models. PMID:25385716

  7. 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

  8. Label-free cytotoxicity screening assay by digital holographic microscopy.

    PubMed

    Kühn, Jonas; Shaffer, Etienne; Mena, Julien; Breton, Billy; Parent, Jérôme; Rappaz, Benjamin; Chambon, Marc; Emery, Yves; Magistretti, Pierre; Depeursinge, Christian; Marquet, Pierre; Turcatti, Gerardo

    2013-03-01

    We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z'-factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy. PMID:23062077

  9. Determination of designer drug cross-reactivity on five commercial immunoassay screening kits.

    PubMed

    Regester, Laura E; Chmiel, Jeffrey D; Holler, Justin M; Vorce, Shawn P; Levine, Barry; Bosy, Thomas Z

    2015-03-01

    The detection of new designer drugs is often a difficult issue in forensic urine drug testing as immunoassays are the primary screening methodology for drugs of abuse in many of these laboratories. Cross-reactivity of compounds with immunoassay kits can either aid or complicate the detection of a variety of drug and drug metabolites. For instance, emerging designer drugs that share structural similarities to amphetamines and phencyclidine (PCP) have the potential to cross-react with assays designed to detect these compounds. This study evaluates the cross-reactivity of five commercially available immunoassay reagent kits for 94 designer drugs on a Roche/Hitachi Modular P automated screening instrument. The compounds used in this study are grouped by structural class as follows: 2,5-dimethoxyamphetamines, 2C (2,5-dimethoxyphenethylamines), β-keto amphetamines, substituted amphetamines, piperazines, α-pyrrolidinopropiophenones, tryptamines and PCP analogs. A drug concentration of 100 µg/mL was used to determine cross-reactivity for each assay and resulted in the following positive rates: Microgenics DRI(®) Ecstasy enzyme assay (19%), Microgenics DRI(®) Phencyclidine enzyme assay (20%), Lin-Zhi Methamphetamine enzyme immunoassay (39%), Siemens/Syva(®) EMIT(®)II Plus Amphetamines assay (43%) and CEDIA(®) DAU Amphetamine/Ecstasy assay (57%). Of the 94 designer drugs tested, 14% produced a negative response for all five kits. No designer drug used in this study generated a positive result for all five immunoassay kits. PMID:25492523

  10. Bioluminescent, Nonlytic, Real-Time Cell Viability Assay and Use in Inhibitor Screening

    PubMed Central

    Zhou, Wenhui; Meisenheimer, Poncho; Vidugiris, Gediminas; Cali, James J.; Gautam, Prson; Wennerberg, Krister; Vidugiriene, Jolanta

    2015-01-01

    Abstract Real-time continuous monitoring of cellular processes offers distinct advantages over traditional endpoint assays. A comprehensive representation of the changes occurring in live cells over the entire length of an experiment provides information about the biological status of the cell and informs decisions about the timing of treatments or the use of other functional endpoint assays. We describe a homogeneous, nonlytic, bioluminescent assay that measures cell viability in real time. This time-dependent measurement allowed us to monitor cell health for 72 h from the same test samples, distinguish differential cell growth, and investigate drug mechanism of action by analyzing time- and dose-dependent drug effects. The real-time measurements also allowed us to detect cell death immediately (>75% signal decrease within 15 min of digitonin addition), analyze drug potency versus efficacy, and identify cytostatic versus toxic drug effects. We screened an oncology compound library (Z′ = 0.7) and identified compounds with varying activity at different time points (1.6% of the library showed activity within 3 h, whereas 35.4% showed a response by 47 h). The assay compared well with orthogonal endpoint cell viability assays and additionally provided data at multiple time points and the opportunity to multiplex assays on the same cells. To test the advantage of time-dependent measurements to direct optimal timing of downstream applications, we used the real-time cell viability assay to determine the ideal time to measure caspase activity by monitoring the onset of cell death and multiplexing a luminescent caspase activation assay on the same test samples. PMID:26383544

  11. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

    PubMed Central

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  12. Developmental toxicity assay using high content screening of zebrafish embryos

    PubMed Central

    Lantz-McPeak, Susan; Guo, Xiaoqing; Cuevas, Elvis; Dumas, Melanie; Newport, Glenn D.; Ali, Syed F.; Paule, Merle G.; Kanungo, Jyotshna

    2016-01-01

    Typically, time-consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post-fertilization. Here we describe an automated image-based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post-acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth-retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. PMID:24871937

  13. Development of a cell viability assay to assess drug metabolite structure-toxicity relationships.

    PubMed

    Rana, Payal; Will, Yvonne; Nadanaciva, Sashi; Jones, Lyn H

    2016-08-15

    Many adverse drug reactions are caused by the cytochrome P450 (CYP)-dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-induced toxicity and to improve the safety of drug candidates, we developed a simple cell viability assay by combining a bioactivation system (human CYP3A4, CYP2D6 and CYP2C9) with Hep3B cells. We screened a series of drugs to explore structural motifs that may be responsible for CYP450-dependent activation caused by reactive metabolite formation, which highlighted specific liabilities regarding certain phenols and anilines. PMID:27397500

  14. Comparison of rapid screening assays using organic chemicals

    SciTech Connect

    Beach, S.A.; Robideau, R.R.

    1994-12-31

    In a continuation of a study presented last year using metals, the sensitivity of short term toxicity tests is examined using common organic chemicals. In toxicity testing, the focus has shifted from the traditional long-term studies utilizing the mortality of complex, multicellular eukaryotic organisms as the endpoint towards short-term studies in which transformation of biochemical pathways are monitored. The relative sensitivity of aquatic screening techniques are compared to the standardized 48-hr Daphnia magna and Ceriodaphnia dubia, 96-hr fathead minnow and 96-hr algal acute assays. The short-term test procedures investigated are: dehydrogenase enzyme activity assays utilizing triphenyltetrazolium chloride (TTC) and resazurin as the calorimetric indicators; TOXI-Chromotest, inhibition of {beta}-galactosidase; reduction in bioluminescence output utilizing the Microtox{reg_sign} test; nitrification inhibition assays with a commercial preparation of nitrifying bacteria (Nitroseed{trademark}) and municipal activated sludge; respiration inhibition assays with a commercial preparation of heterotrophic bacteria (Polytox{reg_sign}) and activated sludge; inhibition of root growth in terrestrial plants; and galactosidase inhibition through the use of a fluorometrically tagged substrate with the Daphnia magna IQ{trademark} test. Toxicity values generated by this laboratory on commonly used organic chemicals are compared.

  15. Inhibition of Microglia Activation as a Phenotypic Assay in Early Drug Discovery

    PubMed Central

    Figuera-Losada, Mariana; Rojas, Camilo; Slusher, Barbara S.

    2014-01-01

    Complex biological processes such as inflammation, cell death, migration, proliferation, and the release of biologically active molecules can be used as outcomes in phenotypic assays during early stages of drug discovery. Although target-based approaches have been widely used over the past decades, a disproportionate number of first-in-class drugs have been identified using phenotypic screening. This review details phenotypic assays based on inhibition of microglial activation and their utility in primary and secondary screening, target validation, and pathway elucidation. The role of microglia, both in normal as well as in pathological conditions such as chronic neurodegenerative diseases, is reviewed. Methodologies to assess microglia activation in vitro are discussed in detail, and classes of therapeutic drugs known to decrease the proinflammatory and cytotoxic responses of activated microglia are appraised, including inhibitors of glutaminase, cystine/glutamate antiporter, nuclear factor κB, and mitogen-activated protein kinases. PMID:23945875

  16. Mini-column screening assay for tetracyclines in chicken.

    PubMed

    Shalaby, Ali R

    2015-01-01

    A simple, rapid, reliable and economical mini-column (MC) method for the detection of tetracyclines (TCs) residues in chicken meat was developed. The method employs a commonly available Pasteur pipette which is tightly packed with silica gel and anhydrous sodium sulfate. Clean-up and detection of illegal levels can be achieved on the same column. Viewing the developed MC under an ultraviolet lamp revealed that TCs can be detected as a compact golden yellow fluorescent band at the junction between the anhydrous sodium sulfate and silica gel layers. Comparing the yellow band of control extracts with those fortified (100 ng ml(-1)) showed no overlap between analyte and impurities. The limit of detection (LOD) of the MC assay was 1 ng, indicating that the chicken sample containing 10 µg TCs kg(-1) sample could be easily detected. Moreover, the intensity of the yellow band increased whenever TCs levels in the extract increased. Evaluation utility of the method with blind samples as controls or samples fortified with total TCs at various levels indicated that the total blank and spiked samples at levels equal or below the permissible limits were assessed as accepted. The method can provide an alternative to microbial screening assays and could be used as an effective pre-screening technique in public health laboratories. PMID:25430068

  17. A Quantitative Microfluidic Angiogenesis Screen for Studying Anti-Angiogenic Therapeutic Assay

    PubMed Central

    Kim, Choong; Kasuya, Junichi; Jeon, Jessie; Chung, Seok; Kamm, Roger D.

    2015-01-01

    Anti-angiogenic therapy, which suppresses tumor growth by disrupting oxygen and nutrient supply from blood to the tumor, is now widely accepted as a treatment for cancer. To investigate the mechanisms of action of these anti-angiogenesis drugs, new three dimensional (3D) cell culture-based drug screening models are increasingly employed. However, there is no in vitro high-throughput screening (HTS) angiogenesis assay that can provide uniform culture conditions for quantitative assessment of physiological responses to chemoattractant reagents under various concentrations of anti-angiogenesis drugs. Here we describe a method for screening and quantifying the vascular endothelial growth factor (VEGF)-induced chemotactic response on human umbilical vein endothelial cells (HUVECs) cultured under different concentrations of bortezomib, a selective 26S proteasome inhibitor. With this quantitative microfluidic angiogenesis screen (QMAS), we demonstrate that bortezomib-induced endothelial cell death was preceded by a series of morphological changes that develop over several days. We also explore the mechanisms by which bortezomib can inhibit angiogenesis. PMID:25370780

  18. Disagreement between Human Papillomavirus Assays: An Unexpected Challenge for the Choice of an Assay in Primary Cervical Screening

    PubMed Central

    Ejegod, Ditte Møller; Rygaard, Carsten; Lynge, Elsebeth; Bonde, Jesper

    2014-01-01

    We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41% tested positive on all four. Agreement was lower in women aged ≥30 years (30%, vs. 49% at <30 years), in primary screening samples (29%, vs. 38% in follow-up samples), and in women with concurrent normal cytology (22%, vs. 68% with abnormal cytology). Among primary screening samples from women aged 30–65 years (n = 2,881), 23% tested positive on at least one assay, and 42 to 58% of these showed positive agreement on any compared pair of the assays. While 4% of primary screening samples showed abnormal cytology, 6 to 10% were discordant on any pair of assays. A literature review corroborated our findings of considerable disagreement between human papillomavirus assays. This suggested that the extent of disagreement in primary screening is neither population- nor storage media-specific, leaving assay design differences as the most probable cause. The substantially different selection of women testing positive on the various human papillomavirus assays represents an unexpected challenge for the choice of an assay in primary cervical screening, and for follow up of in particular HPV positive/cytology normal women. PMID:24466262

  19. Precision multidimensional assay for high-throughput microRNA drug discovery

    PubMed Central

    Haefliger, Benjamin; Prochazka, Laura; Angelici, Bartolomeo; Benenson, Yaakov

    2016-01-01

    Development of drug discovery assays that combine high content with throughput is challenging. Information-processing gene networks can address this challenge by integrating multiple potential targets of drug candidates' activities into a small number of informative readouts, reporting simultaneously on specific and non-specific effects. Here we show a family of networks implementing this concept in a cell-based drug discovery assay for miRNA drug targets. The networks comprise multiple modules reporting on specific effects towards an intended miRNA target, together with non-specific effects on gene expression, off-target miRNAs and RNA interference pathway. We validate the assays using known perturbations of on- and off-target miRNAs, and evaluate an ∼700 compound library in an automated screen with a follow-up on specific and non-specific hits. We further customize and validate assays for additional drug targets and non-specific inputs. Our study offers a novel framework for precision drug discovery assays applicable to diverse target families. PMID:26880188

  20. Precision multidimensional assay for high-throughput microRNA drug discovery.

    PubMed

    Haefliger, Benjamin; Prochazka, Laura; Angelici, Bartolomeo; Benenson, Yaakov

    2016-01-01

    Development of drug discovery assays that combine high content with throughput is challenging. Information-processing gene networks can address this challenge by integrating multiple potential targets of drug candidates' activities into a small number of informative readouts, reporting simultaneously on specific and non-specific effects. Here we show a family of networks implementing this concept in a cell-based drug discovery assay for miRNA drug targets. The networks comprise multiple modules reporting on specific effects towards an intended miRNA target, together with non-specific effects on gene expression, off-target miRNAs and RNA interference pathway. We validate the assays using known perturbations of on- and off-target miRNAs, and evaluate an ∼700 compound library in an automated screen with a follow-up on specific and non-specific hits. We further customize and validate assays for additional drug targets and non-specific inputs. Our study offers a novel framework for precision drug discovery assays applicable to diverse target families. PMID:26880188

  1. A Simple Assay to Screen Antimicrobial Compounds Potentiating the Activity of Current Antibiotics

    PubMed Central

    Iqbal, Junaid; Kazmi, Shahana Urooj; Khan, Naveed Ahmed

    2013-01-01

    Antibiotic resistance continues to pose a significant problem in the management of bacterial infections, despite advances in antimicrobial chemotherapy and supportive care. Here, we suggest a simple, inexpensive, and easy-to-perform assay to screen antimicrobial compounds from natural products or synthetic chemical libraries for their potential to work in tandem with the available antibiotics against multiple drug-resistant bacteria. The aqueous extract of Juglans regia tree bark was tested against representative multiple drug-resistant bacteria in the aforementioned assay to determine whether it potentiates the activity of selected antibiotics. The aqueous extract of J. regia bark was added to Mueller-Hinton agar, followed by a lawn of multiple drug-resistant bacteria, Salmonella typhi or enteropathogenic E. coli. Next, filter paper discs impregnated with different classes of antibiotics were placed on the agar surface. Bacteria incubated with extract or antibiotics alone were used as controls. The results showed a significant increase (>30%) in the zone of inhibition around the aztreonam, cefuroxime, and ampicillin discs compared with bacteria incubated with the antibiotics/extract alone. In conclusion, our assay is able to detect either synergistic or additive action of J. regia extract against multiple drug-resistant bacteria when tested with a range of antibiotics. PMID:23865073

  2. Toxicology screen

    MedlinePlus

    Barbiturates - screen; Benzodiazepines - screen; Amphetamines - screen; Analgesics - screen; Antidepressants - screen; Narcotics - screen; Phenothiazines - screen; Drug abuse screen; Blood alcohol test

  3. Chemical interrogation of the neuronal kinome using a primary cell-based screening assay.

    PubMed

    Al-Ali, Hassan; Schürer, Stephan C; Lemmon, Vance P; Bixby, John L

    2013-05-17

    A fundamental impediment to functional recovery from spinal cord injury (SCI) and traumatic brain injury is the lack of sufficient axonal regeneration in the adult central nervous system. There is thus a need to develop agents that can stimulate axon growth to re-establish severed connections. Given the critical role played by protein kinases in regulating axon growth and the potential for pharmacological intervention, small molecule protein kinase inhibitors present a promising therapeutic strategy. Here, we report a robust cell-based phenotypic assay, utilizing primary rat hippocampal neurons, for identifying small molecule kinase inhibitors that promote neurite growth. The assay is highly reliable and suitable for medium-throughput screening, as indicated by its Z'-factor of 0.73. A focused structurally diverse library of protein kinase inhibitors was screened, revealing several compound groups with the ability to strongly and consistently promote neurite growth. The best performing bioassay hit robustly and consistently promoted axon growth in a postnatal cortical slice culture assay. This study can serve as a jumping-off point for structure activity relationship (SAR) and other drug discovery approaches toward the development of drugs for treating SCI and related neurological pathologies. PMID:23480631

  4. Comprehensive drug screening in blood for detecting abused drugs or drugs potentially hazardous for traffic safety.

    PubMed

    Lillsunde, P; Michelson, L; Forsstrom, T; Korte, T; Schultz, E; Ariniemi, K; Portman, M; Sihvonen, M L; Seppala, T

    1996-02-01

    A comprehensive drug screening procedure for detecting drugs in the blood samples of car drivers suspected of driving under the influence of drugs, is presented. Amphetamines, cannabinoids, opioids, cocaine and benzodiazepines were screened by an immunological EMIT ETS system after acetone precipitation. Gas chromatographic methods were used to screen and quantitate basic, neutral and acidic drugs. The free amino groups of basic drugs were derivatized with heptafluorobutyric anhydride. Analysis was performed by a dual channel gas chromatograph combined with a nitrogen phosphorus and an electron capture detector. Phenyltrimethylammonium hydroxide was used as a methylathing agent for acidic substances before analysis with a gas chromatograph connected to a nitrogen phosphorus detector. A gas chromatograph/mass spectrometry was used as a common confirmation method. Tetrahydrocannabinol was quantitated after bis(trimethylsilyl)trifluoroacetamide derivatization, opiates after pentafluoropropionic anhydride derivatization and benzoylecgonine after pentafluoropropionic anhydride and pentafluoropropanol derivatization. Excluding benzodiazepines, which were confirmed with a gas chromatograph connected to a nitrogen phosphorus and an electron capture detector, the other basic drugs as well as the acidic drugs were confirmed after the same derivatization procedures as in the screening methods. Alcohols were quantitated in triplicate by gas chromatography using three different kinds of columns. Although urine is the most important specimen for screening abused drugs, it has only limited use in forensic toxicology. The described system is most useful for analyzing a wide range of substances, including illicit drugs, benzodiazepines, barbiturates, antidepressants and phenothiazenes in forensic samples when urine is not available. PMID:8819994

  5. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    SciTech Connect

    Morton, M.J.; Armstrong, D.; Abi Gerges, N.; Bridgland-Taylor, M.; Pollard, C.E.; Bowes, J.; Valentin, J.-P.

    2014-09-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

  6. 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

  7. A new in vitro hemagglutinin inhibitor screening system based on a single-vesicle fusion assay.

    PubMed

    Lee, Hanki; Jin, Wook; Jeong, Byeong-Chul; Suh, Joo-Won

    2016-01-01

    Hemagglutinin (HA) from the influenza virus plays a pivotal role in the infection of host mammalian cells and is, therefore, a druggable target, similar to neuraminidase. However, research involving the influenza virus must be conducted in facilities certified at or above Biosafety Level 2 because of the potential threat of the contagiousness of this virus. To develop a new HA inhibitor screening system without intact influenza virus, we conceived a single-vesicle fusion assay using full-length recombinant HA. In this study, we first showed that full-length recombinant HA can mediate membrane fusion in ensemble and single-vesicle fusion assays. The fluorescence resonance energy transfer (FRET) frequency pattern of single-vesicle complexes completely differed when the inhibitors targeted the HA1 or HA2 domain of HA. This result indicates that analysing the FRET patterns in this assay can provide information regarding the domains of HA inhibited by compounds and compounds' inhibitory activities. Therefore, our results suggest that the assay developed here is a promising tool for the discovery of anti-influenza virus drug candidates as a new in vitro inhibitor screening system against HA from the influenza virus. PMID:27469068

  8. 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

  9. A new in vitro hemagglutinin inhibitor screening system based on a single-vesicle fusion assay

    PubMed Central

    Lee, Hanki; Jin, Wook; Jeong, Byeong-Chul; Suh, Joo-Won

    2016-01-01

    Hemagglutinin (HA) from the influenza virus plays a pivotal role in the infection of host mammalian cells and is, therefore, a druggable target, similar to neuraminidase. However, research involving the influenza virus must be conducted in facilities certified at or above Biosafety Level 2 because of the potential threat of the contagiousness of this virus. To develop a new HA inhibitor screening system without intact influenza virus, we conceived a single-vesicle fusion assay using full-length recombinant HA. In this study, we first showed that full-length recombinant HA can mediate membrane fusion in ensemble and single-vesicle fusion assays. The fluorescence resonance energy transfer (FRET) frequency pattern of single-vesicle complexes completely differed when the inhibitors targeted the HA1 or HA2 domain of HA. This result indicates that analysing the FRET patterns in this assay can provide information regarding the domains of HA inhibited by compounds and compounds’ inhibitory activities. Therefore, our results suggest that the assay developed here is a promising tool for the discovery of anti-influenza virus drug candidates as a new in vitro inhibitor screening system against HA from the influenza virus. PMID:27469068

  10. 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

  11. Tango assay for ligand-induced GPCR-β-arrestin2 interaction: Application in drug discovery.

    PubMed

    Dogra, Shalini; Sona, Chandan; Kumar, Ajeet; Yadav, Prem N

    2016-01-01

    G protein-coupled receptors (GPCRs) are widely known to modulate almost all physiological functions and have been demonstrated over the time as therapeutic targets for wide gamut of diseases. The design and implementation of high-throughput GPCR-based assays that permit the efficient screening of large compound libraries to discover novel drug candidates are essential for a successful drug discovery endeavor. Usually, GPCR-based functional assays depend primarily on the measurement of G protein-mediated second messenger generation. However, with advent of advanced molecular biology tools and increased understanding of GPCR signal transduction, many G protein-independent pathways such as β-arrestin translocation are being utilized to detect the activity of GPCRs. These assays provide additional information on functional selectivity (also known as biased agonism) of compounds that could be harnessed to develop pathway-selective drug candidates to reduce the adverse effects associated with given GPCR target. In this chapter, we describe the basic principle, detailed methodologies and assay setup, result analysis and data interpretations of the β-arrestin2 Tango assay, and its comparison with cell-based G protein-dependent GPCR assays, which could be employed in a simple academic setup to facilitate GPCR-based drug discovery. PMID:26928547

  12. Development and utilization of activated STAT3 detection assays for screening a library of secreted proteins.

    PubMed

    Fursov, Natalie; Gates, Irina V; Panavas, Tadas; Giles-Komar, Jill; Powers, Gordon

    2011-08-01

    Interleukin-6 (IL-6) family of cytokines are multifunctional proteins that play an important role in host defenses, acute phase reactions, immune responses, hematopoiesis, and tumorigenesis. The cytokines are produced by various lymphoid and nonlymphoid cells and mediate their biological activity through initial low-affinity binding to cell surface receptors, which are specific for their respective ligands. Ligand-specific receptor binding results in the receptor heterodimerization with ubiquitously expressed signal-transducing transmembrane component gp130 followed by activation of the gp130-associated Janus kinase, which, in turn, phosphorylates signal transducer and activator of transcription 3 (STAT3). Phosphorylated STAT3 (pSTAT3) dimerizes and translocates to the nucleus, where it activates gene transcription. Activation of STAT3 is essential to IL-6 family-associated physiological effects. Therefore, the ability to assess STAT3 phosphorylation is important for drug discovery efforts targeting IL-6 family cytokines. Various reagents and technologies are available to detect the effect of IL-6 type cytokines in treated cells. The present study describes the development of two pSTAT3 detection assays: the high-throughput screening assay based on Meso-Scale Discovery technology, which utilizes electrochemoluminescent signal measurements for the detection of pSTAT3 in treated cell extracts, and the secondary characterization assay based on fluorescent imaging analysis, which monitors pSTAT3 nuclear translocation in cells after activation. We have successfully utilized these assays to screen a small library of secreted proteins and identified inducers of STAT3 phosphorylation. The results obtained in this study demonstrate that both assays are robust, reliable, and amenable to high-throughput screening applications. PMID:21294636

  13. Cross-reactivity of designer drugs, including cathinone derivatives, in commercial enzyme-linked immunosorbent assays.

    PubMed

    Swortwood, Madeleine J; Hearn, W Lee; DeCaprio, Anthony P

    2014-01-01

    Since the introduction of synthetic heroin, designer drugs have been increasing in prevalence in the United States drug market over the past few decades. Recently, 'legal highs' sold as 'bath salts' have become a household term for one such class of designer drugs. While a number of federal and state bans have been enacted, the abuse of these designer drugs still continues. Few assays have been developed for the comprehensive detection of such compounds, so it is important to investigate how they may or may not react in presumptive screens, i.e. pre-existing commercial immunoassays. In this experiment, 16 different ELISA reagents were evaluated to determine the cross-reactivity of 30 designer drugs, including 24 phenylethylamines (including 8 cathinone derivatives), 3 piperazines, and 3 tryptamines. Cross-reactivity towards most drugs was <4% in assays targeting amphetamine or methamphetamine. Compounds such as MDA, MDMA, ethylamphetamine, and α-methyltryptamine demonstrated cross-reactivities in the range of 30-250%, but data were consistent with both manufacturer's inserts and published literature. When tested against the Randox Mephedrone/Methcathinone kit, cathinone derivatives demonstrated cross-reactivity at concentrations as low as 150 ng/ml. Since this same reagent did not cross-react with other amphetamine-like compounds, it opens the possibility to screen post-mortem specimens without the interference of putrefactive amines. All other assays demonstrated essentially no cross-reactivity towards any of the analytes evaluated. Given these results, a great need exists for more broad-range screening techniques to be applied when analyzing biological specimens by immunoassays for drugs of abuse, specifically the more recent designer drugs. PMID:23677923

  14. 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

  15. Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni

    PubMed Central

    2013-01-01

    Background Echinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays. Methods Larvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method. Results The chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5–7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications. Conclusion NEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro

  16. Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines

    PubMed Central

    Wu, Qi; Liu, Shanshan; Coster, Adam D.; Posner, Bruce A.; Altschuler, Steven J.; Wu, Lani F.

    2015-01-01

    High-content, image-based screens enable the identification of compounds that induce cellular responses similar to those of known drugs but through different chemical structures or targets. A central challenge in designing phenotypic screens is choosing suitable imaging biomarkers. Here we present a method for systematically identifying optimal reporter cell lines for annotating compound libraries (ORACLs), whose phenotypic profiles most accurately classify a training set of known drugs. We generate a library of fluorescently tagged reporter cell lines, and let analytical criteria determine which among them—the ORACL—best classifies compounds into multiple, diverse drug classes. We demonstrate that an ORACL can functionally annotate large compound libraries across diverse drug classes in a single-pass screen and confirm high prediction accuracy via orthogonal, secondary validation assays. Our approach will increase the efficiency, scale and accuracy of phenotypic screens by maximizing their discriminatory power. PMID:26655497

  17. Improving drug discovery with high-content phenotypic screens by systematic selection of reporter cell lines.

    PubMed

    Kang, Jungseog; Hsu, Chien-Hsiang; Wu, Qi; Liu, Shanshan; Coster, Adam D; Posner, Bruce A; Altschuler, Steven J; Wu, Lani F

    2016-01-01

    High-content, image-based screens enable the identification of compounds that induce cellular responses similar to those of known drugs but through different chemical structures or targets. A central challenge in designing phenotypic screens is choosing suitable imaging biomarkers. Here we present a method for systematically identifying optimal reporter cell lines for annotating compound libraries (ORACLs), whose phenotypic profiles most accurately classify a training set of known drugs. We generate a library of fluorescently tagged reporter cell lines, and let analytical criteria determine which among them--the ORACL--best classifies compounds into multiple, diverse drug classes. We demonstrate that an ORACL can functionally annotate large compound libraries across diverse drug classes in a single-pass screen and confirm high prediction accuracy by means of orthogonal, secondary validation assays. Our approach will increase the efficiency, scale and accuracy of phenotypic screens by maximizing their discriminatory power. PMID:26655497

  18. 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

  19. Convenient cell fusion assay for rapid screening for HIV entry inhibitors

    NASA Astrophysics Data System (ADS)

    Jiang, Shibo; Radigan, Lin; Zhang, Li

    2000-03-01

    Human immunodeficiency viruses (HIV)-induced cell fusion is a critical pathway of HIV spread from infected cells to uninfected cells. A rapid and simple assay was established to measure HIV-induce cell fusion. This study is particularly useful to rapid screen for HIV inhibitors that block HIV cell-to-cell transmission. Present study demonstrated that coculture of HIV-infected cells with uninfected cells at 37 degree(s)C for 2 hours resulted in the highest cell fusion rate. Using this cell fusion assay, we have identified several potent HIV inhibitors targeted to the HIV gp41 core. These antiviral agents can be potentially developed as antiviral drugs for chemotherapy and prophylaxis of HIV infection and AIDS.

  20. Multidimensional GPCR profiling and screening using impedance-based label-free and real-time assay.

    PubMed

    Ke, Ning; Nguyen, Khanh; Irelan, Jeffery; Abassi, Yama A

    2015-01-01

    GPCRs constitute one of the most sought-after targets in drug discovery because they are associated with conditions ranging from cardiovascular diseases, autoimmune diseases, inflammation, cancer, and diseases of the nervous system. Moreover, they are one of the most amenable targets for drug discovery because they can be modulated by small molecules, peptides, proteins, and antibodies. Therefore it may not come as a surprise that close to 40 % of the drugs that are currently on the market are targeting GPCRs. It has become evident that GPCR signaling is highly complex and may involve multiple or a subset of pathways depending on the interaction of a GPCR with an agonist or antagonist. It is imperative that any functional screening for GPCR activity integrates this complexity. In this assay protocol, we describe how the xCELLigence RTCA HT impedance-based platform which can be used for functional cell-based GPCR assays can be utilized for GPCR screening. PMID:25563187

  1. An LC-MS assay for the screening of cardiovascular medications in human samples.

    PubMed

    Dias, Eduardo; Hachey, Brian; McNaughton, Candace; Nian, Hui; Yu, Chang; Straka, Brittany; Brown, Nancy J; Caprioli, Richard M

    2013-10-15

    Cardiovascular drugs are the most commonly prescribed medications. Some prior assays successfully detect cardiovascular drugs among multiple classes using a single sample. Here, we develop an assay to detect a broad range of cardiovascular drug classes to include commonly used cardiovascular drugs and evaluate the assay's analytical and statistical properties in a clinical setting. We describe a protocol for drug detection that encompasses 34 commonly prescribed cardiovascular drugs or drug metabolites with a single LC-MS/MS method using 100μL of serum or plasma. Drug classes monitored by this assay include: anticoagulants, angiotensin converting enzyme inhibitors (ACEI), angiotensin II receptor blockers (ARB), beta blockers, calcium channel blockers, diuretics, statins, and vasodilators, as well as digoxin, fenofibrate, and niacin. Analytical accuracy and precision for each drug were evaluated by repeating the assay on spiked samples at low, medium, and high concentrations. In 294 clinical samples obtained from hospitalized patients for whom medication administration was recorded, we evaluated the assay's statistical sensitivity, specificity, and accuracy. For the 34 drugs or drug metabolites, the assay was statistically sensitive (>0.90) for all drugs except captopril (0.25), isosorbide (0.81), and niacin (0.89). The assay was statistically specific for all drugs, with a minimum specificity of 0.94 (aspirin). To our knowledge, this method is the first method of simultaneous analysis of 34 cardiovascular drugs or drug metabolites from nine drug classes evaluated using clinical samples from hospitalized patients. PMID:24013190

  2. Drug-symptom networking: Linking drug-likeness screening to drug discovery.

    PubMed

    Xu, Xue; Zhang, Chao; Li, PiDong; Zhang, FeiLong; Gao, Kuo; Chen, JianXin; Shang, HongCai

    2016-01-01

    Understanding the relationships between drugs and symptoms has broad medical consequences, yet a comprehensive description of the drug-symptom associations is currently lacking. Here, 1441 FDA-approved drugs were collected, and PCA was used to extract 122 descriptors which explained 91% of the variance. Then, a k-means++ method was employed to partition the drug dataset into 3 clusters, and 3 corresponding SVDD models (drug-likeness screening models) were constructed with an overall accuracy of up to 95.6%. Furthermore, 6878 herbal molecules from the TcmSP™ database were screened by the above 3 SVDD model to obtain 5309 candidate drug molecules with highly accept classification of 77.19%. To assess the accuracy of the SVDD models, 8559 herbal molecule-symptom co-occurrences were mined from Pubmed abstracts, involving 697 herbal molecules and 314 symptoms. Most of the 697 herbal molecules could be found in the accepted SVDD data (5309 molecules), showing the potential of the SVDD for the screening of drug candidates. Moreover, a herbal molecule-herbal molecule network and a herbal molecule-symptom were constructed. Overall, the results provided a new drug-likeness screening approach independent to abnormal training data, and the comprehensive collection of herbal molecule-symptom associations formed a new data resource for systematic characterization of the symptom-oriented medicines. PMID:26615785

  3. "Dilute-and-inject" multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing.

    PubMed

    Görgens, Christian; Guddat, Sven; Orlovius, Anne-Katrin; Sigmund, Gerd; Thomas, Andreas; Thevis, Mario; Schänzer, Wilhelm

    2015-07-01

    In the field of LC-MS, reversed phase liquid chromatography is the predominant method of choice for the separation of prohibited substances from various classes in sports drug testing. However, highly polar and charged compounds still represent a challenging task in liquid chromatography due to their difficult chromatographic behavior using reversed phase materials. A very promising approach for the separation of hydrophilic compounds is hydrophilic interaction liquid chromatography (HILIC). Despite its great potential and versatile advantages for the separation of highly polar compounds, HILIC is up to now not very common in doping analysis, although most manufacturers offer a variety of HILIC columns in their portfolio. In this study, a novel multi-target approach based on HILIC high resolution/high accuracy mass spectrometry is presented to screen for various polar stimulants, stimulant sulfo-conjugates, glycerol, AICAR, ethyl glucuronide, morphine-3-glucuronide, and myo-inositol trispyrophosphate after direct injection of diluted urine specimens. The usage of an effective online sample cleanup and a zwitterionic HILIC analytical column in combination with a new generation Hybrid Quadrupol-Orbitrap® mass spectrometer enabled the detection of highly polar analytes without any time-consuming hydrolysis or further purification steps, far below the required detection limits. The methodology was fully validated for qualitative and quantitative (AICAR, glycerol) purposes considering the parameters specificity; robustness (rRT < 2.0%); linearity (R > 0.99); intra- and inter-day precision at low, medium, and high concentration levels (CV < 20%); limit of detection (stimulants and stimulant sulfo-conjugates < 10 ng/mL; norfenefrine; octopamine < 30 ng/mL; AICAR < 10 ng/mL; glycerol 100 μg/mL; ETG < 100 ng/mL); accuracy (AICAR 103.8-105.5%, glycerol 85.1-98.3% at three concentration levels) and ion suppression/enhancement effects. PMID

  4. Drug Discovery for Duchenne Muscular Dystrophy via Utrophin Promoter Activation Screening

    PubMed Central

    Moorwood, Catherine; Lozynska, Olga; Suri, Neha; Napper, Andrew D.; Diamond, Scott L.; Khurana, Tejvir S.

    2011-01-01

    Background Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use. Methodology/Principal Findings We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells. Conclusions/Significance We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD. PMID:22028826

  5. Development and application of an automated solution stability assay for drug discovery.

    PubMed

    Di, Li; Kerns, Edward H; Chen, Hong; Petusky, Susan L

    2006-02-01

    Screening of solution stability provides an early alert on potential liabilities of drug candidates so that strategies can be developed to overcome the challenges. A fully automated solution stability assay has been developed to accelerate traditional manual operation. The assay uses the advanced capabilities of a high-performance liquid chromatography instrument that is present in many pharmaceutical research laboratories. The samples are prepared automatically by a temperature-controlled autosampler. The samples are delivered to the stability matrices, mixed, incubated, and injected at selected time points during the reaction time course. This automated process occurs without operator intervention, thus allowing 96 experiments to be run with 0.5 h of a scientist's time compared to 8 h for the same study when performed manually. Automation not only eliminates the manual operation but also improves accuracy and throughput. The assay protocol has been optimized to achieve homogenous mixing and eliminate carryover. The assay is robust, flexible, and high throughput. It can be used to study stability for a large number of samples under multiple incubation conditions and has a wide range of applications in drug discovery and development, such as screening compound stability in biological assay media, obtaining a stability-pH profile, surveying compound stability in physiological fluids, and performing development forced degradation and excipient compatibility. PMID:16234336

  6. Plateletworks: A screening assay for clopidogrel therapy monitoring in healthy cats

    PubMed Central

    Hamel-Jolette, Avril; Dunn, Marilyn; Bédard, Christian

    2009-01-01

    Plateletworks is a screening assay used in human medicine to monitor platelet-inhibiting drugs. As arterial thromboembolism is a common complication in cats suffering from cardiomyopathy, they are often treated with anti-platelet medication. Clopidogrel (Plavix), an anti-platelet aggregation drug, has recently been evaluated in healthy cats. The purpose of this study was to determine if the Plateletworks method can detect a decrease in platelet aggregation in cats receiving clopidogrel. Nine healthy adult cats were used for this study. Platelet aggregation was measured before and after a 3-day clopidogrel treatment (18.75 mg SID). Platelet aggregation after the clopidogrel treatment was significantly lower (P < 0.01). The Plateletworks method appears to be a promising test to monitor clopidogrel therapy in cats. PMID:19337399

  7. Evaluation of bioluminescence-based assays of anti-malarial drug activity

    PubMed Central

    2013-01-01

    Background Transgenic Plasmodium falciparum expressing luciferase offers an attractive bioluminescence-based assay platform for the investigation of the pharmacological properties of anti-malarial drugs. Here a side-by-side comparison of bioluminescence and fluorescence-based assays, utilizing a luciferase reporter cassette that confers a strong temporal pattern of luciferase expression during the S-phase of intraerythrocytic development, is reported. Methods Key assay parameters for a range of commercially available luminogenic substrates are determined and compared to those measured using a Malaria Sybr Green I fluorescence assay. In addition, the short-term temporal effects of anti-malarial compounds are evaluated using both bioluminescent and fluorescent assay platforms. Results The Z’, % coefficient of variation and 50% inhibition concentrations are essentially the same for bioluminescent and fluorescent assays in transgenic parasites generated in both chloroquine-sensitive and -resistant genetic backgrounds. Bioluminescent assays, irrespective of the luminogenic agent employed, do, however, offer significantly enhanced signal-to-noise ratios. Moreover, the bioluminescent assay is more dynamic in terms of determining temporal effects immediately following drug perturbation. Conclusion This study suggests that opportunities for bioluminescence-based assays lie not in the measurement of 50% inhibition concentrations, where the cheaper fluorescence assay performs excellently and is not restricted by the need to genetically modify the parasite clone under investigation. Instead, assays that use the dynamic response of the luciferase reporter for semi-automated screening of additional pharmacological properties, such as relative rate-of-kill and lethal dose estimation, are a more attractive development opportunity. PMID:23394077

  8. Pressure-driven perfusion culture microchamber array for a parallel drug cytotoxicity assay.

    PubMed

    Sugiura, Shinji; Edahiro, Jun-ichi; Kikuchi, Kyoko; Sumaru, Kimio; Kanamori, Toshiyuki

    2008-08-15

    This article reports a pressure-driven perfusion culture chip developed for parallel drug cytotoxicity assay. The device is composed of an 8 x 5 array of cell culture microchambers with independent perfusion microchannels. It is equipped with a simple interface for convenient access by a micropipette and connection to an external pressure source, which enables easy operation without special training. The unique microchamber structure was carefully designed with consideration of hydrodynamic parameters and was fabricated out of a polydimethylsiloxane by using multilayer photolithography and replica molding. The microchamber structure enables uniform cell loading and perfusion culture without cross-contamination between neighboring microchambers. A parallel cytotoxicity assay was successfully carried out in the 8 x 5 microchamber array to analyze the cytotoxic effects of seven anticancer drugs. The pressure-driven perfusion culture chip, with its simple interface and well-designed microfluidic network, will likely become an advantageous platform for future high-throughput drug screening by microchip. PMID:18553395

  9. Phenotypic Screens in Antimalarial Drug Discovery.

    PubMed

    Hovlid, Marisa L; Winzeler, Elizabeth A

    2016-09-01

    Phenotypic high-throughput screens are a valuable tool for identifying new chemical compounds with antimalarial activity. Traditionally, these screens have focused solely on the symptomatic asexual blood stage of the parasite life cycle; however, to discover new medicines for malaria treatment and prevention, robust screening technologies against other parasite life-cycle stages are required. This review highlights recent advances and progress toward phenotypic screening methodologies over the past several years, with a focus on exoerythrocytic stage screens. PMID:27247245

  10. 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

  11. Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Rezaei Darestani, Reza; Winter, Philip; Kitova, Elena N.; Tuszynski, Jack A.; Klassen, John S.

    2016-05-01

    Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

  12. Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Rezaei Darestani, Reza; Winter, Philip; Kitova, Elena N.; Tuszynski, Jack A.; Klassen, John S.

    2016-03-01

    Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

  13. Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry.

    PubMed

    Rezaei Darestani, Reza; Winter, Philip; Kitova, Elena N; Tuszynski, Jack A; Klassen, John S

    2016-05-01

    Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities. Graphical Abstract ᅟ. PMID:26944280

  14. The validation of an invitro colonic motility assay as a biomarker for gastrointestinal adverse drug reactions

    SciTech Connect

    Keating, Christopher; Martinez, Vicente; Ewart, Lorna; Gibbons, Stephen; Grundy, Luke; Valentin, Jean-Pierre; Grundy, David

    2010-06-15

    Motility-related gastrointestinal adverse drug reactions (GADRs), such as constipation and diarrhea, are some of the most frequently reported adverse events associated with the clinical development of new chemical entities, and for marketed drugs. However, biomarkers capable of detecting such GADRs are lacking. Here, we describe an in vitro assay developed to detect and quantify changes in intestinal motility as a surrogate biomarker for constipation/diarrhea-type GADRs. In vitro recordings of intraluminal pressure were used to monitor the presence of colonic peristaltic motor complexes (CPMCs) in mouse colonic segments. CPMC frequency, contractile and total mechanical activity were assessed. To validate the assay, two experimental protocols were conducted. Initially, five drugs with known gastrointestinal effects were tested to determine optimal parameters describing excitation and inhibition as markers for disturbances in colonic motility. This was followed by a 'blinded' evaluation of nine drugs associated with or without clinically identified constipation/diarrhea-type GADRs. Concentration-response relationships were determined for these drugs and the effects were compared with their maximal free therapeutic plasma concentration in humans. The assay detected stimulatory and inhibitory responses, likely correlating to the occurrence of diarrhea or constipation. Concentration-related effects were identified and potential mechanisms of action were inferred for several drugs. Based on the results from the fourteen drugs assessed, the sensitivity of the assay was calculated at 90%, with a specificity of 75% and predictive capacity of 86%. These results support the potential use of this assay in screening for motility-related GADRs during early discovery phase, safety pharmacology assessment.

  15. TranScreen-N: Method for rapid screening of trans-ungual drug delivery enhancers.

    PubMed

    Murthy, S Narasimha; Vaka, Siva Ram Kiran; Sammeta, Srinivasa Murthy; Nair, Anroop B

    2009-11-01

    Topical monotherapy of nail diseases such as onychomycosis and nail psoriasis has been less successful due to poor permeability of the human nail plate to topically administered drugs. Chemical enhancers are utilized to improve the drug delivery across the nail plate. Choosing the most effective chemical enhancers for the given drug and formulation is highly critical in determining the efficacy of topical therapy of nail diseases. Screening the large pool of enhancers using currently followed diffusion cell experiments would be tedious and expensive. The main objective of this study is to develop TranScreen-N, a high throughput method of screening trans-ungual drug permeation enhancers. It is a rapid microwell plate based method which involves two different treatment procedures; the simultaneous exposure treatment and the sequential exposure treatment. In the present study, several chemicals were evaluated by TranScreen-N and by diffusion studies in the Franz diffusion cell (FDC). Good agreement of in vitro drug delivery data with TranScreen-N data provided validity to the screening technique. In TranScreen-N technique, the enhancers can be grouped according to whether they need to be applied before or simultaneously with drugs (or by either procedures) to enhance the drug delivery across the nail plate. TranScreen-N technique can significantly reduce the cost and duration required to screen trans-ungual drug delivery enhancers. PMID:19363796

  16. Differentiation of drug and non-drug Cannabis using a single nucleotide polymorphism (SNP) assay.

    PubMed

    Rotherham, D; Harbison, S A

    2011-04-15

    Cannabis sativa is both an illegal drug and a legitimate crop. The differentiation of illegal drug Cannabis from non-drug forms of Cannabis is relevant in the context of the growth of fibre and seed oil varieties of Cannabis for commercial purposes. This differentiation is currently determined based on the levels of tetrahydrocannabinol (THC) in adult plants. DNA based methods have the potential to assay Cannabis material unsuitable for analysis using conventional means including seeds, pollen and severely degraded material. The purpose of this research was to develop a single nucleotide polymorphism (SNP) assay for the differentiation of "drug" and "non-drug"Cannabis plants. An assay was developed based on four polymorphisms within a 399 bp fragment of the tetrahydrocannabinolic acid (THCA) synthase gene, utilising the snapshot multiplex kit. This SNP assay was tested on 94 Cannabis plants, which included 10 blind samples, and was able to differentiate between "drug" and "non-drug"Cannabis in all cases, while also differentiating between Cannabis and other species. Non-drug plants were found to be homozygous at the four sites assayed while drug Cannabis plants were either homozygous or heterozygous. PMID:21036496

  17. Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy

    PubMed Central

    Cherry, Jonathan J.; Kobayashi, Dione T.; Lynes, Maureen M.; Naryshkin, Nikolai N.; Tiziano, Francesco Danilo; Zaworski, Phillip G.; Rubin, Lee L.

    2014-01-01

    Abstract Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder resulting in degeneration of α-motor neurons of the anterior horn and proximal muscle weakness. It is the leading cause of genetic mortality in children younger than 2 years. It affects ∼1 in 11,000 live births. In 95% of cases, SMA is caused by homozygous deletion of the SMN1 gene. In addition, all patients possess at least one copy of an almost identical gene called SMN2. A single point mutation in exon 7 of the SMN2 gene results in the production of low levels of full-length survival of motor neuron (SMN) protein at amounts insufficient to compensate for the loss of the SMN1 gene. Although no drug treatments are available for SMA, a number of drug discovery and development programs are ongoing, with several currently in clinical trials. This review describes the assays used to identify candidate drugs for SMA that modulate SMN2 gene expression by various means. Specifically, it discusses the use of high-throughput screening to identify candidate molecules from primary screens, as well as the technical aspects of a number of widely used secondary assays to assess SMN messenger ribonucleic acid (mRNA) and protein expression, localization, and function. Finally, it describes the process of iterative drug optimization utilized during preclinical SMA drug development to identify clinical candidates for testing in human clinical trials. PMID:25147906

  18. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  1. Phenotypic Screening Approaches to Develop Aurora Kinase Inhibitors: Drug Discovery Perspectives.

    PubMed

    Marugán, Carlos; Torres, Raquel; Lallena, María José

    2015-01-01

    Targeting mitotic regulators as a strategy to fight cancer implies the development of drugs against key proteins, such as Aurora-A and -B. Current drugs, which target mitosis through a general mechanism of action (stabilization/destabilization of microtubules), have several side effects (neutropenia, alopecia, and emesis). Pharmaceutical companies aim at avoiding these unwanted effects by generating improved and selective drugs that increase the quality of life of the patients. However, the development of these drugs is an ambitious task that involves testing thousands of compounds through biochemical and cell-based assays. In addition, molecules usually target complex biological processes, involving several proteins and different molecular pathways, further emphasizing the need for high-throughput screening techniques and multiplexing technologies in order to identify drugs with the desired phenotype. We will briefly describe two multiplexing technologies [high-content imaging (HCI) and flow cytometry] and two key processes for drug discovery research (assay development and validation) following our own published industry quality standards. We will further focus on HCI as a useful tool for phenotypic screening and will provide a concrete example of HCI assay to detect Aurora-A or -B selective inhibitors discriminating the off-target effects related to the inhibition of other cell cycle or non-cell cycle key regulators. Finally, we will describe other assays that can help to characterize the in vitro pharmacology of the inhibitors. PMID:26779442

  2. Phenotypic Screening Approaches to Develop Aurora Kinase Inhibitors: Drug Discovery Perspectives

    PubMed Central

    Marugán, Carlos; Torres, Raquel; Lallena, María José

    2016-01-01

    Targeting mitotic regulators as a strategy to fight cancer implies the development of drugs against key proteins, such as Aurora-A and -B. Current drugs, which target mitosis through a general mechanism of action (stabilization/destabilization of microtubules), have several side effects (neutropenia, alopecia, and emesis). Pharmaceutical companies aim at avoiding these unwanted effects by generating improved and selective drugs that increase the quality of life of the patients. However, the development of these drugs is an ambitious task that involves testing thousands of compounds through biochemical and cell-based assays. In addition, molecules usually target complex biological processes, involving several proteins and different molecular pathways, further emphasizing the need for high-throughput screening techniques and multiplexing technologies in order to identify drugs with the desired phenotype. We will briefly describe two multiplexing technologies [high-content imaging (HCI) and flow cytometry] and two key processes for drug discovery research (assay development and validation) following our own published industry quality standards. We will further focus on HCI as a useful tool for phenotypic screening and will provide a concrete example of HCI assay to detect Aurora-A or -B selective inhibitors discriminating the off-target effects related to the inhibition of other cell cycle or non-cell cycle key regulators. Finally, we will describe other assays that can help to characterize the in vitro pharmacology of the inhibitors. PMID:26779442

  3. The changing face of screening and drug discovery.

    PubMed

    Cooper, Matthew A

    2012-04-01

    Screening Asia 2011, Singapore, 22–23 November 2011. The meeting covered traditional topics such as high-content screening and assay development, as well as more contemporary, emergent areas involving novel screening platforms and technologies, strategies to deal with biosimilars and biologics, and natural product diversity. Notably, many talks challenged established screening practices and the use of 'combichem' small-molecule libraries. Instead, speakers offered an alternate view of compound library design and screening strategies that could better mimic the target and cell status found in the relevant disease state. PMID:22462783

  4. Automated fluorescent analysis for drug-induced cytotoxicity assays.

    PubMed

    Funa, K; Dawson, N; Jewett, P B; Agren, H; Ruckdeschel, J C; Bunn, P A; Gazdar, A F

    1986-10-01

    The human tumor clonogenic assay has been reported to predict for sensitivity of human tumors to a variety of drugs. However, this assay requires large numbers of viable cells, is time-consuming, and takes at least 2 weeks before results are available. To circumvent these problems, Weisenthal developed a microscope-based dye exclusion assay. Because this method is also time-consuming and subject to observer error, we have developed an automated method of quantitating drug cytotoxicity using a flow cytometric cell sorter (FCM). After incubation of drug-exposed tumor cells, acetaldehyde-fixed duck red blood cells (DRBC) are added. Dead tumor cells and the fixed DRBC are stained by the fluorescent dye propidium iodide, which penetrates dead cell membranes. A two-parameter analysis (cell size as measured by narrow angle light scatter vs propidium iodide fluorescence) enables determination of the live tumor cell:DRBC ratio. There was a strong correlation between the FCM method and manual counting (r = 0.958 for cell lines, r = 0.831 for fresh leukemic cells, P less than 0.0001 in both cases). We conclude that the automatized FCM method gives compatible results to the manual dye exclusion assay and increases efficiency. PMID:3019545

  5. Open innovation for phenotypic drug discovery: The PD2 assay panel.

    PubMed

    Lee, Jonathan A; Chu, Shaoyou; Willard, Francis S; Cox, Karen L; Sells Galvin, Rachelle J; Peery, Robert B; Oliver, Sarah E; Oler, Jennifer; Meredith, Tamika D; Heidler, Steven A; Gough, Wendy H; Husain, Saba; Palkowitz, Alan D; Moxham, Christopher M

    2011-07-01

    Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD(2)), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery. PMID:21521801

  6. 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

  7. Screening system for drug-induced arrhythmogenic risk combining a patch clamp and heart simulator

    PubMed Central

    Okada, Jun-ichi; Yoshinaga, Takashi; Kurokawa, Junko; Washio, Takumi; Furukawa, Tetsushi; Sawada, Kohei; Sugiura, Seiryo; Hisada, Toshiaki

    2015-01-01

    To save time and cost for drug discovery, a paradigm shift in cardiotoxicity testing is required. We introduce a novel screening system for drug-induced arrhythmogenic risk that combines in vitro pharmacological assays and a multiscale heart simulator. For 12 drugs reported to have varying cardiotoxicity risks, dose-inhibition curves were determined for six ion channels using automated patch clamp systems. By manipulating the channel models implemented in a heart simulator consisting of more than 20 million myocyte models, we simulated a standard electrocardiogram (ECG) under various doses of drugs. When the drug concentrations were increased from therapeutic levels, each drug induced a concentration-dependent characteristic type of ventricular arrhythmia, whereas no arrhythmias were observed at any dose with drugs known to be safe. We have shown that our system combining in vitro and in silico technologies can predict drug-induced arrhythmogenic risk reliably and efficiently. PMID:26601174

  8. Screening strategies to identify new chemical diversity for drug development to treat kinetoplastid infections.

    PubMed

    Don, Rob; Ioset, Jean-Robert

    2014-01-01

    The Drugs for Neglected Diseases initiative (DNDi) has defined and implemented an early discovery strategy over the last few years, in fitting with its virtual R&D business model. This strategy relies on a medium- to high-throughput phenotypic assay platform to expedite the screening of compound libraries accessed through its collaborations with partners from the pharmaceutical industry. We review the pragmatic approaches used to select compound libraries for screening against kinetoplastids, taking into account screening capacity. The advantages, limitations and current achievements in identifying new quality series for further development into preclinical candidates are critically discussed, together with attractive new approaches currently under investigation. PMID:23985066

  9. 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...

  10. USER S GUIDE FOR THE RANDOM DRUG SCREENING SYSTEM

    SciTech Connect

    McNeany, Karen I

    2013-12-01

    The Random Drug Screening System (RDSS) is a desktop computing application designed to assign nongameable drug testing dates to each member in a population of employees, within a specific time line. The program includes reporting capabilities, test form generation, unique test ID number assignment, and the ability to flag high-risk employees for a higher frequency of drug testing than the general population.

  11. A Different Approach to Validating Screening Assays for Developmental Toxicity

    EPA Science Inventory

    BACKGROUND: There continues to be many efforts around the world to develop assays that are shorter than the traditional embryofetal developmental toxicity assay, or use fewer or no mammals, or use less compound, or have all three attributes. Each assay developer needs to test th...

  12. Ca2+ mobilization assays in GPCR drug discovery.

    PubMed

    Woszczek, Grzegorz; Fuerst, Elisabeth

    2015-01-01

    Intracellular calcium mobilization can be measured using several methods varying in indicator dyes and devices used. In this chapter, we describe the fluorescence-based method (FLIPR Calcium 4 Assay) developed by Molecular Devices for a FlexStation and routinely used in our laboratory for detecting intracellular calcium changes. The assay is designed to study calcium mobilization induced by majority of GPCRs and calcium channels and allows for simultaneous concentration-dependent analysis of several receptor agonists and antagonists, useful in receptor characterization and drug discovery projects. PMID:25563178

  13. 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

  14. 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

  15. 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

  16. High-throughput screening of FDA-approved drugs using oxygen biosensor plates reveals secondary mitofunctional effects

    PubMed Central

    Sahdeo, Sunil; Tomilov, Alexey; Komachi, Kelly; Iwahashi, Christine; Datta, Sandipan; Hughes, Owen; Hagerman, Paul; Cortopassi, Gino

    2014-01-01

    Repurposing of FDA-approved drugs with effects on mitochondrial function might shorten the critical path to mitochondrial disease drug development. We improved a biosensor-based assay of mitochondrial O2 consumption, and identified mitofunctional defects in cell models of LHON and FXTAS. Using this platform, we screened a 1600-compound library of clinically used drugs. The assay identified drugs known to affect mitochondrial function, such as metformin and decoquinate. We also identified several drugs not previously known to affect mitochondrial respiration including acarbose, metaraminol, gallamine triethiodide, and acamprosate. These previously unknown ‘mitoactives’ represent novel links to targets for mitochondrial regulation and potentially therapy, for mitochondrial disease. PMID:25034306

  17. Establishment of a cell model for screening antibody drugs against rheumatoid arthritis with ADCC and CDC.

    PubMed

    Yan, Li; Hu, Rui; Tu, Song; Cheng, Wen-Jun; Zheng, Qiong; Wang, Jun-Wen; Kan, Wu-Sheng; Ren, Yi-Jun

    2015-01-01

    TNFα played a dominant role in the development and progression of rheumatoid arthritis (RA). Clinical trials proved the efficacies of anti-TNFα agents for curing RA. However, most researchers were concentrating on their abilities of neutralizing TNFα, the potencies of different anti-TNFα agents varied a lot due to the antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). For better understanding and differentiating the potentiality of various candidate anti-TNF reagents at the stage of new drug research and development, present study established a cell model expressing the transmembrane TNFα for usage in in vitro ADCC or CDC assay, meanwhile, the assay protocol described here could provide guidelines for screening macromolecular antibody drugs. A stable cell subline bearing transmembrane TNFα was first established by conventional transfection method, the expression of transmembrane TNFα was approved by flow cytometer, and the performance of the stable subline in ADCC and CDC assay was evaluated, using human peripheral blood mononuclear cells as effector cells, and Adalimumab as the anti-TNFα reagent. The stable cell subline demonstrated high level of surface expression of transmembrane TNFα, and Adalimumab exerted both ADCC and CDC effects on this cell model. In conclusion, the stable cell line we established in present research could be used in ADCC or CDC assay for screening antibody drugs, which would provide in-depth understanding of the potencies of candidate antibody drugs in addition to the traditional TNFα neutralizing assay. PMID:26884918

  18. 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

  19. Drug screening and confirmation by GC-MS: comparison of EMIT II and Online KIMS against 10 drugs between US and England laboratories.

    PubMed

    Lu, Natalie T; Taylor, Bruce G

    2006-03-10

    Drug screening through urinalysis is a widely accepted tool for rapid detection of potential drug use at a relatively low cost. It is, therefore, a potentially useful method for detecting and monitoring drug use in a variety of contexts such as the criminal justice system, pre-employment screening and a variety of treatment centers. This article explores the efficacy of two commercially available drug-screening assays: Online KIMS assay (Roche) and EMIT II assays. First, we evaluate the sensitivity and specificity of two immunoassays. A total of 738 urine samples were collected among adult arrestee populations from Chicago, New Orleans and Seattle through the Arrestee Drug Abuse Monitoring (ADAM) program. Partial samples were split within one laboratory and analyzed by both enzymes multiplied immunoassay technique (EMIT) II and kinetic interaction of microparticle in solution (KIMS) assays for a 10-drug panel (amphetamine, barbiturates, benzodiazepines, marijuana, cocaine, methadone, methaqualone, opiate, phencyclidine and propoxyphene). Gas chromatography-mass spectrometry (GC-MS) was used as a confirmation method for all positives from either EMIT II or KIMS for all experiments. Second, the paper examines whether using different testing laboratories plays a role in the final results. The same experiments were repeated at two different testing locations: one in California and one in London and England. Third, the paper studies whether drug testing results vary between two laboratories when each of them had used their own routine screening method: the Forensic Science Service (FSS) at Birmingham, United Kingdom with KIMS assay and Medscreen Limited at London, United Kingdom with EMIT II. In summary, both EMIT II and KIMS assays generate fairly consistent results. The concordance rate against each of the 10 drugs tested is relatively high (97.4-100%). The discrepancies, in most cases, occurred at drug concentrations near the cut-off levels. There were more

  20. Defining balanced conditions for inhibitor screening assays that target bisubstrate enzymes.

    PubMed

    Yang, Jingsong; Copeland, Robert A; Lai, Zhihong

    2009-02-01

    High-throughput screening (HTS) is a common mechanism for identifying lead compounds for drug discovery efforts. Small molecules can inhibit enzymes by a variety of mechanisms, such as competitive, noncompetitive, and uncompetitive with respect to the substrate(s) of the catalytic reaction. To optimize the chances of finding the broadest diversity of inhibitor modalities during screening, one must run assays under ;;balanced'' conditions where the potency of inhibitors with various modes of action falls within a similar range. When an enzyme reaction involves more than one substrate, the definition and assessment of the apparent potency of inhibitors (IC(50)), in relation to their true potency (K(i)), can be nontrivial. This article provides a theoretical analysis, on the basis of the Cheng-Prusoff derivation, of the IC(50)/K( i) relationship of bisubstrate enzyme reactions following various sequential kinetic mechanisms, as well as the application and limitations of this information for defining optimal screening conditions for such enzymes. PMID:19196704

  1. 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

  2. A high-content screening assay in transgenic zebrafish identifies two novel activators of fgf signaling.

    PubMed

    Saydmohammed, Manush; Vollmer, Laura L; Onuoha, Ezenwa Obi; Vogt, Andreas; Tsang, Michael

    2011-09-01

    Zebrafish have become an invaluable vertebrate animal model to interrogate small molecule libraries for modulators of complex biological pathways and phenotypes. We have recently described the implementation of a quantitative, high-content imaging assay in multi-well plates to analyze the effects of small molecules on Fibroblast Growth Factor (FGF) signaling in vivo. Here we have evaluated the capability of the assay to identify compounds that hyperactivate FGF signaling from a test cassette of agents with known biological activities. Using a transgenic zebrafish reporter line for FGF activity, we screened 1040 compounds from an annotated library of known bioactive agents, including FDA-approved drugs. The assay identified two molecules, 8-hydroxyquinoline sulfate and pyrithione zinc, that enhanced FGF signaling in specific areas of the brain. Subsequent studies revealed that both compounds specifically expanded FGF target gene expression. Furthermore, treatment of early stage embryos with either compound resulted in dorsalized phenotypes characteristic of hyperactivation of FGF signaling in early development. Documented activities for both agents included activation of extracellular signal-related kinase (ERK), consistent with FGF hyperactivation. To conclude, we demonstrate the power of automated quantitative high-content imaging to identify small molecule modulators of FGF. PMID:21932436

  3. Development, validation and quantitative assessment of an enzymatic assay suitable for small molecule screening and profiling: A case-study

    PubMed Central

    Sancenon, Vicente; Goh, Wei Hau; Sundaram, Aishwarya; Er, Kai Shih; Johal, Nidhi; Mukhina, Svetlana; Carr, Grant; Dhakshinamoorthy, Saravanakumar

    2015-01-01

    The successful discovery and subsequent development of small molecule inhibitors of drug targets relies on the establishment of robust, cost-effective, quantitative, and physiologically relevant in vitro assays that can support prolonged screening and optimization campaigns. The current study illustrates the process of developing and validating an enzymatic assay for the discovery of small molecule inhibitors using alkaline phosphatase from bovine intestine as model target. The assay development workflow includes an initial phase of optimization of assay materials, reagents, and conditions, continues with a process of miniaturization and automation, and concludes with validation by quantitative measurement of assay performance and signal variability. The assay is further evaluated for dose–response and mechanism-of-action studies required to support structure–activity-relationship studies. Emphasis is placed on the most critical aspects of assay optimization and other relevant considerations, including the technology, assay materials, buffer constituents, reaction conditions, liquid handling equipment, analytical instrumentation, and quantitative assessments. Examples of bottlenecks encountered during assay development and strategies to address them are provided. PMID:27077032

  4. 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

  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. Identifying GSK-3β kinase inhibitors of Alzheimer's disease: Virtual screening, enzyme, and cell assays.

    PubMed

    Lin, Chih-Hsin; Hsieh, Yu-Shao; Wu, Yih-Ru; Hsu, Chia-Jen; Chen, Hsuan-Chiang; Huang, Wun-Han; Chang, Kuo-Hsuan; Hsieh-Li, Hsiu Mei; Su, Ming-Tsan; Sun, Ying-Chieh; Lee, Guan-Chiun; Lee-Chen, Guey-Jen

    2016-06-30

    Glycogen synthase kinase 3β (GSK-3β) is widely known as a critical target protein for treating Alzheimer's disease (AD). We utilized virtual screening to search databases for compounds with the potential to be used in drugs targeting GSK-3β kinase, and kinase as well as cell assays to investigate top-scored, selected compounds. Virtual screening of >1.1 million compounds in the ZINC and in-house databases was conducted using an optimized computational protocol in the docking program GOLD. Of the top-ranked compounds, 16 underwent a luminescent kinase assay and a cell assay using HEK293 cells expressing DsRed-tagged ΔK280 in the repeat domain of tau (tauRD). The compounds VB-003 (a potent GSK-3β inhibitor) and VB-008 (AM404, an anandamide transport inhibitor), with determined IC50 values of 0.25 and 5.4μM, respectively, were identified as reducing tau aggregation. Both compounds increased expression of phospho-GSK-3β (Ser9) and reduced endogenous tau phosphorylation at the sites of Ser202, Thr231, and Ser396. In the ∆K280 tauRD-DsRed SH-SY5Y cells, VB-008, but not VB-003, enhanced HSPB1 and GRP78 expression, increased ∆K280 tauRD-DsRed solubility, and promoted neurite outgrowth. Thus VB-008 performed best to the end of the present study. The identified compound VB-008 may guide the identification and synthesis of potential inhibitors analogous to this compound. PMID:27094783

  7. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  8. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  9. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  10. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  11. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  12. The QDREC web server: determining dose–response characteristics of complex macroparasites in phenotypic drug screens

    PubMed Central

    Asarnow, Daniel; Rojo-Arreola, Liliana; Suzuki, Brian M.; Caffrey, Conor R.; Singh, Rahul

    2015-01-01

    Summary: Neglected tropical diseases (NTDs) caused by helminths constitute some of the most common infections of the world’s poorest people. The etiological agents are complex and recalcitrant to standard techniques of molecular biology. Drug screening against helminths has often been phenotypic and typically involves manual description of drug effect and efficacy. A key challenge is to develop automated, quantitative approaches to drug screening against helminth diseases. The quantal dose–response calculator (QDREC) constitutes a significant step in this direction. It can be used to automatically determine quantitative dose–response characteristics and half-maximal effective concentration (EC50) values using image-based readouts from phenotypic screens, thereby allowing rigorous comparisons of the efficacies of drug compounds. QDREC has been developed and validated in the context of drug screening for schistosomiasis, one of the most important NTDs. However, it is equally applicable to general phenotypic screening involving helminths and other complex parasites. Availability and implementation: QDREC is publically available at: http://haddock4.sfsu.edu/qdrec2/. Source code and datasets are at: http://tintin.sfsu.edu/projects/phenotypicAssays.html. Contact: rahul@sfsu.edu. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25540182

  13. Comparison of three quantification methods for the TZM-bl pseudovirus assay for screening of anti-HIV-1 agents.

    PubMed

    Xing, Liying; Wang, Shunyi; Hu, Qin; Li, Jingtao; Zeng, Yi

    2016-07-01

    The TZM-bl pseudovirus assay is commonly used to evaluate the efficacy of neutralizing antibodies and small molecular inhibitors in HIV-1 research. Here, to determine the optimal measurement method for screening anti-HIV-1 inhibitors, we compared three measurement methods based on firefly luciferase and β-galactosidase activities. The 50% tissue culture infective doses (TCID50) of the pseudoviruses were determined using the luciferase, β-galactosidase colorimetric, and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining assays. Three commercial reverse-transcriptase inhibitors (azidothymidine, nevirapine, and lamivudine) were tested as reference drugs to compare the reproducibility, linear correlation, and half maximal inhibitory concentration (IC50) values determined using these methods. In the TCID50 assay, the sensitivity of β-galactosidase colorimetric assay was almost 562 times lower than that of the other two methods. Reproducible dose-response curves were obtained for the inhibitors with all methods; the IC50 values of the inhibitors were not significantly different. Linear regression analysis showed linear correlation between methods. Compared to the β-galactosidase colorimetric assay, the other two methods have the advantage of high sensitivity and are less affected by interference. In conclusion, the luciferase and X-gal staining assays, which can be applied either alone or combined, are recommended for anti-HIV-1 inhibitor screening. PMID:27016178

  14. 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

  15. A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

    PubMed Central

    Matsunaga, Satoko; Masaoka, Takashi; Sawasaki, Tatsuya; Morishita, Ryo; Iwatani, Yasumasa; Tatsumi, Masashi; Endo, Yaeta; Yamamoto, Naoki; Sugiura, Wataru; Ryo, Akihide

    2015-01-01

    Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1. PMID:26583013

  16. A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani.

    PubMed

    Tegazzini, Diana; Díaz, Rosario; Aguilar, Fernando; Peña, Imanol; Presa, Jesús L; Yardley, Vanessa; Martin, Julio J; Coteron, Jose M; Croft, Simon L; Cantizani, Juan

    2016-06-01

    The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay. PMID:27021313

  17. 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

  18. Application of ORAL.screen saliva drug test for the screening of methamphetamine, MDMA, and MDEA incorporated in hair.

    PubMed

    Miki, Akihiro; Katagi, Munehiro; Shima, Noriaki; Tsuchihashi, Hitoshi

    2004-03-01

    By the use of a one-step immunoassay drug test for oral fluid, a convenient and fairly sensitive screening method has been devised for methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) incorporated in hair. These drugs, in a 10-mg portion of hair, were extracted into 5M HCl/methanol (1:20, v/v), and the extract reconstituted in 100 micro L water was assayed with the saliva drug test ORAL.screen trade mark. The limits of detection were 0.5 ng/mg hair for d-MA, 0.8 ng/mg for dl-MDMA, and 1.0 ng/mg for dl-MDEA. The results are in good agreement with those of gas chromatography-mass spectrometry (GC-MS) determination. Although all positive results must be confirmed by either GC-MS or a specific alternative methodology, this method provided a simple screening, suitable for drug enforcement purposes, while requiring only a 10-mg hair specimen. PMID:15068568

  19. Thermodynamic Studies for Drug Design and Screening

    PubMed Central

    Garbett, Nichola C.; Chaires, Jonathan B.

    2012-01-01

    Introduction A key part of drug design and development is the optimization of molecular interactions between an engineered drug candidate and its binding target. Thermodynamic characterization provides information about the balance of energetic forces driving binding interactions and is essential for understanding and optimizing molecular interactions. Areas covered This review discusses the information that can be obtained from thermodynamic measurements and how this can be applied to the drug development process. Current approaches for the measurement and optimization of thermodynamic parameters are presented, specifically higher throughput and calorimetric methods. Relevant literature for this review was identified in part by bibliographic searches for the period 2004 – 2011 using the Science Citation Index and PUBMED and the keywords listed below. Expert opinion The most effective drug design and development platform comes from an integrated process utilizing all available information from structural, thermodynamic and biological studies. Continuing evolution in our understanding of the energetic basis of molecular interactions and advances in thermodynamic methods for widespread application are essential to realize the goal of thermodynamically-driven drug design. Comprehensive thermodynamic evaluation is vital early in the drug development process to speed drug development towards an optimal energetic interaction profile while retaining good pharmacological properties. Practical thermodynamic approaches, such as enthalpic optimization, thermodynamic optimization plots and the enthalpic efficiency index, have now matured to provide proven utility in design process. Improved throughput in calorimetric methods remains essential for even greater integration of thermodynamics into drug design. PMID:22458502

  20. Microfluidics-assisted in vitro drug screening and carrier production

    PubMed Central

    Tsui, Jonathan H.; Lee, Woohyuk; Pun, Suzie H.; Kim, Jungkyu; Kim, Deok-Ho

    2013-01-01

    Microfluidic platforms provide several unique advantages for drug development. In the production of drug carriers, physical properties such as size and shape, and chemical properties such as drug composition and pharmacokinetic parameters, can be modified simply and effectively by tuning the flow rate and geometries. Large numbers of carriers can then be fabricated with minimal effort and with little to no batch-to-batch variation. Additionally, cell or tissue culture models in microfluidic systems can be used as in vitro drug screening tools. Compared to in vivo animal models, microfluidic drug screening platforms allow for high-throughput and reproducible screening at a significantly lower cost, and when combined with current advances in tissue engineering, are also capable of mimicking native tissues. In this review, various microfluidic platforms for drug and gene carrier fabrication are reviewed to provide guidelines for designing appropriate carriers. In vitro microfluidic drug screening platforms designed for high-throughput analysis and replication of in vivo conditions are also reviewed to highlight future directions for drug research and development. PMID:23856409

  1. Label-free imaging and temporal signature in phenotypic cellular assays: a new approach to high-content screening.

    PubMed

    Martin, Julio

    2010-09-01

    Some drug targets are not amenable to screening because of the lack of a practical or validated biological assay. Likewise, some screening assays may not be predictive of compound activity in a more disease-relevant scenario, or assay development may demand excessive allocation of resources (i.e., time, money or personnel) with limited knowledge of the actual tractability of the target. Label-free methodologies, implemented in microtiter plate format, may help address these issues and complement, simplify, or facilitate assays. Label-free biosensors, based on grating resonance or electrical impedance, are versatile platforms for detecting phenotypic changes in both engineered and native cells. Their non-invasive nature allows for the kinetic monitoring of multiple real-time cellular responses to external stimuli, as well as for the use of successive pharmacological challenges. The temporal signature recorded for a particular stimulus is characteristic of the cell type and the signaling pathway activated upon binding of a ligand to its receptor. Cellular label-free technology is an important technical advance in the study of functional pharmacological selectivity. Described in this overview are some of the hurdles encountered in modern drug discovery and the ways in which label-free technologies can be used to overcome these obstacles. PMID:22294376

  2. 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

  3. Pathophysiologically relevant in vitro tumor models for drug screening.

    PubMed

    Das, Viswanath; Bruzzese, Francesca; Konečný, Petr; Iannelli, Federica; Budillon, Alfredo; Hajdúch, Marián

    2015-07-01

    The alarming rate of failure of clinical trials is a major hurdle in cancer therapy that partly results from the inadequate use of in vitro tumor models for the screening of promising hits and leads in preclinical studies. 2D cultures of cancer cell lines that are primarily used for drug screening do not adequately recapitulate tumor microenvironment (TME) complexities compared with 3D cancer cell cultures and tumor-derived primary cell cultures. In this review, we focus on the potential use of in vitro tumor models that reproduce in vivo tumor complexities for effective drug selection in the preclinical stages of drug development. PMID:25908576

  4. A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

    PubMed

    Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

    2015-02-01

    Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. PMID:25296660

  5. Evaporative edge lithography of a liposomal drug microarray for cell migration assays

    PubMed Central

    Vafai, Nicholas; Lowry, Troy W.; Wilson, Korey A.; Davidson, Michael W.; Lenhert, Steven

    2016-01-01

    Lipid multilayer microarrays are a promising approach to miniaturize laboratory procedures by taking advantage of the microscopic compartmentalization capabilities of lipids. Here, we demonstrate a new method to pattern lipid multilayers on surfaces based on solvent evaporation along the edge where a stencil contacts a surface called evaporative edge lithography (EEL). As an example of an application of this process, we use EEL to make microarrays suitable for a cell-based migration assay. Currently existing cell migration assays require a separate compartment for each drug which is dissolved at a single concentration in solution. An advantage of the lipid multilayer microarray assay is that multiple compounds can be tested on the same surface. We demonstrate this by testing the effect of two different lipophilic drugs, Taxol and Brefeldin A, on collective cell migration into an unpopulated area. This particular assay should be scalable to test of 2000 different lipophilic compounds or dosages on a standard microtiter plate area, or if adapted for individual cell migration, it would allow for high-throughput screening of more than 50,000 compounds per plate.

  6. Progesterone receptor chaperone complex-based highthroughput screening assay: identification of capsaicin as inhibitor of Hsp90 machine

    PubMed Central

    Patwardhan, Chaitanya A.; Alfa, Eyad; Lu, Su; Chadli, Ahmed

    2016-01-01

    Hsp90 and its co-chaperones are known to be important for cancer cell survival. The N-terminal inhibitors of Hsp90 that are in ongoing clinical trials as anti-tumor agents have unfortunately shown disappointing efficacies in the clinic. Thus, novel inhibitors of the Hsp90 machine with different mechanism of action are urgently needed. We report here the development of a novel high-throughput drug-screening (HTS) assay platform to identify small molecule inhibitors of Hsp90 and its co-chaperones. This assay quantitatively measures the ability of Hsp90 and its co-chaperones to refold/protect the progesterone receptor (PR), a physiological client of Hsp90, in 96-well plate format. We screened the NIH clinical collection drug library and identified capsaicin as a hit molecule. Capsaicin is an FDA-approved drug for topical use in pain management. Cell survival assays showed that capsaicin selectively kills cancer cells and destabilizes several Hsp90 client proteins. Thus, our data may explain the seemingly pleotropic effect of capsaicin. PMID:25184514

  7. Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs

    PubMed Central

    Kouznetsova, Jennifer; Sun, Wei; Martínez-Romero, Carles; Tawa, Gregory; Shinn, Paul; Chen, Catherine Z; Schimmer, Aaron; Sanderson, Philip; McKew, John C; Zheng, Wei; García-Sastre, Adolfo

    2014-01-01

    In light of the current outbreak of Ebola virus disease, there is an urgent need to develop effective therapeutics to treat Ebola infection, and drug repurposing screening is a potentially rapid approach for identifying such therapeutics. We developed a biosafety level 2 (BSL-2) 1536-well plate assay to screen for entry inhibitors of Ebola virus-like particles (VLPs) containing the glycoprotein (GP) and the matrix VP40 protein fused to a beta-lactamase reporter protein and applied this assay for a rapid drug repurposing screen of Food and Drug Administration (FDA)-approved drugs. We report here the identification of 53 drugs with activity of blocking Ebola VLP entry into cells. These 53 active compounds can be divided into categories including microtubule inhibitors, estrogen receptor modulators, antihistamines, antipsychotics, pump/channel antagonists, and anticancer/antibiotics. Several of these compounds, including microtubule inhibitors and estrogen receptor modulators, had previously been reported to be active in BSL-4 infectious Ebola virus replication assays and in animal model studies. Our assay represents a robust, effective and rapid high-throughput screen for the identification of lead compounds in drug development for the treatment of Ebola virus infection. PMID:26038505

  8. Screening and Brief Intervention for Drug Use in Primary Care

    PubMed Central

    Saitz, Richard; Palfai, Tibor P. A.; Cheng, Debbie M.; Alford, Daniel P.; Bernstein, Judith A.; Lloyd-Travaglini, Christine A.; Meli, Seville M.; Chaisson, Christine E.; Samet, Jeffrey H.

    2015-01-01

    IMPORTANCE The United States has invested substantially in screening and brief intervention for illicit drug use and prescription drug misuse, based in part on evidence of efficacy for unhealthy alcohol use. However, it is not a recommended universal preventive service in primary care because of lack of evidence of efficacy. OBJECTIVE To test the efficacy of 2 brief counseling interventions for unhealthy drug use (any illicit drug use or prescription drug misuse)—a brief negotiated interview (BNI) and an adaptation of motivational interviewing (MOTIV)—compared with no brief intervention. DESIGN, SETTING, AND PARTICIPANTS This 3-group randomized trial took place at an urban hospital-based primary care internal medicine practice; 528 adult primary care patients with drug use (Alcohol, Smoking, and Substance Involvement Screening Test [ASSIST] substance-specific scores of $4) were identified by screening between June 2009 and January 2012 in Boston, Massachusetts. INTERVENTIONS Two interventions were tested: the BNI is a 10- to 15-minute structured interview conducted by health educators; the MOTIV is a 30- to 45-minute intervention based on motivational interviewing with a 20- to 30-minute booster conducted by master’s-level counselors. All study participants received a written list of substance use disorder treatment and mutual help resources. MAIN OUTCOMES AND MEASURES Primary outcome was number of days of use in the past 30 days of the self-identified main drug as determined by a validated calendar method at 6 months. Secondary outcomes included other self-reported measures of drug use, drug use according to hair testing, ASSIST scores (severity), drug use consequences, unsafe sex, mutual help meeting attendance, and health care utilization. RESULTS At baseline, 63% of participants reported their main drug was marijuana, 19% cocaine, and 17% opioids. At 6 months, 98% completed follow-up. Mean adjusted number of days using the main drug at 6 months was 12 for

  9. COMPARISON OF AN IN VIVO FISH VTG ASSAY WITH YES AND E-SCREEN

    EPA Science Inventory

    This study compares the efficacy of two in vitro, estrogen-sensitive bioassays to rank the "relative estrogenicity" of five natural, pharmaceutical and xenoestrogens with a newly developed in vivo bioassay. The E-SCREEN (MCF-7 tumor cells) and YES (Yeast Estrogen Screen) assays w...

  10. CELL-FREE NEUROCHEMICAL SCREENING ASSAYS TO PREDICT ADVERSE EFFECTS IN MAMMALS, FISH, AND BIRDS

    EPA Science Inventory

    This work will result in the establishment of a high-throughput screening assay that can be used to predict reproductive impairment across multiple ecologically relevant species (birds, fish, mammals). Resources exist to adapt this platform to screen 1,000s of toxicants. It...

  11. 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...

  12. 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

  13. Potent Human Telomerase Inhibitors: Molecular Dynamic Simulations, Multiple Pharmacophore-Based Virtual Screening, and Biochemical Assays.

    PubMed

    Shirgahi Talari, Faezeh; Bagherzadeh, Kowsar; Golestanian, Sahand; Jarstfer, Michael; Amanlou, Massoud

    2015-12-28

    Telomere maintenance is a universal cancer hallmark, and small molecules that disrupt telomere maintenance generally have anticancer properties. Since the vast majority of cancer cells utilize telomerase activity for telomere maintenance, the enzyme has been considered as an anticancer drug target. Recently, rational design of telomerase inhibitors was made possible by the determination of high resolution structures of the catalytic telomerase subunit from a beetle and subsequent molecular modeling of the human telomerase complex. A hybrid strategy including docking, pharmacophore-based virtual screening, and molecular dynamics simulations (MDS) were used to identify new human telomerase inhibitors. Docking methodology was applied to investigate the ssDNA telomeric sequence and two well-known human telomerase inhibitors' (BIBR1532 and MST-312) modes of interactions with hTERT TEN domain. Subsequently molecular dynamic simulations were performed to monitor and compare hTERT TEN domain, TEN-ssDNA, TEN-BIBR1532, TEN-MST-312, and TEN-ssDNA-BIBR1532 behavior in a dynamic environment. Pharmacophore models were generated considering the inhibitors manner in the TEN domain anchor site. These exploratory studies identified several new potent inhibitors whose IC50 values were generated experimentally in a low micromolar range with the aid of biochemical assays, including both the direct telomerase and the telomeric repeat amplification protocol (TRAP) assays. The results suggest that the current models of human telomerase are useful templates for rational inhibitor design. PMID:26529120

  14. 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

  15. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue.

    PubMed

    Mikus, J; Steverding, D

    2000-01-01

    A quantitative colorimetric assay using the oxidation-reduction indicator Alamar Blue was developed to measure cytotoxicity of compounds against the protozoan parasite Leishmania. Absorbance increased linearly with the plating density of promastigotes of L. major MRHO/IR/76 vaccine strain up to at least 2.5 x 10(6) cells/ml when parasites were incubated for 72 h in the presence of 10% Alamar Blue. The 50% effective dose values of common drugs (amphotericin B, pentostam and paromomycin) obtained by this assay were in the same range as previously determined by other methods. The Alamar Blue assay permits a simple, reproducible and reliable method for screening antileishmanial drugs. PMID:11227767

  16. Highly sensitive and selective fluorescence assays for rapid screening of endothelin-converting enzyme inhibitors.

    PubMed Central

    Luciani, N; de Rocquigny, H; Turcaud, S; Romieu, A; Roques, B P

    2001-01-01

    The highly potent vasoconstrictor peptide endothelin (ET) is generated from an inactive precursor, big endothelin (bET), by endothelin-converting enzyme (ECE). ECE is a phosphoramidon-sensitive zinc metallopeptidase, which is closely related to neprilysin (neutral endopeptidase). It is possible that compounds which inhibit the formation of ET may be used as new drugs for the treatment of cardiovascular diseases. Such an approach requires a fast, simple and selective assay to measure ECE activity, allowing rapid screening of inhibitors. We describe here two new ECE substrates based on the concept of 'intramolecularly quenched fluorescence' which may fulfill this aim. One, S(1) [Pya(21)-Nop(22)-bET-1(19--35)], is the (19--35) fragment of the natural peptide big-ET-1(1--38), which is modified by introducing the fluorescent amino acid, pyrenylalanine (Pya), in position 21 and a quencher, p-nitrophenylalanine (Nop), in position 22. The second substrate (S(2)) is a small peptide, Ac-Ser-Gly-Pya-Lys-Ala-Phe-Ala-Nop-Gly-Lys-NH(2), from a biased substrate peptide library. The recombinant, hECE-1c, cleaved both Pya(21)-Nop(22)-bET-1(19--35) and the natural substrate selectively between residues 21 and 22, whereas cleavage occurred between alanine and phenylalanine in the small peptide. In both cases, this generated intense fluorescence emission. The synthesis and kinetic parameters of these substrates are described. These assays, which can be used directly on tissue homogenates, are the most sensitive and selective described to date for ECE, and are easily automated for a high-throughput screening of inhibitors. PMID:11389689

  17. Fluorimetric screening assay for protein carbonyl evaluation in biological samples.

    PubMed

    Stocker, P; Ricquebourg, E; Vidal, N; Villard, C; Lafitte, D; Sellami, L; Pietri, S

    2015-08-01

    Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments. PMID:25933703

  18. Drug screening for hearing loss: using the zebrafish lateral line to screen for drugs that prevent and cause hearing loss

    PubMed Central

    Santos, Felipe; Raible, David W.; Simon, Julian A.; Rubel, Edwin W.

    2010-01-01

    Several animal models have been used for the study of mechanosensory hair cells and hearing loss. Because of the difficulty of tissue acquisition and large animal size, these traditional models are impractical for high-throughput screening. The zebrafish has emerged as a powerful animal model for screening drugs that cause or prevent hair cell death. The unique characteristics of the zebrafish enable rapid in vivo imaging of hair cells and hair cell death. We have used this model to screen for and identify multiple drugs that protect hair cells from aminoglycoside-induced death. Identification of multiple drugs and drug-like compounds that inhibit multiple hair cell death pathways might enable the development of protective cocktails to achieve complete hair cell protection. PMID:20096805

  19. Cellular Biomechanics in Drug Screening and Evaluation: Mechanopharmacology

    PubMed Central

    Krishnan, Ramaswamy; Park, Jin-Ah; Seow, Chun Y.; Lee, Peter V-S.; Stewart, Alastair G.

    2016-01-01

    The study of mechanobiology is now widespread. The impact of cell and tissue mechanics on cellular responses is well appreciated. However, knowledge of the impact of cell and tissue mechanics on pharmacological responsiveness, and its application to drug screening and mechanistic investigations, have been very limited in scope. We emphasize the need for a heightened awareness of the important bidirectional influence of drugs and biomechanics in all living systems. We propose that the term ‘mechanopharmacology’ be applied to approaches that employ in vitro systems, biomechanically appropriate to the relevant (patho)physiology, to identify new drugs and drug targets. This article describes the models and techniques that are being developed to transform drug screening and evaluation, ranging from a 2D environment to the dynamic 3D environment of the target expressed in the disease of interest. PMID:26651416

  20. Virtual screening and its integration with modern drug design technologies.

    PubMed

    Guido, Rafael V C; Oliva, Glaucius; Andricopulo, Adriano D

    2008-01-01

    Drug discovery is a highly complex and costly process, which demands integrated efforts in several relevant aspects involving innovation, knowledge, information, technologies, expertise, R&D investments and management skills. The shift from traditional to genomics- and proteomics-based drug research has fundamentally transformed key R&D strategies in the pharmaceutical industry addressed to the design of new chemical entities as drug candidates against a variety of biological targets. Therefore, drug discovery has moved toward more rational strategies based on our increasing understanding of the fundamental principles of protein-ligand interactions. The combination of available knowledge of several 3D protein structures with hundreds of thousands of small-molecules have attracted the attention of scientists from all over the world for the application of structure- and ligand-based drug design approaches. In this context, virtual screening technologies have largely enhanced the impact of computational methods applied to chemistry and biology and the goal of applying such methods is to reduce large compound databases and to select a limited number of promising candidates for drug design. This review provides a perspective of the utility of virtual screening in drug design and its integration with other important drug discovery technologies such as high-throughput screening (HTS) and QSAR, highlighting the present challenges, limitations, and future perspectives in medicinal chemistry. PMID:18220761

  1. 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

  2. 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.

  3. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    NASA Astrophysics Data System (ADS)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  4. 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.

  5. 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

  6. 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

  7. A chromogenic cephalosporin for β-lactamase inhibitor screening assays.

    PubMed

    Yu, Sophia; Vosbeek, Amy; Corbella, Katherine; Severson, Jonathan; Schesser, Jacob; Sutton, Larry D

    2012-09-15

    Production of β-lactamases is the primary mechanism of antibiotic resistance employed by gram-negative pathogens. Chromogenic β-lactams are important reagents for detection and assay of β-lactamases, but limited commercial availability and exorbitant pricing of these compounds are prohibitive. Here we describe a straightforward synthesis of a chromogenic cephalosporin for β-lactamase assay that gives an overall yield of 74%. On hydrolysis, its λ(max) undergoes a bathochromic shift that is easy to see and measure spectrophotometrically with a Δε(442 nm) of 14,500 cm⁻¹ M⁻¹. This compound was shown to be a substrate for a variety of β-lactamases. PMID:22709853

  8. In situ hybridization assay-based small molecule screening in zebrafish

    PubMed Central

    Jing, Lili; Durand, Ellen M.; Ezzio, Catherine; Pagliuca, Stephanie M.; Zon, Leonard I.

    2012-01-01

    In vitro biochemical and cell-based small molecule screens have been widely used to identify compounds that target specific signaling pathways. But the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism and toxicity of chemicals. Zebrafish has recently emerged as a vertebrate whole organism model for small molecule screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for chemical screens at a large scale similar to cellular assays. This protocol describes an approach for in situ hybridization (ISH)-based chemical screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts. PMID:23001521

  9. Assessment of the Microscreen phage-induction assay for screening hazardous wastes

    SciTech Connect

    Houk, V.S.; DeMarini, D.M.

    1987-09-01

    The Microscreen phage-induction assay, which quantitatively measures the induction of prophage lambda in Escherichia coli WP2s(lambda), was used to test 14 crude (unfractionated) hazardous industrial waste samples for genotoxic activity in the presence and absence of metabolic activation. Eleven of the 14 wastes induced prophage, and induction was observed at concentrations as low as 0.4 picograms per ml. Comparisons between the mutagenicity of these waste samples in Salmonella and their ability to induce prophage lambda indicate that the Microscreen phage-induction assay detected genotoxic activity in all but one of the wastes that were mutagenic in Salmonella. Moreover, the Microscreen assay detected as genotoxic 5 additional wastes that were not detected in the Salmonella assay. The applicability of the Microscreen phage-induction assay for screening hazardous wastes for genotoxic activity is discussed along with some of the problems associated with screening highly toxic wastes containing toxic volatile compounds.

  10. Syphilis detection: evaluation of serological screening and pilot reverse confirmatory assay algorithm in blood donors.

    PubMed

    Sommese, Linda; Paolillo, Rossella; Sabia, Chiara; Costa, Dario; De Pascale, Maria Rosaria; Iannone, Carmela; Esposito, Antonella; Schiano, Concetta; Napoli, Claudio

    2016-07-01

    Serological assays are still considered the most useful tests in the diagnosis of syphilis. Since no single serological assay is able to provide a satisfactory result, in our laboratory we have evaluated the usefulness of a commercially-available immunoblot to diagnose syphilis infection among blood donors. From October 2012 to June 2013, 4572 blood donors were screened for syphilis with an automated chemiluminescent microparticle immunoassay (CMIA). To confirm the presence of treponemal antibodies, CMIA-reactive sera were tested by standard Treponema pallidum haemagglutination assay (TPHA). In addition, an alternative confirmatory test - the immunoblot INNO-LIA assay was introduced in our laboratory. Since two additional positives among CMIA-reactive-TPHA-negative samples were found, we concluded that the INNO-LIA immunoblot allowed a better detection of syphilis compared to TPHA. A confirmatory strategy based on the use of two treponemal assays could meet the screening requirements for blood donors as well as in our centre. PMID:26068964

  11. Comparative drug screening in NUT midline carcinoma

    PubMed Central

    Beesley, A H; Stirnweiss, A; Ferrari, E; Endersby, R; Howlett, M; Failes, T W; Arndt, G M; Charles, A K; Cole, C H; Kees, U R

    2014-01-01

    Background: The NUT midline carcinoma (NMC) is a rare but fatal cancer for which systematic testing of therapy options has never been performed. Methods: On the basis of disease biology, we compared the efficacy of the CDK9 inhibitor flavopiridol (FP) with a panel of anticancer agents in NMC cell lines and mouse xenografts. Results: In vitro anthracyclines, topoisomerase inhibitors, and microtubule poisons were among the most cytotoxic drug classes for NMC cells, while efficacy of the bromodomain inhibitor JQ1 varied considerably between lines carrying different BRD4 (bromodomain-containing protein 4)–NUT (nuclear protein in testis) translocations. Efficacy of FP was comparable to vincristine and doxorubicin, drugs that have been previously used in NMC patients. All three compounds showed significantly better activity than etoposide and vorinostat, agents that have also been used in NMC patients. Statins and antimetabolites demonstrated intermediate single-agent efficacy. In vivo, vincristine significantly inhibited tumour growth in two different NMC xenografts. Flavopiridol in vivo was significantly effective in one of the two NMC xenograft lines, demonstrating the biological heterogeneity of this disease. Conclusions: These results demonstrate that FP may be of benefit to a subset of patients with NMC, and warrant a continued emphasis on microtubule inhibitors, anthracyclines, and topoisomerase inhibitors as effective drug classes in this disease. PMID:24518598

  12. Multiple animal studies for medical chemical defense program in soldier/patient decontamination and drug development on task 88-36: Development of in vitro screening assays for candidate pretreatment and treatment compounds. Final report, 1 July 1988-1 July 1989

    SciTech Connect

    Joiner, R.; Dill, G.; Hobson, D.; Blank, J.

    1990-03-01

    A task was instituted at the Medical Research and Evaluation Facility (MREF) to develop in vitro assays to screen pretreatment and treatment compounds for their ability to protect or reverse the toxic effects of organophosphates and vesicants. Four vesicant assays and three nerve agent assays were developed. Two of the vesicant assays were for cell viability of keratinocyte, one in the presence of distilled mustard and one lewisite. One assay determines the effect of vesicants on keratinocyte reproduction and the other the effect of distilled mustard on cellular coenzyme nicotinamide adenine dinucleotide content. The organophosphate assays measure the effects on acetylcholinesterase of selected compounds measured by ability to reactivate, effect on aging rate, and directly. In vitro screen; HD; L; Cellular NAD+ cellular viability; GA; GD; VX; Acetylcholinesterase inhibition; Reactivators; RA 5; Aging rate; Keratinocytes; Treatment and pretreatments; Assaying; Tabun (GA); Sarin (GB); Soman (GD); Organoarsenic; Organophosphates; Chemical Surety Material (CSM); Blisters; Toxicity; Toxic agents; Nerve agents; Chemical warfare agents; G Agents; V Agents; Vesicants; Mustard agents.

  13. An Escherichia coli Expression Assay and Screen for Human Immunodeficiency Virus Protease Variants with Decreased Susceptibility to Indinavir

    PubMed Central

    Melnick, Laurence; Yang, Shiow-Shong; Rossi, Rick; Zepp, Charlie; Heefner, Donald

    1998-01-01

    We have developed a recombinant Escherichia coli screening system for the rapid detection and identification of amino acid substitutions in the human immunodeficiency virus (HIV) protease associated with decreased susceptibility to the protease inhibitor indinavir (MK-639; Merck & Co.). The assay depends upon the correct processing of a segment of the HIV-1 HXB2 gag-pol polyprotein followed by detection of HIV reverse transcriptase activity by a highly sensitive, colorimetric enzyme-linked immunosorbent assay. The highly sensitive system detects the contributions of single substitutions such as I84V, L90M, and L63P. The combination of single substitutions further decreases the sensitivity to indinavir. We constructed a library of HIV protease variant genes containing dispersed mutations and, using the E. coli recombinant system, screened for mutants with decreased indinavir sensitivity. The discovered HIV protease variants contain amino acid substitutions commonly associated with indinavir resistance in clinical isolates, including the substitutions L90M, L63P, I64V, V82A, L24I, and I54T. One substitution, W6R, is also frequently found by the screen and has not been reported elsewhere. Of a total of 12,000 isolates that were screened, 12 protease variants with decreased sensitivity to indinavir were found. The L63P substitution, which is also associated with indinavir resistance, increases the stability of the isolated protease relative to that of the native HXB2 protease. The rapidity, sensitivity, and accuracy of this screen also make it useful for screening for novel inhibitors. We have found the approach described here to be useful for the detection of amino acid substitutions in HIV protease that have been associated with drug resistance as well as for the screening of novel compounds for inhibitory activity. PMID:9835523

  14. 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

  15. 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.

  16. Recommended Immunological Assays to Screen for Ricin-Containing Samples.

    PubMed

    Simon, Stéphanie; Worbs, Sylvia; Avondet, Marc-André; Tracz, Dobryan M; Dano, Julie; Schmidt, Lisa; Volland, Hervé; Dorner, Brigitte G; Corbett, Cindi R

    2015-12-01

    Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories' capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods "dangerous" samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin. PMID:26703725

  17. Recommended Immunological Assays to Screen for Ricin-Containing Samples

    PubMed Central

    Simon, Stéphanie; Worbs, Sylvia; Avondet, Marc-André; Tracz, Dobryan M.; Dano, Julie; Schmidt, Lisa; Volland, Hervé; Dorner, Brigitte G.; Corbett, Cindi R.

    2015-01-01

    Ricin, a toxin from the plant Ricinus communis, is one of the most toxic biological agents known. Due to its availability, toxicity, ease of production and absence of curative treatments, ricin has been classified by the Centers for Disease Control and Prevention (CDC) as category B biological weapon and it is scheduled as a List 1 compound in the Chemical Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection and quantification capabilities of 17 expert laboratories. In this exercise one goal was to analyse the laboratories’ capacity to detect and differentiate ricin and the less toxic, but highly homologuous protein R. communis agglutinin (RCA120). Six analytical strategies are presented in this paper based on immunological assays (four immunoenzymatic assays and two immunochromatographic tests). Using these immunological methods “dangerous” samples containing ricin and/or RCA120 were successfully identified. Based on different antibodies used the detection and quantification of ricin and RCA120 was successful. The ricin PT highlighted the performance of different immunological approaches that are exemplarily recommended for highly sensitive and precise quantification of ricin. PMID:26703725

  18. Advantageous use of HepaRG cells for the screening and mechanistic study of drug-induced steatosis.

    PubMed

    Tolosa, Laia; Gómez-Lechón, M José; Jiménez, Nuria; Hervás, David; Jover, Ramiro; Donato, M Teresa

    2016-07-01

    Only a few in vitro assays have been proposed to evaluate the steatotic potential of new drugs. The present study examines the utility of HepaRG cells as a cell-based assay system for screening drug-induced liver steatosis. A high-content screening assay was run to evaluate multiple toxicity-related cell parameters in HepaRG cells exposed to 28 compounds, including drugs reported to cause steatosis through different mechanisms and non-steatotic compounds. Lipid content was the most sensitive parameter for all the steatotic drugs, whereas no effects on lipid levels were produced by non-steatotic compounds. Apart from fat accumulation, increased ROS production and altered mitochondrial membrane potential were also found in the cells exposed to steatotic drugs, which indicates that all these cellular events contributed to drug-induced hepatotoxicity. These findings are of clinical relevance as most effects were observed at drug concentrations under 100-fold of the therapeutic peak plasmatic concentration. HepaRG cells showed increased lipid overaccumulation vs. HepG2 cells, which suggests greater sensitivity to drug-induced steatosis. An altered expression profile of transcription factors and the genes that code key proteins in lipid metabolism was also found in the cells exposed to drugs capable of inducing liver steatosis. Our results generally indicate the value of HepaRG cells for assessing the risk of liver damage associated with steatogenic compounds and for investigating the molecular mechanisms involved in drug-induced steatosis. PMID:27089845

  19. An FDA-Drug Library Screen for Compounds with Bioactivities against Meticillin-Resistant Staphylococcus aureus (MRSA)

    PubMed Central

    Lau, Qiu Ying; Tan, Yoke Yan Fion; Goh, Vanessa Chai Yin; Lee, David Jing Qin; Ng, Fui Mee; Ong, Esther H. Q.; Hill, Jeffrey; Chia, Cheng San Brian

    2015-01-01

    The lack of new antibacterial drugs entering the market and their misuse have resulted in the emergence of drug-resistant bacteria, posing a major health crisis worldwide. In particular, meticillin-resistant Staphylococcus aureus (MRSA), a pathogen responsible for numerous human infections, has become endemic in hospitals worldwide. Drug repurposing, the finding of new therapeutic indications for approved drugs, is deemed a plausible solution to accelerate drug discovery and development in this area. Towards this end, we screened 1163 drugs approved by the Food and Drug Administration (FDA) for bioactivities against MRSA in a 10 μM single-point assay. After excluding known antibiotics and antiseptics, six compounds were identified and their MICs were determined against a panel of clinical MRSA strains. A toxicity assay using human keratinocytes was also conducted to gauge their potential for repurposing as topical agents for treating MRSA skin infections. PMID:27025633

  20. Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

    PubMed Central

    Zellefrow, Crystal D.; Sharlow, Elizabeth R.; Epperly, Michael W.; Reese, Celeste E.; Shun, Tongying; Lira, Ana; Greenberger, Joel S.; Lazo, John S.

    2013-01-01

    Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D0 = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D0 = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D0 = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation

  1. Identification of druggable targets for radiation mitigation using a small interfering RNA screening assay.

    PubMed

    Zellefrow, Crystal D; Sharlow, Elizabeth R; Epperly, Michael W; Reese, Celeste E; Shun, Tongying; Lira, Ana; Greenberger, Joel S; Lazo, John S

    2012-09-01

    Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation

  2. High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes

    PubMed Central

    Grimm, Fabian Alexander; Iwata, Yasuhiro; Sirenko, Oksana; Bittner, Michael

    2015-01-01

    Abstract Cell-based high-content screening (HCS) assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. The time- and cost-effectiveness of HCS assays, combined with the organotypic nature of human induced pluripotent stem cell (iPSC)-derived cells, open new opportunities to employ physiologically relevant in vitro model systems to improve screening for potential chemical hazards. In this study, we used two human iPSC types, cardiomyocytes and hepatocytes, to test various high-content and molecular assay combinations for their applicability in a multiparametric screening format. Effects on cardiomyocyte beat frequency were characterized by calcium flux measurements for up to 90 min. Subsequent correlation with intracellular cAMP levels was used to determine if the effects on cardiac physiology were G-protein-coupled receptor dependent. In addition, we utilized high-content cell imaging to simultaneously determine cell viability, mitochondrial integrity, and reactive oxygen species (ROS) formation in both cell types. Kinetic analysis indicated that ROS formation is best detectable 30 min following initial treatment, whereas cytotoxic effects were most stable after 24 h. For hepatocytes, high-content imaging was also used to evaluate cytotoxicity and cytoskeletal integrity, as well as mitochondrial integrity and the potential for lipid accumulation. Lipid accumulation, a marker for hepatic steatosis, was most reliably detected 48 h following treatment with test compounds. Overall, our results demonstrate how a compendium of assays can be utilized for quantitative screening of chemical effects in iPSC cardiomyocytes and hepatocytes and enable rapid and cost-efficient multidimensional biological profiling of toxicity. PMID:26539751

  3. Yeast as a Model for Studies on Aβ Aggregation Toxicity in Alzheimer's Disease, Autophagic Responses, and Drug Screening.

    PubMed

    Porzoor, Afsaneh; Macreadie, Ian

    2016-01-01

    The Aβ peptide is widely considered a major cause of Alzheimer's disease since it causes neuronal death in an oligomerisation-dependent manner. In order to identify new inhibitors of Aβ that may be chemo preventative for Alzheimer's disease, a yeast assay that qualitatively determines the amounts and state of the human Aβ42 peptide has been developed. Yeast assays such as this can be applied to studies on aggregation toxicity, autophagic responses and drug screening in Alzheimer's disease. PMID:26235069

  4. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    PubMed

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples. PMID:25822163

  5. Fluorescence anisotropy (polarization): from drug screening to precision medicine

    PubMed Central

    Zhang, Hairong; Wu, Qian; Berezin, Mikhail Y.

    2016-01-01

    Introduction Fluorescence anisotropy (FA) is one of the major established methods accepted by industry and regulatory agencies for understanding the mechanisms of drug action and selecting drug candidates utilizing a high-throughput format. Areas covered This review covers the basics of FA and complementary methods, such as fluorescence lifetime anisotropy and their roles in the drug discovery process. The authors highlight the factors affecting FA readouts, fluorophore selection, and instrumentation. Furthermore, the authors describe the recent development of a successful, commercially valuable FA assay for Long QT syndrome drug toxicity to illustrate the role that FA can play in the early stages of drug discovery. Expert opinion Despite the success in drug discovery, the FA-based technique experiences competitive pressure from other homogeneous assays. That being said, FA is an established yet rapidly developing technique, recognized by academic institutions, the pharmaceutical industry, and regulatory agencies across the globe. The technical problems encountered in working with small molecules in homogeneous assays are largely solved, and new challenges come from more complex biological molecules and nanoparticles. With that, FA will remain one of the major work-horse techniques leading to precision (personalized) medicine. PMID:26289575

  6. A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection.

    PubMed

    Barrows, Nicholas J; Campos, Rafael K; Powell, Steven T; Prasanth, K Reddisiva; Schott-Lerner, Geraldine; Soto-Acosta, Ruben; Galarza-Muñoz, Gaddiel; McGrath, Erica L; Urrabaz-Garza, Rheanna; Gao, Junling; Wu, Ping; Menon, Ramkumar; Saade, George; Fernandez-Salas, Ildefonso; Rossi, Shannan L; Vasilakis, Nikos; Routh, Andrew; Bradrick, Shelton S; Garcia-Blanco, Mariano A

    2016-08-10

    Currently there are no approved vaccines or specific therapies to prevent or treat Zika virus (ZIKV) infection. We interrogated a library of FDA-approved drugs for their ability to block infection of human HuH-7 cells by a newly isolated ZIKV strain (ZIKV MEX_I_7). More than 20 out of 774 tested compounds decreased ZIKV infection in our in vitro screening assay. Selected compounds were further validated for inhibition of ZIKV infection in human cervical, placental, and neural stem cell lines, as well as primary human amnion cells. Established anti-flaviviral drugs (e.g., bortezomib and mycophenolic acid) and others that had no previously known antiviral activity (e.g., daptomycin) were identified as inhibitors of ZIKV infection. Several drugs reduced ZIKV infection across multiple cell types. This study identifies drugs that could be tested in clinical studies of ZIKV infection and provides a resource of small molecules to study ZIKV pathogenesis. PMID:27476412

  7. Mind the gap: Concerns using endpoints from endocrine screening assays in risk assessment.

    PubMed

    Wheeler, James R; Weltje, Lennart; Green, Richard M

    2014-08-01

    Endocrine screening assays not only provide mechanistic information on the potential of a substance to interact with the endocrine system, but also data potentially relevant for risk assessment. However, these screening assays have a number of limitations that should be considered before the direct use of such data for risk assessment purposes. This paper discusses the limitations that should be considered for both human and environmental risk assessment. A proposal is made to provide an objective and transparent process in order to consider which endpoint(s) might be incorporated into a risk assessment, and when more definitive studies may be of value. The proposal is complemented with an easy-to-follow flowchart to aid industry scientists and regulators when evaluating the relevance of these data. Such an approach is necessary to ensure the appropriate use of screening data to further our understanding of the eco/toxicological profile of substances undergoing screening. PMID:24887212

  8. Development of a Screening Assay for Microbial Community Profiling

    NASA Astrophysics Data System (ADS)

    Miracle, A. L.; Tilton, F.; Bonheyo, G. T.; McDermott, J.

    2010-12-01

    Remediation of subsurface contaminant plumes has been challenging in the aspects of site characterization, design for treatability, and monitoring of treatment efficacy, to name a few. Characterization of physical and geochemical properties can be achieved through advances in sensor technologies, modeling, and well placement. However, the biotic composition within the subsurface is also an important component that adds an additional biochemical contribution that is not currently being assessed. Changes in the environment have impacts to the composition of microbial communities at this solid/fluid phase interface. The introduction of a remediative treatment may provide an abundant food source for microorganisms in the subsurface and alter the community dynamics. Such changes to the microbial community composition may have dramatic effects on bulk community biochemistry, which in turn may affect the quality of the remediative treatment in terms of effectiveness and transport through alteration of the environment. A screening array is being developed based on DNA sequence information from indigenous microorganisms within target sediments to be used to assess microbial community changes throughout remediative treatments and through time. Integration of physical, chemical, and biotic community information will be assessed to determine efficacy of treatment before, during, and after treatment to assess success of treatment, and measure any post-treatment changes.

  9. Which high-risk HPV assays fulfil criteria for use in primary cervical cancer screening?

    PubMed

    Arbyn, M; Snijders, P J F; Meijer, C J L M; Berkhof, J; Cuschieri, K; Kocjan, B J; Poljak, M

    2015-09-01

    Several countries are in the process of switching to high-risk human papillomavirus (hrHPV) testing for cervical cancer screening. Given the multitude of available tests, validated assays which assure high-quality screening need to be identified. A systematic review was conducted to answer the question which hrHPV tests fulfil the criteria defined by an international expert team in 2009, based on reproducibility and relative sensitivity and specificity compared to Hybrid Capture-2 or GP5+/6+ PCR-enzyme immunoassay. These latter two hrHPV DNA assays were validated in large randomized trials and cohorts with a follow-up duration of 8 years or more. Eligible studies citing the 2009 guideline were retrieved from Scopus (http://www.scopus.com) and from a meta-analysis assessing the relative accuracy of new hrHPV assays versus the standard comparator tests to detect high-grade cervical intraepithelial neoplasia or cancer in primary screening. The cobas 4800 HPV test and Abbott RealTime High Risk HPV test were consistently validated in two and three studies, respectively, whereas the PapilloCheck HPV-screening test, BD Onclarity HPV assay and the HPV-Risk assay were validated each in one study. Other tests which partially fulfil the 2009 guidelines are the following: Cervista HPV HR Test, GP5+/6+ PCR-LMNX, an in-house E6/E7 RT quantitative PCR and MALDI-TOF (matrix-assisted laser desorption-ionization time-of-flight). The APTIMA HPV assay targeting E6/E7 mRNA of hrHPV was also fully validated. However, the cross-sectional equivalency criteria of the 2009 guidelines were set up for HPV DNA assays. Demonstration of a low risk of CIN3+ after a negative APTIMA test over a longer period is awaited to inform us about its utility in cervical cancer screening at 5-year or longer intervals. PMID:25936581

  10. 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

  11. 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

  12. Phospholipid vesicle-based permeation assay and EpiSkin® in assessment of drug therapies destined for skin administration.

    PubMed

    Engesland, André; Škalko-Basnet, Nataša; Flaten, Gøril Eide

    2015-03-01

    Cost-effective and efficient methods for permeability screening are crucial during early development of drugs, drug formulations, and cosmeceuticals. Alternatives to animal experiments are impelled for both economical and ethical reasons. The aim of this study was to determine the ability of the phospholipid vesicle-based permeation assay (PVPA) to assess the effect of different formulations on drug permeability and thus establish its utility in formulation development. Three model drugs were tested in solutions and as liposomal formulations. The permeability results for the PVPA models were compared with the results for the reconstructed human skin model, EpiSkin(®). The drugs were ranked based on their estimated penetration potentials, and the results were in accordance with what was expected considering the physicochemical properties of the drugs. PVPAs (E-80, ceramide, cholesterol, cholesteryl sulfate, and palmitic acid) was able to distinguish between drug solutions and liposomal formulations; however, EpiSkin(®) detected only small differences between the drugs in solution and formulations. In contrast with EpiSkin(®), which is limited by a 3-day testing window, PVPA barriers can be stored frozen for up to 2 weeks or even up to 16 months, depending on their compositions. The PVPA models are thus more cost effective and efficient than the EpiSkin(®) model for permeability screening during early drug development. PMID:25558045

  13. Performance characteristics of an ELISA screening assay for urinary synthetic cannabinoids.

    PubMed

    Spinelli, Eliani; Barnes, Allan J; Young, Sheena; Castaneto, Marisol S; Martin, Thomas M; Klette, Kevin L; Huestis, Marilyn A

    2015-06-01

    Synthetic cannabinoids are marketed as legal alternatives to cannabis, as routine urine cannabinoid immunoassays do not detect synthetic cannabinoids. Laboratories are challenged to identify these new designer drugs that are widely available and represent a major public health and safety problem. Immunoassay testing offers rapid separation of presumptive positive and negative specimens, prior to more costly and time-consuming chromatographic confirmation. The Neogen SPICE ELISA kit targets JWH-018 N-pentanoic acid as a marker for urinary synthetic cannabinoids. Assay performance was evaluated by analyzing 2469 authentic urine samples with the Neogen immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two immunoassay cut-off concentrations, 5 and 10 µg/L, classified samples as presumptive positive or negative, followed by qualitative LC-MS/MS confirmation for 29 synthetic cannabinoids markers with limits of detection of 0.5-10 µg/L to determine the assay's sensitivity, specificity and efficacy. Challenges at ±25% of each cut-off also were investigated to determine performance around the cut-off and intra- and inter-plate imprecision. The immunoassay was linear from 1 to 250 µg/L (r(2)  = 0.992) with intra- and inter-plate imprecision of ≤5.3% and <9%, respectively. Sensitivity, specificity, and efficiency results with the 5 µg/L cut-off were 79.9%, 99.7%, and 97.4% and with the 10 µg/L cut-off 69.3%, 99.8%, and 96.3%, respectively. Cross-reactivity was shown for 18 of 73 synthetic cannabinoids markers evaluated. Good sensitivity, specificity, and efficiency, lack of sample preparation requirements, and rapid semi-automation documented that the Neogen SPICE ELISA kit is a viable method for screening synthetic cannabinoids in urine targeting JWH-018 N-pentanoic acid. PMID:25167963

  14. Discovering novel neuroactive drugs through high-throughput behavior-based chemical screening in the zebrafish

    PubMed Central

    Bruni, Giancarlo; Lakhani, Parth; Kokel, David

    2014-01-01

    Most neuroactive drugs were discovered through unexpected behavioral observations. Systematic behavioral screening is inefficient in most model organisms. But, automated technologies are enabling a new phase of discovery-based research in central nervous system (CNS) pharmacology. Researchers are using large-scale behavior-based chemical screens in zebrafish to discover compounds with new structures, targets, and functions. These compounds are powerful tools for understanding CNS signaling pathways. Substantial differences between human and zebrafish biology will make it difficult to translate these discoveries to clinical medicine. However, given the molecular genetic similarities between humans and zebrafish, it is likely that some of these compounds will have translational utility. We predict that the greatest new successes in CNS drug discovery will leverage many model systems, including in vitro assays, cells, rodents, and zebrafish. PMID:25104936

  15. siRNA Genome Screening Approaches to Therapeutic Drug Repositioning

    PubMed Central

    Perwitasari, Olivia; Bakre, Abhijeet; Tompkins, S. Mark; Tripp, Ralph A.

    2013-01-01

    Bridging high-throughput screening (HTS) with RNA interference (RNAi) has allowed for rapid discovery of the molecular basis of many diseases, and identification of potential pathways for developing safe and effective treatments. These features have identified new host gene targets for existing drugs paving the pathway for therapeutic drug repositioning. Using RNAi to discover and help validate new drug targets has also provided a means to filter and prioritize promising therapeutics. This review summarizes these approaches across a spectrum of methods and targets in the host response to pathogens. Particular attention is given to the utility of drug repurposing utilizing the promiscuous nature of some drugs that affect multiple molecules or pathways, and how these biological pathways can be targeted to regulate disease outcome. PMID:24275945

  16. Virtual screening in drug discovery -- a computational perspective.

    PubMed

    Reddy, A Srinivas; Pati, S Priyadarshini; Kumar, P Praveen; Pradeep, H N; Sastry, G Narahari

    2007-08-01

    Virtual screening emerged as an important tool in our quest to access novel drug like compounds. There are a wide range of comparable and contrasting methodological protocols available in screening databases for the lead compounds. The number of methods and software packages which employ the target and ligand based virtual screening are increasing at a rapid pace. However, the general understanding on the applicability and limitations of these methodologies is not emerging as fast as the developments of various methods. Therefore, it is extremely important to compare and contrast various protocols with practical examples to gauge the strength and applicability of various methods. The review provides a comprehensive appraisal on several of the available virtual screening methods to-date. Recent developments of the docking and similarity based methods have been discussed besides the descriptor selection and pharmacophore based searching. The review touches upon the application of statistical, graph theory based methods machine learning tools in virtual screening and combinatorial library design. Finally, several case studies are undertaken where the virtual screening technology has been applied successfully. A critical analysis of these case studies provides a good platform to estimate the applicability of various virtual screening methods in the new lead identification and optimization. PMID:17696867

  17. A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library

    PubMed Central

    Feng, Jie; Shi, Wanliang; Zhang, Shuo; Sullivan, David; Auwaerter, Paul G.; Zhang, Ying

    2016-01-01

    Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10–20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi

  18. A Drug Combination Screen Identifies Drugs Active against Amoxicillin-Induced Round Bodies of In Vitro Borrelia burgdorferi Persisters from an FDA Drug Library.

    PubMed

    Feng, Jie; Shi, Wanliang; Zhang, Shuo; Sullivan, David; Auwaerter, Paul G; Zhang, Ying

    2016-01-01

    Although currently recommended antibiotics for Lyme disease such as doxycycline or amoxicillin cure the majority of the patients, about 10-20% of patients treated for Lyme disease may experience lingering symptoms including fatigue, pain, or joint and muscle aches. Under experimental stress conditions such as starvation or antibiotic exposure, Borrelia burgdorferi can develop round body forms, which are a type of persister bacteria that appear resistant in vitro to customary first-line antibiotics for Lyme disease. To identify more effective drugs with activity against the round body form of B. burgdorferi, we established a round body persister model induced by exposure to amoxicillin (50 μg/ml) and then screened the Food and Drug Administration drug library consisting of 1581 drug compounds and also 22 drug combinations using the SYBR Green I/propidium iodide viability assay. We identified 23 drug candidates that have higher activity against the round bodies of B. burgdorferi than either amoxicillin or doxycycline. Eleven individual drugs scored better than metronidazole and tinidazole which have been previously described to be active against round bodies. In this amoxicillin-induced round body model, some drug candidates such as daptomycin and clofazimine also displayed enhanced activity which was similar to a previous screen against stationary phase B. burgdorferi persisters not exposure to amoxicillin. Additional candidate drugs active against round bodies identified include artemisinin, ciprofloxacin, nifuroxime, fosfomycin, chlortetracycline, sulfacetamide, sulfamethoxypyridazine and sulfathiozole. Two triple drug combinations had the highest activity against amoxicillin-induced round bodies and stationary phase B. burgdorferi persisters: artemisinin/cefoperazone/doxycycline and sulfachlorpyridazine/daptomycin/doxycycline. These findings confirm and extend previous findings that certain drug combinations have superior activity against B. burgdorferi

  19. A survey of yeast genomic assays for drug and target discovery

    PubMed Central

    Smith, Andrew M.; Ammar, Ron; Nislow, Corey; Giaever, Guri

    2010-01-01

    Over the past decade, the development and application of chemical genomic assays using the model organism Saccharomyces cerevisiae has provided powerful methods to identify the mechanism of action of known drugs and novel small molecules in vivo. These assays identify drug target candidates, genes involved in buffering drug target pathways and also help to define the general cellular response to small molecules. In this review, we examine current yeast chemical genomic assays and summarize the potential applications of each approach. PMID:20546776

  20. DEVELOPMENT, STANDARDIZATION AND VALIDATION OF THE MAMMALIAN IN VIVO ASSAYS IN THE PROPOSED TIER I SCREENING BATTERY FOR ENDOCRINE DISRUPTORS

    EPA Science Inventory

    This research directly supports the development, standardization and validation of several Tier 1 screening mammalian in vivo assays. Through the development and use of many of these assays for testing specific hypothesis in their respective research programs, these investigato...

  1. A quantitative microfluidic angiogenesis screen for studying anti-angiogenic therapeutic drugs.

    PubMed

    Kim, Choong; Kasuya, Junichi; Jeon, Jessie; Chung, Seok; Kamm, Roger D

    2015-01-01

    Anti-angiogenic therapy, which suppresses tumor growth by disrupting oxygen and nutrient supply from blood to the tumor, is now widely accepted as a treatment for cancer. To investigate the mechanisms of action of these anti-angiogenesis drugs, new three dimensional (3D) cell culture-based drug screening models are increasingly employed. However, there is no in vitro high-throughput screening (HTS) angiogenesis assay that can provide uniform culture conditions for the quantitative assessment of physiological responses to chemoattractant reagents under various concentrations of anti-angiogenesis drugs. Here we describe a method for screening and quantifying the vascular endothelial growth factor (VEGF)-induced chemotactic response on human umbilical vein endothelial cells (HUVECs) cultured with different concentrations of bortezomib, a selective 26S proteasome inhibitor. With this quantitative microfluidic angiogenesis screen (QMAS), we demonstrate that bortezomib-induced endothelial cell death is preceded by a series of morphological changes that develop over several days. We also explore the mechanisms by which bortezomib can inhibit angiogenesis. PMID:25370780

  2. Chemical & RNAi screening at MSKCC: a collaborative platform to discover & repurpose drugs to fight disease

    PubMed Central

    Bhinder, Bhavneet; Antczak, Christophe; Shum, David; Radu, Constantin; Mahida, Jeni P.; Liu-Sullivan, Nancy; Ibáñez, Glorymar; Raja, Balajee Somalinga; Calder, Paul A.; Djaballah, Hakim

    2014-01-01

    Memorial Sloan-Kettering Cancer Center (MSKCC) has implemented the creation of a full service state-of-the-art High-throughput Screening Core Facility (HTSCF) equipped with modern robotics and custom-built screening data management resources to rapidly store and query chemical and RNAi screening data outputs. The mission of the facility is to provide oncology clinicians and researchers alike with access to cost-effective HTS solutions for both chemical and RNAi screening, with an ultimate goal of novel target identification and drug discovery. HTSCF was established in 2003 to support the institution’s commitment to growth in molecular pharmacology and in the realm of therapeutic agents to fight chronic diseases such as cancer. This endeavor required broad range of expertise in technology development to establish robust and innovative assays, large collections of diverse chemical and RNAi duplexes to probe specific cellular events, sophisticated compound and data handling capabilities, and a profound knowledge in assay development, hit validation, and characterization. Our goal has been to strive for constant innovation, and we strongly believe in shifting the paradigm from traditional drug discovery towards translational research now, making allowance for unmet clinical needs in patients. Our efforts towards repurposing FDA-approved drugs fructified when digoxin, identified through primary HTS, was administered in the clinic for treatment of stage Vb retinoblastoma. In summary, the overall aim of our facility is to identify novel chemical probes, to study cellular processes relevant to investigator’s research interest in chemical biology and functional genomics, and to be instrumental in accelerating the process of drug discovery in academia. PMID:24661215

  3. A Developmental Toxicology Assay Platform for Screening Teratogenic Liability of Pharmaceutical Compounds.

    PubMed

    Augustine-Rauch, Karen; Zhang, Cindy X; Panzica-Kelly, Julieta M

    2016-02-01

    Increasing need for proactive safety optimization of pharmaceutical compounds has led to generation and/or refinement of in vitro developmental toxicology assays. Our laboratory has developed three in vitro developmental toxicology assays to assess teratogenic liability of pharmaceutical compounds. These assays included a mouse molecular embryonic stem cell assay (MESCA), a dechorionated zebrafish embryo culture (ZEC) assay, and a streamlined rat whole embryo culture (rWEC) assay. Individually, the assays presented good (73-82%) predictivity. However, it remains to be determined whether combining or tiering the assays could enhance performance. Seventy-three compounds representing a broad spectrum of pharmaceutical targets and chemistry were evaluated across the assays to generate testing strategies that optimized performance. The MESCA and ZEC assays were found to have two limitations: compound solubility and frequent misclassification of compounds with H1 receptor or GABAnergic activity. The streamlined rWEC assay was found to be a cost-effective stand-alone assay for supporting poorly soluble compounds and/or ones with H1 or GABAnergic activity. For all other compounds, a tiering strategy using the MESCA and ZEC assays additionally optimized throughput, cost, and minimized animal use. The tiered strategy resulted in improved performance achieving 88% overall predictivity and was comparable with 89% overall predictivity achieved with frequency analysis (final teratogenic classification made from most frequent teratogenic classification from each individual assay). Furthermore there were 21 compounds in the test set characterized as definitive or suspect human teratogens and the multiassay approach achieved 95 and 91% correct classification using the tiered or frequency screening approach, respectively. PMID:26729651

  4. Human Papillomavirus Assays and Cytology in Primary Cervical Screening of Women Aged 30 Years and Above.

    PubMed

    Rebolj, Matejka; Bonde, Jesper; Preisler, Sarah; Ejegod, Ditte; Rygaard, Carsten; Lynge, Elsebeth

    2016-01-01

    In women aged ≥ 30 years, Human Papillomavirus testing will replace cytology for primary cervical screening. We compared Hybrid Capture 2 (HC2), cobas, CLART, and APTIMA HPV assays with cytology on 2869 SurePath samples from women undergoing routine screening at 30-65 years in Copenhagen, Denmark. Women with cytological abnormalities were managed according to routine recommendations, with 92% completeness. Those with cytology-normal/HPV-positive samples (on any of the four assays) were invited for repeated cytology and HPV testing in 1.5 year, and 58% had additional testing. HPV testing detected more ≥ CIN3 than cytology (HC2: 35, cobas, CLART: 37, APTIMA: 34, cytology: 31), although statistically the differences were not significant. Cobas and CLART detected significantly more ≥ CIN2 than cytology (cobas, CLART: 49, cytology: 39). The proportion of women with false-positive test results (positive test results without ≥ CIN3) varied between 3.3% with cytology and 14.9% with cobas. All HPV assays led to significantly more false-positive tests, whereas compared to HC2 cobas and CLART were associated with a significantly higher and APTIMA with a significantly lower proportion. Detection of CIN1 was particularly increased for the three DNA assays. With APTIMA combined with cytological triage, about 20% more women were referred for colposcopy than with cytology screening. With the three DNA assays, the increase was ≥ 50%. The number of women with repeated testing was twice as high with APTIMA and almost five times as high with cobas compared to cytology. To our knowledge, Horizon was the only study set in routine practice that compared more than two HPV assays in the same women while also ascertaining the histological status of women with normal cytology/HPV-positive test results. HPV-based screening of Danish women aged 30-65 detected more high-grade CIN but decreased the screening specificity, and increased the demand for additional testing. PMID:26789267

  5. 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

  6. 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

  7. 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

  8. Screening for Drug Abuse Among College Students: Modification of the Michigan Alcoholism Screening Test

    ERIC Educational Resources Information Center

    Cannell, M. Barry; Favazza, Armando R.

    1978-01-01

    Modified version of the Michigan Alcoholism Screening Test was anonymously given to 245 college students on two Midwestern university campuses. Cutoff score for suspected drug abuse was set at five points. The percent of students scoring five or more points was 25 and 22 from campuses A and B respectively. (Author)

  9. Genome Editing-Enabled HTS Assays Expand Drug Target Pathways for Charcot–Marie–Tooth Disease

    PubMed Central

    2015-01-01

    Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot–Marie–Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin. PMID:25188731

  10. Development of a potato seedling assay to screen for resistance to Verticillium dahliae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A seedling assay was developed for Verticillium wilt (VW) resistance in potato (Solanum tuberosum) in order to provide efficient and rapid screening to identify resistant clones in segregating populations. The method provides uniform inoculum to avoid false negatives and reduces the confusion of sy...

  11. Development of Tyrosinase Promoter-Based Fluorescent Assay for Screening of Anti-melanogenic Agents.

    PubMed

    Lee, JaeHo; Lee, SeungJun; Lee, ByungMan; Roh, KyungBaeg; Park, DeokHoon; Jung, EunSun

    2015-01-01

    For screening of skin-whitening ingredients that modulate inhibition of melanogenesis, tyrosinase promoter-based assay using a three-dimensional (3D) spheroid culture technique is a beneficial tool to improve the accuracy of raw material screening in cosmetics through mimicking of the in vivo microenvironment. Although the advantages of high-throughput screening (HTS) are widely known, there has been little focus on specific cell-based promoter assays for HTS in identifying skin-whitening ingredients that inhibit accumulation of melanin. The aim of this study was therefore to develop a large-scale compatible assay through pTyr-EGFP, an enhanced green fluorescent protein (EGFP)-based tyrosinase-specific promoter, to seek potential melanogenesis inhibitors for cosmetic use. Herein, a stably transfected human melanoma cell line expressing EGFP under the control of a 2.2-kb fragment derived from the tyrosinase gene was generated. Spontaneous induction of the tyrosinase promoter by 3D spheroid culture resulted in increased expression of EGFP, providing a significant correlation with the tyrosinase mRNA level, and subsequent inhibition of tyrosinase activity. Importantly, the pTyr-EGFP system provided successful tracking of the changes in the live image and real-time monitoring. Thus tyrosinase promoter-based fluorescent assay using a 3D spheroid culture can be useful as a screening system for exploring the efficiency of anti-melanogenesis ingredients. PMID:26179334

  12. MAMMALIAN SCREENING ASSAYS FOR THE DETECTION OF POTENTIAL ENDOCRINE DISRUPTING CHEMICALS WITH AN EMPHASIS ON MALES

    EPA Science Inventory

    MAMMALIAN SCREENING ASSAYS FOR THE DETECTION OF POTENTIAL
    ENDOCRINE DISRUPTING CHEMICALS WITH AN EMPHASIS ON MALES.
    Authors: L E Gray 1 , J Furr 1 , M G Price 2 , C J Wolf 3 and J S Ostby 1
    Institutions: 1. Endocrinology Branch, Reproductive Toxicology Division, NH...

  13. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection

    PubMed Central

    Kremastinou, J.; Polymerou, V.; Lavranos, D.; Aranda Arrufat, A.; Harwood, J.; Martínez Lorenzo, M. J.; Ng, K. P.; Queiros, L.; Vereb, I.

    2016-01-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum. The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results. PMID:27358468

  14. Evaluation of Elecsys Syphilis Assay for Routine and Blood Screening and Detection of Early Infection.

    PubMed

    Kremastinou, J; Polymerou, V; Lavranos, D; Aranda Arrufat, A; Harwood, J; Martínez Lorenzo, M J; Ng, K P; Queiros, L; Vereb, I; Cusini, M

    2016-09-01

    Treponema pallidum infections can have severe complications if not diagnosed and treated at an early stage. Screening and diagnosis of syphilis require assays with high specificity and sensitivity. The Elecsys Syphilis assay is an automated treponemal immunoassay for the detection of antibodies against T. pallidum The performance of this assay was investigated previously in a multicenter study. The current study expands on that evaluation in a variety of diagnostic settings and patient populations, at seven independent laboratories. The samples included routine diagnostic samples, blood donation samples, samples from patients with confirmed HIV infections, samples from living organ or bone marrow donors, and banked samples, including samples previously confirmed as syphilis positive. This study also investigated the seroconversion sensitivity of the assay. With a total of 1,965 syphilis-negative routine diagnostic samples and 5,792 syphilis-negative samples collected from blood donations, the Elecsys Syphilis assay had specificity values of 99.85% and 99.86%, respectively. With 333 samples previously identified as syphilis positive, the sensitivity was 100% regardless of disease stage. The assay also showed 100% sensitivity and specificity with samples from 69 patients coinfected with HIV. The Elecsys Syphilis assay detected infection in the same bleed or earlier, compared with comparator assays, in a set of sequential samples from a patient with primary syphilis. In archived serial blood samples collected from 14 patients with direct diagnoses of primary syphilis, the Elecsys Syphilis assay detected T. pallidum antibodies for 3 patients for whom antibodies were not detected with the Architect Syphilis TP assay, indicating a trend for earlier detection of infection, which may have the potential to shorten the time between infection and reactive screening test results. PMID:27358468

  15. Phenotypic assays to identify agents that induce reactive gliosis: a counter-screen to prioritize compounds for preclinical animal studies.

    PubMed

    Beckerman, Samuel R; Jimenez, Joaquin E; Shi, Yan; Al-Ali, Hassan; Bixby, John L; Lemmon, Vance P

    2015-09-01

    Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton, adoption of stellate morphologies, and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial, but in the chronic injury phase, reactive astrocytes produce inhibitory factors (i.e., chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS, we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However, the effects these compounds have on astrocytes are unknown. Specifically, we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis, we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for six-point dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Z-factor of 0.44±0.03 (mean±standard error of the mean; N=3 biological replicates). The CSPG assay is reproducible and informative, but does not satisfy common metrics for a "screenable" assay. As proof of principle, we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis. PMID:26230074

  16. Saliva Polymerase-Chain-Reaction Assay for Cytomegalovirus Screening in Newborns

    PubMed Central

    Boppana, Suresh B.; Ross, Shannon A.; Shimamura, Masako; Palmer, April L.; Ahmed, Amina; Michaels, Marian G.; Sánchez, Pablo J.; Bernstein, David I.; Tolan, Robert W.; Novak, Zdenek; Chowdhury, Nazma; Britt, William J.; Fowler, Karen B.

    2011-01-01

    BACKGROUND Congenital cytomegalovirus (CMV) infection is an important cause of hearing loss, and most infants at risk for CMV-associated hearing loss are not identified early in life because of failure to test for the infection. The standard assay for newborn CMV screening is rapid culture performed on saliva specimens obtained at birth, but this assay cannot be automated. Two alternatives — real-time polymerase-chain-reaction (PCR)–based testing of a liquid-saliva or dried-saliva specimen obtained at birth — have been developed. METHODS In our prospective, multicenter screening study of newborns, we compared real-time PCR assays of liquid-saliva and dried-saliva specimens with rapid culture of saliva specimens obtained at birth. RESULTS A total of 177 of 34,989 infants (0.5%; 95% confidence interval [CI], 0.4 to 0.6) were positive for CMV, according to at least one of the three methods. Of 17,662 newborns screened with the use of the liquid-saliva PCR assay, 17,569 were negative for CMV, and the remaining 85 infants (0.5%; 95% CI, 0.4 to 0.6) had positive results on both culture and PCR assay. The sensitivity and specificity of the liquid-saliva PCR assay were 100% (95% CI, 95.8 to 100) and 99.9% (95% CI, 99.9 to 100), respectively, and the positive and negative predictive values were 91.4% (95% CI, 83.8 to 96.2) and 100% (95% CI, 99.9 to 100), respectively. Of 17,327 newborns screened by means of the dried-saliva PCR assay, 74 were positive for CMV, whereas 76 (0.4%; 95% CI, 0.3 to 0.5) were found to be CMV-positive on rapid culture. Sensitivity and specificity of the dried-saliva PCR assay were 97.4% (95% CI, 90.8 to 99.7) and 99.9% (95% CI, 99.9 to 100), respectively. The positive and negative predictive values were 90.2% (95% CI, 81.7 to 95.7) and 99.9% (95% CI, 99.9 to 100), respectively. CONCLUSIONS Real-time PCR assays of both liquid- and dried-saliva specimens showed high sensitivity and specificity for detecting CMV infection and should be

  17. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  18. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  19. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  20. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  1. 21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Neuroleptic drugs radioreceptor assay test system. 862.3645 Section 862.3645 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical...

  2. Screening for noise in gene expression identifies drug synergies.

    PubMed

    Dar, Roy D; Hosmane, Nina N; Arkin, Michelle R; Siliciano, Robert F; Weinberger, Leor S

    2014-06-20

    Stochastic fluctuations are inherent to gene expression and can drive cell-fate specification. We used such fluctuations to modulate reactivation of HIV from latency-a quiescent state that is a major barrier to an HIV cure. By screening a diverse library of bioactive small molecules, we identified more than 80 compounds that modulated HIV gene-expression fluctuations (i.e., "noise"), without changing mean expression. These noise-modulating compounds would be neglected in conventional screens, and yet, they synergized with conventional transcriptional activators. Noise enhancers reactivated latent cells significantly better than existing best-in-class reactivation drug combinations (and with reduced off-target cytotoxicity), whereas noise suppressors stabilized latency. Noise-modulating chemicals may provide novel probes for the physiological consequences of noise and an unexplored axis for drug discovery, allowing enhanced control over diverse cell-fate decisions. PMID:24903562

  3. Towards novel therapeutics for HIV through fragment-based screening and drug design.

    PubMed

    Tiefendbrunn, Theresa; Stout, C David

    2014-01-01

    Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins. PMID:25455312

  4. A critical evaluation of in vitro cell culture models for high-throughput drug screening and toxicity.

    PubMed

    Astashkina, Anna; Mann, Brenda; Grainger, David W

    2012-04-01

    Drug candidate and toxicity screening processes currently rely on results from early-stage in vitro cell-based assays expected to faithfully represent essential aspects of in vivo pharmacology and toxicology. Several in vitro designs are optimized for high throughput to benefit screening efficiencies, allowing the entire libraries of potential pharmacologically relevant or possible toxin molecules to be screened for different types of cell signals relevant to tissue damage or to therapeutic goals. Creative approaches to multiplexed cell-based assay designs that select specific cell types, signaling pathways and reporters are routine. However, substantial percentages of new chemical and biological entities (NCEs/NBEs) that fail late-stage human drug testing, or receive regulatory "black box" warnings, or that are removed from the market for safety reasons after regulatory approvals all provide strong evidence that in vitro cell-based assays and subsequent preclinical in vivo studies do not yet provide sufficient pharmacological and toxicity data or reliable predictive capacity for understanding drug candidate performance in vivo. Without a reliable translational assay tool kit for pharmacology and toxicology, the drug development process is costly and inefficient in taking initial in vitro cell-based screens to in vivo testing and subsequent clinical approvals. Commonly employed methods of in vitro testing, including dissociated, organotypic, organ/explant, and 3-D cultures, are reviewed here with specific focus on retaining cell and molecular interactions and physiological parameters that determine cell phenotypes and their corresponding responses to bioactive agents. Distinct advantages and performance challenges for these models pertinent to cell-based assay and their predictive capabilities required for accurate correlations to in vivo mechanisms of drug toxicity are compared. PMID:22252140

  5. High efficacy vasopermeability drug candidates identified by screening in an ex ovo chorioallantoic membrane model

    PubMed Central

    Pink, Desmond; Luhrs, Keith A.; Zhou, Longen; Schulte, Wendy; Chase, Jennifer; Frosch, Christian; Haberl, Udo; Nguyen, Van; Roy, Aparna I.; Lewis, John D.; Zijlstra, Andries; Parseghian, Missag H.

    2015-01-01

    The use of rodent models to evaluate efficacy during testing is accompanied by significant economic and regulatory hurdles which compound the costs of screening for promising drug candidates. Vasopermeation Enhancement Agents (VEAs) are a new class of biologics that are designed to increase the uptake of cancer therapeutics at the tumor site by modifying vascular permeability in the tumor to increase the therapeutic index of co-administered drugs. To evaluate the efficacy of a panel of VEA clinical candidates, we compared the rodent Miles assay to an equivalent assay in the ex ovo chicken embryo model. Both model systems identified the same candidate (PVL 10) as the most active promoter of vasopermeation in non-tumor tissues. An ex ovo chicken embryo system was utilized to test each candidate VEA in two human tumor models at a range of concentrations. Vasopermeation activity due to VEA was dependent on tumor type, with HEp3 tumors displaying higher levels of vasopermeation than MDA-MB-435. One candidate (PVL 10) proved optimal for HEp3 tumors and another (PVL 2) for MDA-MB-435. The use of the ex ovo chicken embryo model provides a rapid and less costly alternative to the use of rodent models for preclinical screening of drug candidates. PMID:26510887

  6. Tubulin-binding drug screening by MALDI-TOFMS.

    PubMed

    Hannewald, Paul; Maunit, Benoît; Muller, Jean-François

    2006-07-01

    Despite a large amount of drugs available to treat cancer, none is totally satisfactory with respect to its tolerance or side effects. It is very important to discover new compounds that exhibit specific features such as binding to proteic targets. Given the clinical successes of the poisons of the mitotic spindle chemotherapeutic agent class, it is often considered that tubulin represents one of the best cancer targets identified so far, and it seems likely that discovering new drugs of this class will significantly improve the range of active chemotherapeutic agents. The aim of this work is to present the new screening test that has been developed in our laboratory in order to study the binding of compounds to tubulin. We have developed a screening protocol involving three sampling strategies before the MALDI-TOFMS analysis. The three strategies give very accurate and reproducible results and could therefore possibly be used in screening campaigns. We have also proved that no unspecific binding can provide a loss of specificity of the test. Our protocol presents all the requirements for being a useful tool to screen the binding of compounds to tubulin. PMID:16808446

  7. Building a Tiered Approach to In Vitro Predictive Toxicity Screening: A Focus on Assays with In Vivo Relevance

    PubMed Central

    McKim, James M

    2010-01-01

    One of the greatest challenges facing the pharmaceutical industry today is the failure of promising new drug candidates due to unanticipated adverse effects discovered during preclinical animal safety studies and clinical trials. Late stage attrition increases the time required to bring a new drug to market, inflates development costs, and represents a major source of inefficiency in the drug discovery/development process. It is generally recognized that early evaluation of new drug candidates is necessary to improve the process. Building in vitro data sets that can accurately predict adverse effects in vivo would allow compounds with high risk profiles to be deprioritized, while those that possess the requisite drug attributes and a lower risk profile are brought forward. In vitro cytotoxicity assays have been used for decades as a tool to understand hypotheses driven questions regarding mechanisms of toxicity. However, when used in a prospective manner, they have not been highly predictive of in vivo toxicity. Therefore, the issue may not be how to collect in vitro toxicity data, but rather how to translate in vitro toxicity data into meaningful in vivo effects. This review will focus on the development of an in vitro toxicity screening strategy that is based on a tiered approach to data collection combined with data interpretation. PMID:20053163

  8. A stereospecific solid-phase screening assay for colonies expressing both (R)- and (S)-selective ω-aminotransferases.

    PubMed

    Willies, Simon C; Galman, James L; Slabu, Iustina; Turner, Nicholas J

    2016-02-28

    A novel solid-phase screening assay was developed for colonies expressing both (R)- and (S)-selective ω-aminotransferases. This high-throughput assay can be used to screen rapidly large variant libraries with enhanced substrate selectivity and enantioselectivities. PMID:26755753

  9. Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates.

    PubMed Central

    Kellam, P; Larder, B A

    1994-01-01

    Antiviral drug susceptibility assays for clinical human immunodeficiency virus type 1 (HIV-1) isolates are required to monitor the development of drug resistance during clinical trials and antiretroviral drug therapy. First-generation phenotypic assays possess a number of drawbacks, not least the selection of unrepresentative virus populations during cocultivation. Here we describe a rapid phenotypic assay for the assessment of the susceptibility of clinical isolates to reverse transcriptase (RT) inhibitors. This procedure, called the recombinant virus assay, allows the generation of viable virus by homologous recombination of a PCR-derived pool of RT coding sequences into an RT-deleted, noninfectious proviral clone, pHIV delta BstEII. A nested PCR procedure has been optimized to allow the amplification of an RT pool from both uncultured and cocultured infected patient peripheral blood lymphocyte (PBL) DNA for subsequent use in the creation of recombinant viruses. Analysis of two patients during the course of zidovudine therapy showed that this approach produced viruses which accurately exhibited the same genotype and phenotype as that of the original infected PBL DNA. The recombinant virus assay can be performed in approximately 3 weeks without the use of donor PBLs and therefore represents a rapid, nonselective procedure for the assay of clinical isolates. Images PMID:8141575

  10. 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

  11. 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

  12. A parasite rescue and transformation assay for antileishmanial screening against intracellular Leishmania donovani amastigotes in THP1 human acute monocytic leukemia cell line.

    PubMed

    Jain, Surendra K; Sahu, Rajnish; Walker, Larry A; Tekwani, Babu L

    2012-01-01

    Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly(1). Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited (2);current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance (3). The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models. In vitro promastigotes (4) and axenic amastigotes assays(5) are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes. Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2

  13. 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

  14. Comparison of Automated Treponemal and Nontreponemal Test Algorithms as First-Line Syphilis Screening Assays

    PubMed Central

    Chung, Jae-Woo; Park, Seong Yeon; Chae, Seok Lae

    2016-01-01

    Background Automated Mediace Treponema pallidum latex agglutination (TPLA) and Mediace rapid plasma reagin (RPR) assays are used by many laboratories for syphilis diagnosis. This study compared the results of the traditional syphilis screening algorithm and a reverse algorithm using automated Mediace RPR or Mediace TPLA as first-line screening assays in subjects undergoing a health checkup. Methods Samples from 24,681 persons were included in this study. We routinely performed Mediace RPR and Mediace TPLA simultaneously. Results were analyzed according to both the traditional algorithm and reverse algorithm. Samples with discordant results on the reverse algorithm (e.g., positive Mediace TPLA, negative Mediace RPR) were tested with Treponema pallidum particle agglutination (TPPA). Results Among the 24,681 samples, 30 (0.1%) were found positive by traditional screening, and 190 (0.8%) by reverse screening. The identified syphilis rate and overall false-positive rate according to the traditional algorithm were lower than those according to the reverse algorithm (0.07% and 0.05% vs. 0.64% and 0.13%, respectively). A total of 173 discordant samples were tested with TPPA by using the reverse algorithm, of which 140 (80.9%) were TPPA positive. Conclusions Despite the increased false-positive results in populations with a low prevalence of syphilis, the reverse algorithm detected 140 samples with treponemal antibody that went undetected by the traditional algorithm. The reverse algorithm using Mediace TPLA as a screening test is more sensitive for the detection of syphilis. PMID:26522755

  15. A 1536-well Fluorescence Polarization Assay to Screen for Modulators of the MUSASHI Family of RNA-Binding Proteins

    PubMed Central

    Minuesa, Gerard; Antczak, Christophe; Shum, David; Radu, Constantin; Bhinder, Bhavneet; Li, Yueming; Djaballah, Hakim; Kharas, Michael G.

    2014-01-01

    RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds, we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10μM. Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will facilitate novel drug discovery for this class of RBPs. PMID:24912481

  16. Key Learnings from the Endocrine Disruptor Screening Program (EDSP) Tier 1 Rodent Uterotrophic and Hershberger Assays

    PubMed Central

    Marty, M Sue; O'Connor, John C

    2014-01-01

    In 2009, companies began screening compounds using the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP). EDSP has two tiers: Tier 1 includes 11 assays to identify compounds with potential endocrine activity. This article describes two laboratories' experiences conducting Tier 1 uterotrophic and Hershberger assays. The uterotrophic assay detects estrogen receptor agonists through increases in uterine weight. The advantages of the uterotrophic rat models (immature vs. adult ovariectomized) and exposure routes are discussed. Across 29 studies, relative differences in uterine weights in the vehicle control group and 17α-ethynylestradiol–positive control group were reasonably reproducible. The Hershberger assay detects androgen receptor (AR) agonists, antagonists, and 5α-reductase inhibitors through changes in accessory sex tissue (AST) weights. Across 23 studies, AST weights were relatively reproducible for the vehicle groups (baseline), testosterone propionate (TP) groups (androgenic response), and flutamide + TP groups (antiandrogenic response). In one laboratory, one and four compounds were positive in the androgenic and antiandrogenic portions of the assay, respectively. Each compound was also positive for AR binding. In the other laboratory, three compounds showed potential antiandrogenic activity, but each compound was negative for AR binding and did not fit the profile for 5α-reductase inhibition. These compounds induced hepatic enzymes that enhanced testosterone metabolism/clearance, resulting in lower testosterone and decreased capacity to maintain AST weights. The Hershberger androgenic and antiandrogenic performance criteria were generally attainable. Overall, the uterotrophic and Hershberger assays were easily adopted and function as described for EDSP screening, although the mode of action for positive results may not be easily determined. PMID:24515841

  17. 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

  18. 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

  19. Evaluation of the HISCL Anti-Treponema pallidum Assay as a Screening Test for Syphilis.

    PubMed

    An, Jingna; Chen, Qixia; Liu, Qianqian; Rao, Chenli; Li, Dongdong; Wang, Tingting; Tao, Chuanmin; Wang, Lanlan

    2015-07-01

    The resurgence of syphilis in recent years has become a serious threat to public health worldwide, and the serological detection of specific antibodies against Treponema pallidum remains the most reliable method for laboratory diagnosis of syphilis. This study examined the performance of the recently launched HISCL anti-Treponema pallidum (anti-TP) assay as a screening test for syphilis in a high-volume laboratory. The HISCL anti-TP assay was tested in 300 preselected syphilis-positive samples, 704 fresh syphilis-negative samples, 48 preselected potentially interfering samples, and 30 "borderline" samples and was compared head to head with the commercially available Lumipulse G TP-N. In this study, the HISCL anti-TP assay was in perfect agreement with the applied testing algorithms with an overall agreement of 100%, comparable to that of Lumipulse G TP-N (99.63%). The sensitivity and specificity of the HISCL anti-TP assay were 100% (95% confidence interval [CI], 98.42% to 100%) and 100% (95% CI, 99.37% to 100%), respectively. Considering the excellent ease of use and automation, high throughput, and its favorable sensitivity and specificity, the HISCL anti-TP assay may represent a new choice for syphilis screening in high-volume laboratories. PMID:25972403

  20. Tissue spheroid fusion-based in vitro screening assays for analysis of tissue maturation.

    PubMed

    Hajdu, Zoltan; Mironov, Vladimir; Mehesz, Agnes Nagy; Norris, Russell A; Markwald, Roger R; Visconti, Richard P

    2010-12-01

    Organ printing or computer-aided robotic layer-by-layer additive biofabrication of thick three-dimensional (3D) living tissue constructs employing self-assembling tissue spheroids is a rapidly evolving alternative to classic solid scaffold-based approaches in tissue engineering. However, the absence of effective methods of accelerated tissue maturation immediately after bioprinting is the main technological imperative and potential impediment for further progress in the development of this emerging organ printing technology. Identification of the optimal combination of factors and conditions that accelerate tissue maturation ('maturogenic' factors) is an essential and necessary endeavour. Screening of maturogenic factors would be most efficiently accomplished using high-throughput quantitative in vitro tissue maturation assays. We have recently reviewed the formation of solid scaffold-free tissue constructs through the fusion of bioprinted tissue spheroids that have measurable material properties. We hypothesize that the fusion kinetics of these tissue spheroids will provide an efficacious in vitro assay of the level of tissue maturation. We report here the results of experimental testing of two simple quantitative tissue spheroid fusion-based in vitro high-throughput screening assays of tissue maturation: (a) a tissue spheroid envelopment assay; and (b) a tissue spheroid fusion kinetics assay. PMID:20603872

  1. Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.

    PubMed

    Krug, Anne K; Balmer, Nina V; Matt, Florian; Schönenberger, Felix; Merhof, Dorit; Leist, Marcel

    2013-12-01

    Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants. PMID:23670202

  2. Overcoming compound fluorescence in the FLiK screening assay with red-shifted fluorophores.

    PubMed

    Schneider, Ralf; Gohla, Anne; Simard, Jeffrey R; Yadav, Dharmendra B; Fang, Zhizhou; van Otterlo, Willem A L; Rauh, Daniel

    2013-06-01

    In the attempt to discover novel chemical scaffolds that can modulate the activity of disease-associated enzymes, such as kinases, biochemical assays are usually deployed in high-throughput screenings. First-line assays, such as activity-based assays, often rely on fluorescent molecules by measuring a change in the total emission intensity, polarization state, or energy transfer to another fluorescent molecule. However, under certain conditions, intrinsic compound fluorescence can lead to difficult data analysis and to false-positive, as well as false-negative, hits. We have reported previously on a powerful direct binding assay called fluorescent labels in kinases ('FLiK'), which enables a sensitive measurement of conformational changes in kinases upon ligand binding. In this assay system, changes in the emission spectrum of the fluorophore acrylodan, induced by the binding of a ligand, are translated into a robust assay readout. However, under the excitation conditions of acrylodan, intrinsic compound fluorescence derived from highly conjugated compounds complicates data analysis. We therefore optimized this method by identifying novel fluorophores that excite in the far red, thereby avoiding compound fluorescence. With this advancement, even rigid compounds with multiple π-conjugated ring systems can now be measured reliably. This study was performed on three different kinase constructs with three different labeling sites, each undergoing distinct conformational changes upon ligand binding. It may therefore serve as a guideline for the establishment of novel fluorescence-based detection assays. PMID:23672540

  3. Ethical aspects of workplace urine screening for drug abuse.

    PubMed Central

    Forrest, A R

    1997-01-01

    OBJECTIVE: To review the ethical and legal implications of the involvement of medical practitioners in workplace screening for drug misuse. CONCLUSIONS: Workplace screening for drugs of abuse raises many ethical issues. If screening is considered as being part of medical practice with the involvement of occupational health physicians, as suggested by the Faculty of Occupational Medicine, then the ethical requirements of a normal medical consultation are fully applicable. The employee's full and informed consent to the process must be obtained and the employee should have an unfettered right of access to all the relevant records and to the urine sample he/she has provided in the event that he/she wishes to challenge the opinion expressed by the physician. If the process is not part of medical practice then employees should have the same rights as they would have if required to provide intimate body samples in the course of a criminal investigation, given the potentially serious consequences of an erroneous positive finding for their livelihood. PMID:9055156

  4. Newborn Hearing Screening in Neonates Exposed to Psychoactive Drugs

    PubMed Central

    Rocha, Bruna Salazar Castro da; Machado, Márcia Salgado; Zanini, Cláudia Fernandes Costa; Paniz, Tatiana de Carvalho; Menegotto, Isabela Hoffmeister

    2013-01-01

    Introduction In pregnancy, the mother and fetus share body structures based on the maternal organism. Exposure to psychoactive drugs in this period may have repercussions on the baby's hearing. Therefore, it is necessary to investigate this association. Aim Analyze the results of newborn hearing screening (NHS), the occurrence of associated risk factors, and the incidence of hearing loss in newborn exposed to psychoactive drugs during pregnancy. Methods This is an observational retrospective study done from a database analysis. From this database, records were selected about the use of psychoactive drugs by mothers during pregnancy, then the neonates were divide into two groups: the study group (146 babies exposed to drugs) and the control group (500 babies not exposed to drugs). The NHS failure rate, the presence of risk factors for hearing loss, and need for audiological diagnosis were analyzed in both groups. From these variables, absolute frequency and prevalence rates were calculated and the results compared between groups. Results There was no statistically significant difference in the comparison of NHS failure rates between the groups (p = 0.267). The occurrence of risk factors for hearing loss was greater in babies exposed to drugs (p < 0.0001). There was only one diagnosis of hearing loss, which occurred in the control group (p = 0.667). Conclusion The use of psychoactive drugs by mothers during pregnancy did not affect the NHS failure rate of this sample. However, the occurrence of significant risk factors in the study group showed a possible sensitivity of babies exposed to psychoactive drugs during pregnancy. PMID:25992062

  5. Magnetically optimized SERS assay for rapid detection of trace drug-related biomarkers in saliva and fingerprints.

    PubMed

    Yang, Tianxi; Guo, Xiaoyu; Wang, Hui; Fu, Shuyue; Wen, Ying; Yang, Haifeng

    2015-06-15

    New developments in the fields of human healthcare and social security call for the exploration of an easy and on-field method to detect drug-related biomarkers. In this paper, Au nanoparticles dotted magnetic nanocomposites (AMN) modified with inositol hexakisphosphate (IP6) were used as surface-enhanced Raman scattering (SERS) substrate to quickly monitor trace drug-related biomarkers in saliva and to on-site screen a trace drug biomarker in fingerprints. Due to inducing with an external magnet, such substrate presented a huge SERS activity, which has met the sensitivity requirement for assay to detect the drug biomarkers in saliva from the U.S. Substance Abuse and Mental Health Services Administration, and also the limit of detection for drug biomarker in fingerprint reached 100 nM. In addition, this AMN-based SERS assay was successfully conducted using a portable Raman spectrometer, which could be used to on-site and accurately differentiate between the smokers and drug addicts in near future. PMID:25603400

  6. Antiprotozoan lead discovery by aligning dry and wet screening: prediction, synthesis, and biological assay of novel quinoxalinones.

    PubMed

    Martins Alho, Miriam A; Marrero-Ponce, Yovani; Barigye, Stephen J; Meneses-Marcel, Alfredo; Machado Tugores, Yanetsy; Montero-Torres, Alina; Gómez-Barrio, Alicia; Nogal, Juan J; García-Sánchez, Rory N; Vega, María Celeste; Rolón, Miriam; Martínez-Fernández, Antonio R; Escario, José A; Pérez-Giménez, Facundo; Garcia-Domenech, Ramón; Rivera, Norma; Mondragón, Ricardo; Mondragón, Mónica; Ibarra-Velarde, Froylán; Lopez-Arencibia, Atteneri; Martín-Navarro, Carmen; Lorenzo-Morales, Jacob; Cabrera-Serra, Maria Gabriela; Piñero, Jose; Tytgat, Jan; Chicharro, Roberto; Arán, Vicente J

    2014-03-01

    which the individual QSAR outputs are the inputs of the aforementioned fusion approach. Finally, the fusion model was used for the identification of a novel generation of lead-like antiprotozoan compounds by using ligand-based virtual screening of 'available' small molecules (with synthetic feasibility) in our 'in-house' library. A new molecular subsystem (quinoxalinones) was then theoretically selected as a promising lead series, and its derivatives subsequently synthesized, structurally characterized, and experimentally assayed by using in vitro screening that took into consideration a battery of five parasite-based assays. The chemicals 11(12) and 16 are the most active (hits) against apicomplexa (sporozoa) and mastigophora (flagellata) subphylum parasites, respectively. Both compounds depicted good activity in every protozoan in vitro panel and they did not show unspecific cytotoxicity on the host cells. The described technical framework seems to be a promising QSAR-classifier tool for the molecular discovery and development of novel classes of broad-antiprotozoan-spectrum drugs, which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of protozoan illnesses. PMID:24513185

  7. Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs

    PubMed Central

    Sewing, Sabine; Boess, Franziska; Moisan, Annie; Bertinetti-Lapatki, Cristina; Minz, Tanja; Hedtjaern, Maj; Tessier, Yann; Schuler, Franz; Singer, Thomas; Roth, Adrian B.

    2016-01-01

    Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development. PMID:27442522

  8. Establishment of a Predictive In Vitro Assay for Assessment of the Hepatotoxic Potential of Oligonucleotide Drugs.

    PubMed

    Sewing, Sabine; Boess, Franziska; Moisan, Annie; Bertinetti-Lapatki, Cristina; Minz, Tanja; Hedtjaern, Maj; Tessier, Yann; Schuler, Franz; Singer, Thomas; Roth, Adrian B

    2016-01-01

    Single stranded oligonucleotides (SSO) represent a novel therapeutic modality that opens new space to address previously undruggable targets. In spite of their proven efficacy, the development of promising SSO drug candidates has been limited by reported cases of SSO-associated hepatotoxicity. The mechanisms of SSO induced liver toxicity are poorly understood, and up to now no preclinical in vitro model has been established that allows prediction of the hepatotoxicity risk of a given SSO. Therefore, preclinical assessment of hepatic liability currently relies on rodent studies that require large cohorts of animals and lengthy protocols. Here, we describe the establishment and validation of an in vitro assay using primary hepatocytes that recapitulates the hepatotoxic profile of SSOs previously observed in rodents. In vitro cytotoxicity upon unassisted delivery was measured as an increase in extracellular lactate dehydrogenase (LDH) levels and concomitant reduction in intracellular glutathione and ATP levels after 3 days of treatment. Furthermore, toxic, but not safe, SSOs led to an increase in miR-122 in cell culture supernatants after 2 days of exposure, revealing the potential use of miR122 as a selective translational biomarker for detection of SSO-induced hepatotoxicity. Overall, we have developed and validated for the first time a robust in vitro screening assay for SSO liver safety profiling which allows rapid prioritization of candidate molecules early on in development. PMID:27442522

  9. 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

  10. Human Papillomavirus Assays and Cytology in Primary Cervical Screening of Women Aged 30 Years and Above

    PubMed Central

    Rebolj, Matejka; Bonde, Jesper; Preisler, Sarah; Ejegod, Ditte; Rygaard, Carsten; Lynge, Elsebeth

    2016-01-01

    In women aged ≥30 years, Human Papillomavirus testing will replace cytology for primary cervical screening. We compared Hybrid Capture 2 (HC2), cobas, CLART, and APTIMA HPV assays with cytology on 2869 SurePath samples from women undergoing routine screening at 30–65 years in Copenhagen, Denmark. Women with cytological abnormalities were managed according to routine recommendations, with 92% completeness. Those with cytology-normal/HPV-positive samples (on any of the four assays) were invited for repeated cytology and HPV testing in 1.5 year, and 58% had additional testing. HPV testing detected more ≥CIN3 than cytology (HC2: 35, cobas, CLART: 37, APTIMA: 34, cytology: 31), although statistically the differences were not significant. Cobas and CLART detected significantly more ≥CIN2 than cytology (cobas, CLART: 49, cytology: 39). The proportion of women with false-positive test results (positive test results without ≥CIN3) varied between 3.3% with cytology and 14.9% with cobas. All HPV assays led to significantly more false-positive tests, whereas compared to HC2 cobas and CLART were associated with a significantly higher and APTIMA with a significantly lower proportion. Detection of CIN1 was particularly increased for the three DNA assays. With APTIMA combined with cytological triage, about 20% more women were referred for colposcopy than with cytology screening. With the three DNA assays, the increase was ≥50%. The number of women with repeated testing was twice as high with APTIMA and almost five times as high with cobas compared to cytology. To our knowledge, Horizon was the only study set in routine practice that compared more than two HPV assays in the same women while also ascertaining the histological status of women with normal cytology/HPV-positive test results. HPV-based screening of Danish women aged 30–65 detected more high-grade CIN but decreased the screening specificity, and increased the demand for additional testing. PMID:26789267

  11. 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

  12. Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays.

    PubMed

    Lee, Jonathan A; Shinn, Paul; Jaken, Susan; Oliver, Sarah; Willard, Francis S; Heidler, Steven; Peery, Robert B; Oler, Jennifer; Chu, Shaoyou; Southall, Noel; Dexheimer, Thomas S; Smallwood, Jeffrey; Huang, Ruili; Guha, Rajarshi; Jadhav, Ajit; Cox, Karen; Austin, Christopher P; Simeonov, Anton; Sittampalam, G Sitta; Husain, Saba; Franklin, Natalie; Wild, David J; Yang, Jeremy J; Sutherland, Jeffrey J; Thomas, Craig J

    2015-01-01

    Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS) pharmaceutical collection (NPC) is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD) phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (AID 1117321). Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben. PMID:26177200

  13. Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays

    PubMed Central

    Oliver, Sarah; Willard, Francis S.; Heidler, Steven; Peery, Robert B.; Oler, Jennifer; Chu, Shaoyou; Southall, Noel; Dexheimer, Thomas S.; Smallwood, Jeffrey; Huang, Ruili; Guha, Rajarshi; Jadhav, Ajit; Cox, Karen; Austin, Christopher P.; Simeonov, Anton; Sittampalam, G. Sitta; Husain, Saba; Franklin, Natalie; Wild, David J.; Yang, Jeremy J.; Sutherland, Jeffrey J.; Thomas, Craig J.

    2015-01-01

    Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS) pharmaceutical collection (NPC) is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD) phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (AID 1117321). Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben. PMID:26177200

  14. Quantification of cell viability and rapid screening anti-cancer drug utilizing nanomechanical fluctuation.

    PubMed

    Wu, Shangquan; Liu, Xiaoli; Zhou, Xiarong; Liang, Xin M; Gao, Dayong; Liu, Hong; Zhao, Gang; Zhang, Qingchuan; Wu, Xiaoping

    2016-03-15

    Cancer is a serious threat to human health. Although numerous anti-cancer drugs are available clinically, many have shown toxic side effects due to poor tumor-selectivity, and reduced effectiveness due to cancers rapid development of resistance to treatment. The development of new highly efficient and practical methods to quantify cell viability and its change under drug treatment is thus of significant importance in both understanding of anti-cancer mechanism and anti-cancer drug screening. Here, we present an approach of utilizing a nanomechanical fluctuation based highly sensitive microcantilever sensor, which is capable of characterizing the viability of cells and quantitatively screening (within tens of minutes) their responses to a drug with the obvious advantages of a rapid, label-free, quantitative, noninvasive, real-time and in-situ assay. The microcantilever sensor operated in fluctuation mode was used in evaluating the paclitaxel effectiveness on breast cancer cell line MCF-7. This study demonstrated that the nanomechanical fluctuations of the microcantilever sensor are sensitive enough to detect the dynamic variation in cellular force which is provided by the cytoskeleton, using cell metabolism as its energy source, and the dynamic instability of microtubules plays an important role in the generation of the force. We propose that cell viability consists of two parts: biological viability and mechanical viability. Our experimental results suggest that paclitaxel has little effect on biological viability, but has a significant effect on mechanical viability. This new method provides a new concept and strategy for the evaluation of cell viability and the screening of anti-cancer drugs. PMID:26406457

  15. Application of luciferase assay for ATP to antimicrobial drug susceptibility

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Vellend, H.; Tuttle, S. A.; Barza, M. J.; Weinstein, L. (Inventor)

    1977-01-01

    The susceptibility of bacteria, particularly those derived from body fluids, to antimicrobial agents is determined in terms of an ATP index measured by culturing a bacterium in a growth medium. The amount of ATP is assayed in a sample of the cultured bacterium by measuring the amount of luminescent light emitted when the bacterial ATP is reacted with a luciferase-luciferin mixture. The sample of the cultured bacterium is subjected to an antibiotic agent. The amount of bacterial adenosine triphosphate is assayed after treatment with the antibiotic by measuring the luminescent light resulting from the reaction. The ATP index is determined from the values obtained from the assay procedures.

  16. 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

  17. A TR-FRET-based functional assay for screening activators of CARM1.

    PubMed

    Zeng, Hao; Wu, Jiacai; Bedford, Mark T; Sbardella, Gianluca; Hoffmann, F Michael; Bi, Kun; Xu, Wei

    2013-05-10

    Epigenetics is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR-FRET assay uses MCF7 cells expressing GFP-PABP1 fusion protein through BacMam gene delivery system, methyl-PABP1 specific antibody, and terbium-labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions. PMID:23585185

  18. 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

  19. Application of the E-screen assay to test for oestrogenically active substances in swine feed.

    PubMed

    Bitsch, N; Körner, W; Postupka, S; Brunn, H

    2001-12-01

    A pig breeder in central Hesse (Germany) noticed the occurrence of enlarged vulvae in female piglets. Intoxication with oestrogenically active substances by contamination of two feed mixes ingested by the mother sows appeared to be a possible cause. Using a combined technique of the DFG analytical method S19 and the E-screen assay, two feed samples were found to contain powerful oestrogenically active compounds. By co-incubation with the anti-oestrogen tamoxifen it could be clearly demonstrated that the oestrogenic activity was mediated by the oestrogen receptor. These results demonstrate that use of the E-screen assay in combination with the DFG analytical method S19 provides a simple and readily usable prescreening method for the routine detection of oestrogenically active compounds in animal feed. The results from the E-screen assay show that the sows ingested 10-80 microg oestradiol equivalents per day in their feed. Because of the bioavailability of these substances, the oestrogenic active compounds seem to be transferred into the milk and passed to the piglets via suckling. The milk of the dam appears to contain this substance in biologically active form and at such high concentrations that the female piglets had enlarged vulvae. PMID:11906561

  20. Tumorsphere as an effective in vitro platform for screening anti-cancer stem cell drugs

    PubMed Central

    Lee, Che-Hsin; Yu, Cheng-Chia; Wang, Bing-Yen; Chang, Wen-Wei

    2016-01-01

    Cancer stem cells (CSCs) are a sub-population of cells within cancer tissues with tumor initiation, drug resistance and metastasis properties. CSCs also have been considered as the main cause of cancer recurrence. Targeting CSCs have been suggested as the key for successful treatment against cancer. Tumorsphere cultivation is based on culturing cancer cells onto ultralow attachment surface in serum-free media under the supplementation with growth factors such as epidermal growth factor and basic fibroblast growth factor. Tumorsphere cultivation is widely used to analyze the self-renewal capability of CSCs and to enrich these cells from bulk cancer cells. This method also provides a reliable platform for screening potential anti-CSC agents. The in vitro anti-proliferation activity of potential agents selected from tumorsphere assay is more translatable into in vivo anti-tumorigenic activity compared with general monolayer culture. Tumorsphere assay can also measure the outcome of clinical trials for potential anti-cancer agents. In addition, tumorsphere assay may be a promising strategy in the innovation of future cancer therapeutica and may help in the screening of anti-cancer small-molecule chemicals. PMID:26527320

  1. Screening pharmaceuticals for possible carcinogenic effects: initial positive results for drugs not previously screened

    PubMed Central

    Friedman, Gary D.; Udaltsova, Natalia; Chan, James; Quesenberry, Charles P; Habel, Laurel A.

    2010-01-01

    Objective We screened commonly used prescription drugs for possible carcinogenic effects. Methods In a large health care program we identified 105 commonly used drugs, not previously screened. Recipients were followed for up to 12½ years for incident cancer. Nested case-control analyses of 55 cancer sites and all combined included up to ten matched controls per case, with lag of at least two years between drug dispensing and cancer. Positive associations entailed a relative risk (RR) of 1.50, with p≤ 0.01 and higher risk for three or more, than for one prescription. Evaluation included further analyses, searches of the literature, and clinical judgment. Results There were 101 associations of interest for 61 drugs. Sixty-six associations were judged to have involved substantial confounding. We found evidence that of the remaining 35, the following associations may not be due to chance: sulindac with gallbladder cancer and leukemia, hyoscyamine with non-Hodgkin lymphoma, nortriptyline with esophageal and hepatic cancer, oxazepam with lung cancer, both fluoxetine and paroxetine with testicular cancer, hydrochlorothiazide with renal and lip cancer, and nifedipine with lip cancer. Conclusions These preliminary findings suggest that further studies are indicated regarding sulindac, hyoscyamine, nortriptyline, oxazepam, fluoxetine, paroxetine, hydrochlorothiazide and nifedipine. PMID:19582585

  2. 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

  3. 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

  4. 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.

  5. Screening and identification of inhibitors against influenza A virus from a US drug collection of 1280 drugs.

    PubMed

    An, Liwei; Liu, Rui; Tang, Wei; Wu, Jian-Guo; Chen, Xulin

    2014-09-01

    Infection with influenza A virus is still a global concern since it causes significant mortality, morbidity and economic loss. New burst pandemics and rapid emergence of drug-resistance strains in recent years call for novel antiviral therapies. One promising way to overcome this problem is searching new inhibitors among thousands of drugs approved in the clinic for the treatment of different diseases or approved to be safe by clinical trials. In the present work, a collection of 1280 compounds, most of which have been clinically used in human or animal, were screened for anti-influenza activity and 41 hits (SI>4.0) were obtained. Next the 18 hit compounds with SI >10.0 were tested for antiviral activity against 7 other influenza virus strains in canine-originated MDCK cells, 9 compounds exhibited broad antiviral spectrum. The antiviral effects of the 9 compounds were also confirmed in human-originated A549 cells and chicken-originated DF1 cells, by infectious virus yield reduction assay and indirect immunofluorescent assay. Results from the time of addition assay showed that the 9 candidates impaired different stages of influenza virus life cycle, indicating they are novel inhibitors with different mechanisms compared with the existing M2 ion-channel blockers or neuraminidase (NA) inhibitors. Taken together, our findings provide 9 novel drug candidates for the treatment of influenza virus infection. Further mechanism of action study of these inhibitors may lead to the discovery of new anti-influenza targets and structure-activity relationship (SAR) study can be initiated to improve the efficacy of these new classes of influenza inhibitors. PMID:24971493

  6. Novel screening method for potential skin-whitening compounds by a luciferase reporter assay.

    PubMed

    Shirasugi, Ichiro; Sakakibara, Yoichi; Yamasaki, Masao; Nishiyama, Kazuo; Matsui, Takashi; Liu, Ming-Cheh; Suiko, Masahito

    2010-01-01

    Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3'-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds. PMID:21071833

  7. High-content screening assay for identification of chemicals impacting spontaneous activity in zebrafish embryos.

    PubMed

    Raftery, Tara D; Isales, Gregory M; Yozzo, Krystle L; Volz, David C

    2014-01-01

    Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration-response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3-100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA's ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate-both potent avermectins-abolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants. PMID:24328182

  8. A novel immunoassay for quantitative drug abuse screening in serum.

    PubMed

    Schumacher, Sarah; Seitz, Harald

    2016-09-01

    An immunoassay was established which enables a reliable quantification of serological drug samples. The assay is based on a competitive ELISA. In total nine drugs (amphetamine, methamphetamine, 3,4-methylenedioxy-methamphetamine (MDMA), tetrahydrocannabinol (THC), phencyclidine (PCP), methadone, morphine, cocaine and benzoylecgonine) were tested. All reagents had to pass through a stringent validation process. Within the established test for three out of the nine drugs no cross-reactivity with any tested compounds, e.g. serum, other antibodies or chemically related molecules was detectable for the tested antibodies. Furthermore, a sensitive and selective detection was possible, even in the presence of up to 9 drugs or of various anti-drug antibodies. After exclusion of cross-reactivities antibodies against three drugs (methadone, MDMA, benzoylecgonine) were validated, which allowed a specific and sensitive quantification. For the competitive measurements CVs in the range of 2-17% could be reached with LLOQs of 10ng/mL and LODs of 150ng/mL for methadone, 250ng/mL for MDMA and 400ng/mL for benzoylecgonine. Anonymized serum samples (n=10) provided by the office of criminal investigation Berlin were analyzed for verification purposes. Evaluation of these data showed a correlation (CV) of ≈0.9 with standard GC-MS methods. A miniaturization on microarray was possible by using the anti-MDMA antibody for the detection of MDMA in serum. The microarray increased the through-put drastically and enabled the simultaneous quantification of various drugs. PMID:27343723

  9. Validation of FRET Assay for the Screening of Growth Inhibitors of Escherichia coli Reveals Elongasome Assembly Dynamics

    PubMed Central

    van der Ploeg, René; Goudelis, Spyridon Theodoros; den Blaauwen, Tanneke

    2015-01-01

    The increase in antibiotic resistant bacteria demands the development of new antibiotics against preferably new targets. The common approach is to test compounds for their ability to kill bacteria or to design molecules that inhibit essential protein activities in vitro. In the first case, the mode of action of the drug is unknown and in the second case, it is not known whether the compound will pass the impermeable barrier of the bacterial envelope. We developed an assay that detects the target of a compound, as well as its ability to pass the membrane(s) simultaneously. The Escherichia coli cytoskeletal protein MreB recruits protein complexes (elongasomes) that are essential for cell envelope growth. An in cell Förster Resonance Energy Transfer (FRET) assay was developed to detect the interaction between MreB molecules and between MreB and the elongasome proteins RodZ, RodA and PBP2. Inhibition of the polymerization of MreB by S-(3,4-dichlorobenzyl) isothiourea (A22) or of the activity of PBP2 by mecilinam resulted in loss or reduction of all measured interactions. This suggests that the interactions between the elongasome proteins are governed by a combination of weak affinities and substrate availability. This validated in cell FRET assay can be used to screen for cell envelope growth inhibitors. PMID:26263980

  10. 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

  11. Investigation of the incidence of "undesirable" molecular moieties for high-throughput screening compound libraries in marketed drug compounds.

    PubMed

    Axerio-Cilies, Peter; Castañeda, Ivan P; Mirza, Amin; Reynisson, Jóhannes

    2009-03-01

    A database of 1070 marketed drug compounds was compiled and analyzed in order to assess the occurrence of moieties described in the literature as "undesirable" for high-throughput screening compound libraries due to their ability to perturb assay formats. The study revealed a total of 277 compounds, 26% of the database, contained at least one of the moieties. As some of the drug compounds contained more than one "undesirable" moiety, the total number was 352. Electrophilic reactive groups, particularly aliphatic esters, were the most abundant type with 55% of the total. Half of the drug compounds incorporating the "undesirable" moieties were synthetic organic molecules. These findings suggest that "undesirable" moieties do not pose a major hindrance during clinical trials, the most expensive phase of drug development. In addition, their early elimination in the preclinical stage excludes large regions of known drug space due to the reliance on biochemical and cell-based assays. In general, it can be concluded that compounds with "undesirable" moieties should not simply be eliminated from compound screening libraries but rather be flagged as potentially problematic. A possible solution is to segregate the compounds containing suspect moieties and screen them when deemed appropriate. PMID:18692938

  12. Optical diagnostics of osteoblast cells and osteogenic drug screening

    NASA Astrophysics Data System (ADS)

    Kolanti, Elayaraja; Veerla, Sarath C.; Khajuria, Deepak K.; Roy Mahapatra, D.

    2016-02-01

    Microfluidic device based diagnostics involving optical fibre path, in situ imaging and spectroscopy are gaining importance due to recent advances in diagnostics instrumentation and methods, besides other factors such as low amount of reagent required for analysis, short investigation times, and potential possibilities to replace animal model based study in near future. It is possible to grow and monitor tissues in vitro in microfluidic lab-on-chip. It may become a transformative way of studying how cells interact with drugs, pathogens and biomaterials in physiologically relevant microenvironments. To a large extent, progress in developing clinically viable solutions has been constrained because of (i) contradiction between in vitro and in vivo results and (ii) animal model based and clinical studies which is very expensive. Our study here aims to evaluate the usefulness of microfluidic device based 3D tissue growth and monitoring approach to better emulate physiologically and clinically relevant microenvironments in comparison to conventional in vitro 2D culture. Moreover, the microfluidic methodology permits precise high-throughput investigations through real-time imaging while using very small amounts of reagents and cells. In the present study, we report on the details of an osteoblast cell based 3D microfluidic platform which we employ for osteogenic drug screening. The drug formulation is functionalized with fluorescence and other biomarkers for imaging and spectroscopy, respectively. Optical fibre coupled paths are used to obtain insight regarding the role of stress/flow pressure fluctuation and nanoparticle-drug concentration on the osteoblast growth and osteogenic properties of bone.

  13. Genetic toxicity assessment: employing the best science for human safety evaluation part III: the comet assay as an alternative to in vitro clastogenicity tests for early drug candidate selection.

    PubMed

    Witte, Irene; Plappert, Ulla; de Wall, Hartmut; Hartmann, Andreas

    2007-05-01

    Early screening of drug candidates for genotoxicity typically includes an analysis for mutagenicity in bacteria and for clastogenicity in cultured mammalian cells. In addition, in recent years, an early assessment of photogenotoxicity potential has become increasingly important. Also, for screening purposes, expert computer systems can be used to identify structural alerts. In cases where structural alerts are identified, mutagenicity testing limited to bacteria can be conducted. The sequence of computer-aided analysis and limited testing using bacteria allows for screening a comparatively large number of drug candidates. In contrast, considerably more resources, in terms of supplies, technical time, and the amount of a test substance needed, are required when screening for clastogenic activity in mammalian cells. In addition, the relatively large percentage of false positive results for rodent carcinogenicity associated with clastogenicity assays is of considerable concern. As a consequence, mammalian cell-based alternatives to clastogenicity assays are needed for early screening of mammalian genotoxicity. The comet assay is a relatively fast, simple, and sensitive technique for the analysis of DNA damage in mammalian cells. This assay seems especially useful for screening purposes because false positives associated with excessive toxicity appear to occur less frequently, only relatively small amounts of a test compound are needed, and certain steps of the test procedure can be automated. Therefore, the in vitro comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates. PMID:17204584

  14. A Systematic Screen of FDA-Approved Drugs for Inhibitors of Biological Threat Agents

    PubMed Central

    Madrid, Peter B.; Chopra, Sidharth; Manger, Ian D.; Gilfillan, Lynne; Keepers, Tiffany R.; Shurtleff, Amy C.; Green, Carol E.; Iyer, Lalitha V.; Dilks, Holli Hutcheson; Davey, Robert A.; Kolokoltsov, Andrey A.; Carrion, Ricardo; Patterson, Jean L.; Bavari, Sina; Panchal, Rekha G.; Warren, Travis K.; Wells, Jay B.; Moos, Walter H.; Burke, RaeLyn L.; Tanga, Mary J.

    2013-01-01

    Background The rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since currently approved drugs already have well-established safety and pharmacokinetic profiles in patients, as well as manufacturing and distribution networks. Therefore, approved drugs could rapidly be made available for a new indication in an emergency. Methodology/Principal Findings A large systematic effort to determine whether existing drugs can be used against high containment bacterial and viral pathogens is described. We assembled and screened 1012 FDA-approved drugs for off-label broad-spectrum efficacy against Bacillus anthracis; Francisella tularensis; Coxiella burnetii; and Ebola, Marburg, and Lassa fever viruses using in vitro cell culture assays. We found a variety of hits against two or more of these biological threat pathogens, which were validated in secondary assays. As expected, antibiotic compounds were highly active against bacterial agents, but we did not identify any non-antibiotic compounds with broad-spectrum antibacterial activity. Lomefloxacin and erythromycin were found to be the most potent compounds in vivo protecting mice against Bacillus anthracis challenge. While multiple virus-specific inhibitors were identified, the most noteworthy antiviral compound identified was chloroquine, which disrupted entry and replication of two or more viruses in vitro and protected mice against Ebola virus challenge in vivo. Conclusions/Significance The feasibility of repurposing existing drugs to face novel threats is demonstrated and this represents the first effort to apply this approach to high containment bacteria and viruses. PMID:23577127

  15. Use of external metabolizing systems when testing for endocrine disruption in the T-screen assay

    SciTech Connect

    Taxvig, Camilla Olesen, Pelle Thonning; Nellemann, Christine

    2011-02-01

    Although, it is well-established that information on the metabolism of a substance is important in the evaluation of its toxic potential, there is limited experience with incorporating metabolic aspects into in vitro tests for endocrine disrupters. The aim of the current study was a) to study different in vitro systems for biotransformation of ten known endocrine disrupting chemicals (EDs): five azole fungicides, three parabens and 2 phthalates, b) to determine possible changes in the ability of the EDs to bind and activate the thyroid receptor (TR) in the in vitro T-screen assay after biotransformation and c) to investigate the endogenous metabolic capacity of the GH3 cells, the cell line used in the T-screen assay, which is a proliferation assay used for the in vitro detection of agonistic and antagonistic properties of compounds at the level of the TR. The two in vitro metabolizing systems tested the human liver S9 mix and the PCB-induced rat microsomes gave an almost complete metabolic transformation of the tested parabens and phthalates. No marked difference the effects in the T-screen assay was observed between the parent compounds and the effects of the tested metabolic extracts. The GH3 cells themselves significantly metabolized the two tested phthalates dimethyl phthalate (DMP) and diethyl phthalate (DEP). Overall the results and qualitative data from the current study show that an in vitro metabolizing system using liver S9 or microsomes could be a convenient method for the incorporation of metabolic and toxicokinetic aspects into in vitro testing for endocrine disrupting effects.

  16. A three-stage biophysical screening cascade for fragment-based drug discovery.

    PubMed

    Mashalidis, Ellene H; Śledź, Paweł; Lang, Steffen; Abell, Chris

    2013-11-01

    This protocol describes the screening of a library of low-molecular-weight compounds (fragments) using a series of biophysical ligand-binding assays. Fragment-based drug discovery (FBDD) has emerged as a successful method to design high-affinity ligands for biomacromolecules of therapeutic interest. It involves detecting relatively weak interactions between the fragments and a target macromolecule using sensitive biophysical techniques. These weak binders provide a starting point for the development of inhibitors with submicromolar affinity. Here we describe an efficient fragment screening cascade that can identify binding fragments (hits) within weeks. It is divided into three stages: (i) preliminary screening using differential scanning fluorimetry (DSF), (ii) validation by NMR spectroscopy and (iii) characterization of binding fragments by isothermal titration calorimetry (ITC) and X-ray crystallography. Although this protocol is readily applicable in academic settings because of its emphasis on low cost and medium-throughput early-stage screening technologies, the core principle of orthogonal validation makes it robust enough to meet the quality standards of an industrial laboratory. PMID:24157549

  17. Screening applications in drug discovery based on microfluidic technology.

    PubMed

    Eribol, P; Uguz, A K; Ulgen, K O

    2016-01-01

    Microfluidics has been the focus of interest for the last two decades for all the advantages such as low chemical consumption, reduced analysis time, high throughput, better control of mass and heat transfer, downsizing a bench-top laboratory to a chip, i.e., lab-on-a-chip, and many others it has offered. Microfluidic technology quickly found applications in the pharmaceutical industry, which demands working with leading edge scientific and technological breakthroughs, as drug screening and commercialization are very long and expensive processes and require many tests due to unpredictable results. This review paper is on drug candidate screening methods with microfluidic technology and focuses specifically on fabrication techniques and materials for the microchip, types of flow such as continuous or discrete and their advantages, determination of kinetic parameters and their comparison with conventional systems, assessment of toxicities and cytotoxicities, concentration generations for high throughput, and the computational methods that were employed. An important conclusion of this review is that even though microfluidic technology has been in this field for around 20 years there is still room for research and development, as this cutting edge technology requires ingenuity to design and find solutions for each individual case. Recent extensions of these microsystems are microengineered organs-on-chips and organ arrays. PMID:26865904

  18. Screening and quantitative determination of drugs of abuse in diluted urine by UPLC-MS/MS.

    PubMed

    Hegstad, Solfrid; Hermansson, Sigurd; Betnér, Ingvar; Spigset, Olav; Falch, Berit Margrethe Hasle

    2014-02-01

    The purpose of this work was to develop and evaluate a fast, robust and specific UPLC-MS/MS screening platform for the determination and quantification of a variety of commonly used drugs of abuse in urine, i.e. a high-throughput quantitative analysis. Substances in the drug classes opioids, central nervous system stimulants and benzodiazepines and related agents were included in addition to cannabis and pregabalin, a total of 35 different analytes. Based on the concentrations and the physico-chemical properties of the substances, three UPLC-MS/MS methods were developed in parallel. Prior to analysis, sample preparation consisted of two different simple dilutions with 60 and 100 μL urine, respectively, using a Tecan Freedom Evo pipetting robot platform. A Waters Xevo TQ-S tandem quadrupole mass spectrometer coupled to a Waters I-class UPLC was used for quantitative analysis of one quantitative and one qualifying MRM transition for each analyte, except for tramadol for which the metabolite O-desmethyl-tramadol was included in the MRM method to confirm tramadol identity. Deuterated analogs were included as internal standards. The between-assay relative standard deviations varied from 2% to 11% and the limits of quantification were in the range 1-200 ng/mL for the various analytes. After development and initial testing, the method has been successfully implemented and routinely used at our hospital for quantitative screening of drugs of abuse in more than 35,000 urinary samples. PMID:24413020

  19. Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

    PubMed Central

    Creek, Darren J.; Nijagal, Brunda; Kim, Dong-Hyun; Rojas, Federico; Matthews, Keith R.

    2013-01-01

    In vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening. PMID:23571546

  20. Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening.

    PubMed

    Gómez-Lechón, María José; Tolosa, Laia

    2016-09-01

    Drug-induced liver injury (DILI) is a frequent cause of failure in both clinical and post-approval stages of drug development, and poses a key challenge to the pharmaceutical industry. Current animal models offer poor prediction of human DILI. Although several human cell-based models have been proposed for the detection of human DILI, human primary hepatocytes remain the gold standard for preclinical toxicological screening. However, their use is hindered by their limited availability, variability and phenotypic instability. In contrast, pluripotent stem cells, which include embryonic and induced pluripotent stem cells (iPSCs), proliferate extensively in vitro and can be differentiated into hepatocytes by the addition of soluble factors. This provides a stable source of hepatocytes for multiple applications, including early preclinical hepatotoxicity screening. In addition, iPSCs also have the potential to establish genotype-specific cells from different individuals, which would increase the predictivity of toxicity assays allowing more successful clinical trials. Therefore, the generation of human hepatocyte-like cells derived from pluripotent stem cells seems to be promising for overcoming limitations of hepatocyte preparations, and it is expected to have a substantial repercussion in preclinical hepatotoxicity risk assessment in early drug development stages. PMID:27325232

  1. 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

  2. A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α

    PubMed Central

    van der Wel, Tom; Janssen, Freek J.; Baggelaar, Marc P.; Deng, Hui; den Dulk, Hans; Overkleeft, Herman S.; van der Stelt, Mario

    2015-01-01

    The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors. PMID:25684760

  3. Comparison of three assays for genetic effects of antineoplastic drugs on cancer patients and their nurses

    SciTech Connect

    Krepinsky, A. ); Bryant, D.W.; Davison, L.; McCalla, D.R. ); Young, B. ); Heddle, J. ); Douglas, G. ); Michalko, K. )

    1990-01-01

    Three assays have been compared for their ability to detect genetic damage caused by antineoplastic drugs in cancer patients and possible damage in the nurses who administered these drugs. The assays were sister chromatid exchanges (SCE) and chromosomal aberrations in peripheral blood lymphocytes, and the Salmonella/mammalian microsome assay on urine. Three comparisons were made: (1) patients before versus after treatment; (2) the administering nurses immediately after their work period versus after a few days off that followed (work and off-work); (3) the exposed nurses versus other nurses who did not administer antineoplastic drugs (controls). The SCE assay did not distinguish between the work and off-work samples in either the exposed or control nurses. Chromosomal aberration was the only assay which showed significant difference between the two samples of the exposed nurses and, consequently, between the exposed and control nurses. There is no evidence that the increase was connected to occupational exposure.

  4. 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

  5. Drug cytotoxicity and signaling pathway analysis with three-dimensional tumor spheroids in a microwell-based microfluidic chip for drug screening.

    PubMed

    Chen, Yongli; Gao, Dan; Liu, Hongxia; Lin, Shuo; Jiang, Yuyang

    2015-10-22

    Currently, there has been a growing need for developing in vitro models to better reflect organism response to chemotherapy at tissue level. For this reason, a microfluidic platform was developed for mimicking physiological microenvironment of solid tumor with multicellular tumor spheroids (MTS) for anticancer drug screening. Importantly, the power of this system over traditional systems is that it is simple to operate and high integration in a more physiologically relevant context. As a proof of concept, long-term MTS cultures with uniform structure were realized on the microfluidic based platform. The response of doxorubicin and paclitaxel on different types of spheroids were simultaneously performed by in situ Live/Dead fluorescence stain to provide spatial distribution of dead cells as well as cytotoxicity information. In addition, the established platform combined with microplate reader was capable to determine the cytotoxicity of different sized MTS, showing a more powerful tool than cell staining examination at the end-point of assay. The HCT116 spheroids were then lysed on chip followed by signaling transduction pathway analysis. To our knowledge, the on chip drug screening study is the first to address the drug susceptibility testing and the offline detailed drug signaling pathway analysis combination on one system. Thus, this novel microfluidic platform provides a useful tool for drug screening with tumor spheroids, which is crucial for drug discovery and development. PMID:26526913

  6. 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

  7. Identification of APC mutations and evaluation of their expression level using a functional screening assay

    SciTech Connect

    Varesco, L.; Gismondi, V.; Bafico, A.

    1994-09-01

    A functional screen for chain-terminating mutations in the APC gene recently has been developed. It is based on the PCR and cloning of a segment of the gene in-frame with a colorimetric marker gene (lacz) followed by screening for the level of activity of the marker polypeptide (beta-galactosidase). This method scores colony number with different blue colors that are produced by bacteria containing normal and mutant APC segments. In the present work this method was used to screen the entire APC coding region by using eight primer pairs. DNA segments with known APC mutations at different positions in the gene were used as controls and were clearly identifiable with this assay. In addition, the entire APC coding region has been examined in 21 APC patients in whom PCR-SSCP did not identify an APC mutation. Novel mutations (n=14) were identified by the blue/white assay and were all confirmed by sequence analysis. This method also was used to quantitate the expression of paternal and maternal APC alleles taking advantage of an RsaI site polymorphism at position 1458 in a small number of informative individuals. Differential expression of some known mutant APC mRNAs was observed.

  8. Midkine inhibitors: application of a simple assay procedure to screening of inhibitory compounds

    PubMed Central

    2010-01-01

    Background Midkine is a heparin-binding cytokine and is involved in etiology of various diseases. Thus, midkine inhibitors are expected to be helpful in treatment of many diseases. Methods We developed a simple assay for midkine activity based on midkine-dependent migration of osteblastic cells. Midkine inhibitors were searched as materials that inhibit this midkine activity. To develop peptides that inhibit midkine activity, we constructed models in which C-terminal half of midkine interacted with α4β1-integrin. Low molecular weight compounds which are expected to bind to midkine with high affinity were searched by in silico screening with the aid of Presto-X2 program. Results Among peptides in putative binding sites of midkine and the integrin, a peptide derived from β1-integrin and that derived from the first β sheet of the C-terminal half of midkine significantly inhibited midkine activity. Two low molecular weight compounds found by in silico screening exhibited no toxicity to target cells, but inhibited midkine activity. They are trifluoro compounds: one (PubChem 4603792) is 2-(2,6-dimethylpiperidin-1-yl)-4-thiophen-2-yl-6-(trifluoromethy)pyrimidine, and the other has a related structure. Conclusions The assay procedure is helpful in screening midkine inhibitors. All reagents described here might become mother material to develop clinically effective midkine inhibitors. PMID:20565917

  9. Identification of antifungal niphimycin from Streptomyces sp. KP6107 by screening based on adenylate kinase assay.

    PubMed

    Kim, Hye Yoon; Kim, Jeong Do; Hong, Jin Sung; Ham, Jong Hyun; Kim, Beom Seok

    2013-07-01

    Microbial culture extracts are used for natural product screening to find antifungal lead compounds. A microbial culture extract library was constructed using 343 actinomycete isolates to examine the value of the adenylate kinase (AK) assay for screening to identify antifungal metabolites that disrupt cell integrity in plant pathogenic fungi. A culture extract of Streptomyces sp. strain KP6107 lysed cells of Fusarium oxysporum f.sp. lycopersici which resulted in high AK activity. The active ingredient N-1 was purified from the culture extract using various chromatographic procedures and identified to be the guanidyl-polyol macrolide antibiotic, niphimycin, which is a potent fungal cell membrane disruptor. Niphimycin showed broad-spectrum antifungal activity against Alternaria mali, Aspergillus oryzae, Colletotrichum coccodes, Colletotrichum gloeosporioides, Cercospora canescens, Cylindrocarpon destructans, F. oxysporum f.sp. cucumerinum, F. oxysporum f.sp. lycopersici, and Rhizoctonia solani at concentrations of 8-64 µg ml(-1). Anthracnose development in pepper plants was completely inhibited by treatment with 50 µg ml(-1) niphimycin, which was as effective as chlorothalonil. These results show that the AK assay is an efficient and selective tool in screening for cell membrane/wall disruptors of plant pathogenic fungi. PMID:22915202

  10. Development of HTS Assays and Pilot Screen for Inhibitors of Metalloproteases Meprin α and β

    PubMed Central

    Madoux, Franck; Tredup, Claudia; Spicer, Timothy P.; Scampavia, Louis; Chase, Peter S.; Hodder, Peter S.; Fields, Gregg B.; Becker-Pauly, Christoph; Minond, Dmitriy

    2015-01-01

    Zinc metalloproteinases meprin α and meprin β are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools we have developed high throughput screening (HTS) assays to enable discovery of inhibitors of both meprin α and meprin β and screened a collection of well characterized pharmaceutical agents (LOPAC, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin β. Kinetic studies revealed competitive (PPNDS) and mixed competitive/non-competitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin β active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin β reported to the date. These results demonstrate the ability of meprin α and β assays to identify selective compounds and discard artifacts of primary screening. PMID:25048711

  11. Population screening for glucose-6-phosphate dehydrogenase deficiencies in Isabel Province, Solomon Islands, using a modified enzyme assay on filter paper dried bloodspots

    PubMed Central

    2010-01-01

    Background Glucose-6-phosphate dehydrogenase deficiency poses a significant impediment to primaquine use for the elimination of liver stage infection with Plasmodium vivax and for gametocyte clearance, because of the risk of life-threatening haemolytic anaemia that can occur in G6PD deficient patients. Although a range of methods for screening G6PD deficiency have been described, almost all require skilled personnel, expensive laboratory equipment, freshly collected blood, and are time consuming; factors that render them unsuitable for mass-screening purposes. Methods A published WST8/1-methoxy PMS method was adapted to assay G6PD activity in a 96-well format using dried blood spots, and used it to undertake population screening within a malaria survey undertaken in Isabel Province, Solomon Islands. The assay results were compared to a biochemical test and a recently marketed rapid diagnostic test. Results Comparative testing with biochemical and rapid diagnostic test indicated that results obtained by filter paper assay were accurate providing that blood spots were assayed within 5 days when stored at ambient temperature and 10 days when stored at 4 degrees. Screening of 8541 people from 41 villages in Isabel Province, Solomon Islands revealed the prevalence of G6PD deficiency as defined by enzyme activity < 30% of normal control was 20.3% and a prevalence of severe deficiency that would predispose to primaquine-induced hemolysis (WHO Class I-II) of 6.9%. Conclusions The assay enabled simple and quick semi-quantitative population screening in a malaria-endemic region. The study indicated a high prevalence of G6PD deficiency in Isabel Province and highlights the critical need to consider G6PD deficiency in the context of P. vivax malaria elimination strategies in Solomon Islands, particularly in light of the potential role of primaquine mass drug administration. PMID:20684792

  12. An Efficient and Economical Assay to Screen for Triclosan Binding to FabI.

    PubMed

    Demissie, Robel D; Kabre, Pauline; Tuntland, Micheal L; Fung, Leslie W-M

    2016-04-01

    Triclosan is an effective inhibitor for enoyl acyl carrier protein reductase (ENR) in fatty acid biosynthesis. Triclosan-resistant mutants of ENR have emerged. Thus, it is important to detect these triclosan-resistant mutations in ENR. Generally, enzyme activity assays on the mutants are used to determine the effect of triclosan on ENR activity. Since the substrates are linked to acyl carrier protein (ACP), the assays are challenging due to the need to prepare the ACP and link it to the substrates. Non-ACP-linked (coenzyme A [CoA]-linked) substrates can be used in some ENR, but not in all. Consequently, screening for triclosan-resistant mutants is also challenging. We have developed a simple thermal shift assay, which does not use ACP-linked substrates, to determine the binding ability of triclosan to the ENR active site, and thus it can be used for screening for triclosan-resistant mutants. Staphylococcus aureus FabI enzyme and its mutants were used to demonstrate the binding ability of triclosan with NADP(+) to FabI. The direct correlation between the binding ability and enzyme activity was demonstrated with Francisella tularensis FabI. This method may also be applied to select effective triclosan analogues that inhibit ENR activity. PMID:26538431

  13. Platelet aggregation inhibitors from Philippine marine invertebrate samples screened in a new microplate assay.

    PubMed

    Pimentel, Sheila Marie V; Bojo, Zenaida P; Roberto, Amy V D; Lazaro, Jose Enrico H; Mangalindan, Gina C; Florentino, Leila M; Lim-Navarro, Pilar; Tasdemir, Deniz; Ireland, Chris M; Concepcion, Gisela P

    2003-01-01

    A new microplate assay for Ca(2+)-induced platelet aggregation as detected by Giemsa dye was used to screen marine invertebrate samples from the Philippines for inhibitors of human platelet aggregation. Out of 261 crude methanol extracts of marine sponges and tunicates, 25 inhibited aggregation at 2 mg/ml. Inhibition of agonist-induced aggregation in an aggregometer was used to confirm results of the microplate assay and to determine the specific mode of inhibition of 2 samples. The marine sponge Xestospongia sp. yielded a xestospongin/araguspongine-type molecule that inhibited collagen-induced aggregation by 87% at 2 micro g/ml, and epinephrine-induced aggregation by 78% at 20 micro g/ml, while the marine sponge Aplysina sp. yielded 5,6-dibromotryptamine, which inhibited epinephrine-induced aggregation by 51% at 20 micro g/ml. In this study we have found that the microplate assay is a simple, inexpensive, yet useful preliminary tool to qualitatively screen a large number of marine samples for antiplatelet aggregation activity. PMID:14719168

  14. In Vitro Reporter Assays for Screening of Chemicals That Disrupt Androgen Signaling

    PubMed Central

    Paul Khurana, S. M.

    2014-01-01

    Endocrine disruptive chemicals (EDCs) modulate hormone signaling and cause developmental and reproductive anomalies. Today, there is a global concern regarding endocrine disruption effects, particularly those mediated by the androgen receptor (AR). Androgen or male hormones are critical for the development and maintenance of male characteristics and numerous EDCs exist in the environment with the potential to disrupt androgen action. The threat is more during critical developmental windows when there is increased sensitivity to these compounds. Timely screening and detection of the EDCs is essential to minimize deleterious effects produced by these toxic chemicals. As a first line of screening, in vitro transcription assays are very useful due to their speed, convenience, and cost effectiveness. In this paper, recent in vitro reporter assays for detecting androgenic or antiandrogenic activity of EDCs have been reviewed. Two important cell systems used for this purpose, namely, the mammalian or yeast cell systems, have been discussed. Use of reporter genes such as bacterial luciferase (lux) and green fluorescent protein (gfp) has significantly improved speed and sensitivity of detection. Also, many of the current reporter assay systems can be used in a high throughput format allowing speedy evaluation of multiple potential EDCs at a lower price. PMID:25435875

  15. Screening Oligosaccharide Libraries against Lectins Using the Proxy Protein Electrospray Ionization Mass Spectrometry Assay.

    PubMed

    Han, Ling; Shams-Ud-Doha, Km; Kitova, Elena N; Klassen, John S

    2016-08-16

    An electrospray ionization mass spectrometry (ESI-MS) assay for screening carbohydrate libraries against lectins is described. The assay is based on the proxy protein ESI-MS method, which combines direct ESI-MS protein-ligand binding measurements and competitive protein binding, to simultaneously detect and quantify protein-carbohydrate interactions. Specific interactions between components of the library and the target protein (PT) are identified from changes in the relative abundances (as measured by ESI-MS) of the carbohydrate complexes of a proxy protein (Pproxy), which binds to all components of the library with known affinity, upon addition of PT to the solution. The magnitude of the change in relative abundance of a given Pproxy-ligand complex provides a quantitative measure of the affinity of the corresponding PT-ligand interaction. A mathematical framework for the implementation of the method in the case of monovalent (single binding site) Pproxy and monovalent and multivalent (multiple equivalent and independent binding sites) PT is described. The application of the method to screen small libraries of oligosaccharides, on the basis of human histo-blood group antigens and milk oligosaccharides, against an N-terminal fragment of the family 51 carbohydrate-binding module, a fucose-binding lectin from Ralstonia solanacearum, and human norovirus VA387 P particle (24-mer of the protruding domain of the capsid protein), serves to demonstrate the reliability and versatility of the assay. PMID:27366913

  16. Virtual screen for repurposing approved and experimental drugs for candidate inhibitors of EBOLA virus infection

    PubMed Central

    Veljkovic, Veljko; Loiseau, Philippe M.; Figadere, Bruno; Glisic, Sanja; Veljkovic, Nevena; Perovic, Vladimir R.; Cavanaugh, David P.; Branch, Donald R.

    2015-01-01

    The ongoing Ebola virus epidemic has presented numerous challenges with respect to control and treatment because there are no approved drugs or vaccines for the Ebola virus disease (EVD). Herein is proposed simple theoretical criterion for fast virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection. We performed a repurposing screen of 6438 drugs from DrugBank using this criterion and selected 267 approved and 382 experimental drugs as candidates for treatment of EVD including 15 anti-malarial drugs and 32 antibiotics. An open source Web server allowing screening of molecular libraries for candidate drugs for treatment of EVD was also established. PMID:25717373

  17. Drug search for leishmaniasis: a virtual screening approach by grid computing.

    PubMed

    Ochoa, Rodrigo; Watowich, Stanley J; Flórez, Andrés; Mesa, Carol V; Robledo, Sara M; Muskus, Carlos

    2016-07-01

    The trypanosomatid protozoa Leishmania is endemic in ~100 countries, with infections causing ~2 million new cases of leishmaniasis annually. Disease symptoms can include severe skin and mucosal ulcers, fever, anemia, splenomegaly, and death. Unfortunately, therapeutics approved to treat leishmaniasis are associated with potentially severe side effects, including death. Furthermore, drug-resistant Leishmania parasites have developed in most endemic countries. To address an urgent need for new, safe and inexpensive anti-leishmanial drugs, we utilized the IBM World Community Grid to complete computer-based drug discovery screens (Drug Search for Leishmaniasis) using unique leishmanial proteins and a database of 600,000 drug-like small molecules. Protein structures from different Leishmania species were selected for molecular dynamics (MD) simulations, and a series of conformational "snapshots" were chosen from each MD trajectory to simulate the protein's flexibility. A Relaxed Complex Scheme methodology was used to screen ~2000 MD conformations against the small molecule database, producing >1 billion protein-ligand structures. For each protein target, a binding spectrum was calculated to identify compounds predicted to bind with highest average affinity to all protein conformations. Significantly, four different Leishmania protein targets were predicted to strongly bind small molecules, with the strongest binding interactions predicted to occur for dihydroorotate dehydrogenase (LmDHODH; PDB:3MJY). A number of predicted tight-binding LmDHODH inhibitors were tested in vitro and potent selective inhibitors of Leishmania panamensis were identified. These promising small molecules are suitable for further development using iterative structure-based optimization and in vitro/in vivo validation assays. PMID:27438595

  18. 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

  19. A First Application of Enzyme-Linked Immunosorbent Assay for Screening Cyclodiene Insecticides in Ground Water

    USGS Publications Warehouse

    Dombrowski, T.R.; Thurman, E.M.; Mohrman, G.B.

    1996-01-01

    A commercially available enzyme-linked immunosorbent assay (ELISA) plate kit for screening of cyclodiene insecticides (aldrin, chlordane, dieldrin, endosulfan, endrin, and heptachlor) was evaluated for sensitivity, cross reactivity, and overall performance using groundwater samples from a contaminated site. Ground-water contaminants included several pesticide compounds and their manufacturing byproducts, as well as many other organic and inorganic compounds. Cross-reactivity studies were carried out for the cyclodiene compounds, and results were compared to those listed by the manufacturer. Data obtained were used to evaluate the sensitivity of the ELISA kit to the cyclodiene compounds in ground water samples with a contaminated matrix. The method quantitation limit for the ELISA kit was 15 ??g/L (as chlordane). Of the 56 ground-water samples analyzed using the ELISA plate kits, more than 85% showed cyclodiene insecticide contamination. The ELISA kit showed excellent potential as a screening tool for sites with suspected groundwater contamination by insecticides.

  20. A screening study of drug-drug interactions in cerivastatin users: an adverse effect of clopidogrel.

    PubMed

    Floyd, J S; Kaspera, R; Marciante, K D; Weiss, N S; Heckbert, S R; Lumley, T; Wiggins, K L; Tamraz, B; Kwok, P-Y; Totah, R A; Psaty, B M

    2012-05-01

    An analysis of a case-control study of rhabdomyolysis was conducted to screen for previously unrecognized cytochrome P450 enzyme (CYP) 2C8 inhibitors that may cause other clinically important drug-drug interactions. Medication use in cases of rhabdomyolysis using cerivastatin (n = 72) was compared with that in controls using atorvastatin (n = 287) for the period 1998-2001. The use of clopidogrel was strongly associated with rhabdomyolysis (odds ratio (OR) 29.6; 95% confidence interval (CI), 6.1-143). In a replication effort that used the US Food and Drug Administration (FDA) Adverse Event Reporting System (AERS), it was found that clopidogrel was used more commonly in patients with rhabdomyolysis receiving cerivastatin (17%) than in those receiving atorvastatin (0%, OR infinity; 95% CI = 5.2-infinity). Several medications were tested in vitro for their potential to cause drug-drug interactions. Clopidogrel, rosiglitazone, and montelukast were the most potent inhibitors of cerivastatin metabolism. Clopidogrel and its metabolites also inhibited cerivastatin metabolism in human hepatocytes. These epidemiological and in vitro findings suggest that clopidogrel may cause clinically important, dose-dependent drug-drug interactions with other medications metabolized by CYP2C8. PMID:22419147

  1. Screening for drugs in oral fluid: illicit drug use and drug driving in a sample of Queensland motorists.

    PubMed

    Davey, J; Leal, N; Freeman, J

    2007-05-01

    Police Services in a number of Australian states have indicated random roadside drug testing will be implemented to target drug driving. This paper outlines research conducted to provide an estimate of the prevalence of drug driving in a sample of Queensland drivers. Oral fluid samples were collected from 781 drivers who volunteered to participate at Random Breath Testing (RBT) sites in a large Queensland regional area. Illicit substances tested for included cannabis (delta 9 tetrahydrocannibinol [THC]), amphetamine type substances, heroin and cocaine. Drivers also completed a self-report questionnaire regarding their drug-related driving behaviour. Samples that were drug-positive at initial screening were sent to a government laboratory for confirmation. Oral fluid samples from 27 participants (3.5%) were confirmed positive for at least one illicit substance. The most common drugs detected in oral fluid were cannabis (delta 9 THC) (n = 13) followed by amphetamine type substances (n = 11). A key finding was that cannabis was also confirmed as the most common self-reported drug combined with driving and that individuals who tested positive to any drug through oral fluid analysis were also more likely to report the highest frequency of drug driving. Furthermore, a comparison between drug vs drink driving detection rates for the study period revealed a higher detection rate for drug driving (3.5%) vs drink driving (0.8%). This research provides evidence that drug driving is relatively prevalent on Queensland Roads. The paper will further outline the study findings and present possible directions for future drug driving research. PMID:17454020

  2. Immunomagnetic Reduction Assay on Des-Gamma-Carboxy Prothrombin for Screening of Hepatocellular Carcinoma.

    PubMed

    Chieh, Jen-Jie; Huang, K W; Chuang, C P; Wei, W C; Dong, J J; Lee, Y Y

    2016-08-01

    The accredited biomarker alpha-fetoprotein (AFP) offers limited sensitivity and specificity in the early detection of hepatocellular carcinoma (HCC). To improve the screening performance, des-gamma-carboxy prothrombin (DCP) has been identified as another promising biomarker of HCC, combined with AFP biomarkers. The results of the commercial optical enzyme-linked immunosorbent assay (ELISA) kit easily have the interference problem due to the optical methodology. The immunomagnetic reduction (IMR) assay based on the magnetic measurement was utilized to assay DCP biomarkers without the excellent antiinterference performances. A DCP magnetic reagent, composed of iron-oxide (Fe3O4 ) magnetic nanoparticles coated with anti-DCP antibodies solved in phosphoryl-buffer solution, was synthesized and characterized. In the test of standard DCP antigens, superior antiinterference and sensitivity than optical ELISA were proved. In the animal test, the results indicate good agreement between the IMR assay findings and the tumor sizes of HCC rats at all time points after the HCC implantation. The feasibility of the developed DCP magnetic reagent with the IMR for the detection of DCP is verified, and demonstrates the high potential for future clinical applications. PMID:26415145

  3. The Lumipulse G HBsAg-Quant assay for screening and quantification of the hepatitis B surface antigen.

    PubMed

    Yang, Ruifeng; Song, Guangjun; Guan, Wenli; Wang, Qian; Liu, Yan; Wei, Lai

    2016-02-01

    Qualitative HBsAg assay is used to screen HBV infection for decades. The utility of quantitative assay is also rejuvenated recently. We aimed to evaluate and compare the performance of a novel ultra-sensitive and quantitative assay, the Lumipulse assay, with the Architect and Elecsys assays. As screening methods, specificity was compared using 2043 consecutive clinical routine samples. As quantitative assays, precision and accuracy were assessed. Sera from 112 treatment-naïve chronic hepatitis B patients, four patients undergoing antiviral therapy and one patient with acute infection were tested to compare the correlations. Samples with concurrent HBsAg/anti-HBs were also quantified. The Lumipulse assay precisely quantified ultra-low level of HBsAg (0.004 IU/mL). It identified additional 0.98% (20/2043) clinical samples with trance amount of HBsAg. Three assays displayed excellent linear correlations irrespective of genotypes and S-gene mutations (R(2)>0.95, P<0.0001), while minor quantitative biases existed. The Lumipulse assay did not yield higher HBsAg concentrations in samples with concomitant anti-HBs. Compared with other assays, the Lumipulse assay is sensitive and specific for detecting HBsAg. The interpretation of the extremely low-level results, however, is challenging. Quantitative HBsAg results by different assays are highly correlated, but they should be interpreted interchangeably only after conversion to eliminate the biases. PMID:26615803

  4. 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...

  5. Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s.

    PubMed

    Weissenborn, Martin J; Notonier, Sandra; Lang, Sarah-Luise; Otte, Konrad B; Herter, Susanne; Turner, Nicholas J; Flitsch, Sabine L; Hauer, Bernhard

    2016-05-01

    A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids. PMID:27074906

  6. The Cellular Thermal Shift Assay: A Novel Biophysical Assay for In Situ Drug Target Engagement and Mechanistic Biomarker Studies.

    PubMed

    Martinez Molina, Daniel; Nordlund, Pär

    2016-01-01

    A drug must engage its intended target to achieve its therapeutic effect. However, conclusively measuring target engagement (TE) in situ is challenging. This complicates preclinical development and is considered a key factor in the high rate of attrition in clinical trials. Here, we discuss a recently developed, label-free, biophysical assay, the cellular thermal shift assay (CETSA), which facilitates the direct assessment of TE in cells and tissues at various stages of drug development. CETSA also reveals biochemical events downstream of drug binding and therefore provides a promising means of establishing mechanistic biomarkers. The implementation of proteome-wide CETSA using quantitative mass spectrometry represents a novel strategy for defining off-target toxicity and polypharmacology and for identifying downstream mechanistic biomarkers. The first year of CETSA applications in the literature has focused on TE studies in cell culture systems and has confirmed the broad applicability of CETSA to many different target families. The next phase of CETSA applications will likely encompass comprehensive animal and patient studies, and CETSA will likely serve as a very valuable tool in many stages of preclinical and clinical drug development. PMID:26566155

  7. 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

  8. Small molecule drug screening in Drosophila identifies the 5HT2A receptor as a feeding modulation target

    PubMed Central

    Gasque, Gabriel; Conway, Stephen; Huang, Juan; Rao, Yi; Vosshall, Leslie B.

    2013-01-01

    Dysregulation of eating behavior can lead to obesity, which affects 10% of the adult population worldwide and accounts for nearly 3 million deaths every year. Despite this burden on society, we currently lack effective pharmacological treatment options to regulate appetite. We used Drosophila melanogaster larvae to develop a high-throughput whole organism screen for drugs that modulate food intake. In a screen of 3630 small molecules, we identified the serotonin (5-hydroxytryptamine or 5-HT) receptor antagonist metitepine as a potent anorectic drug. Using cell-based assays we show that metitepine is an antagonist of all five Drosophila 5-HT receptors. We screened fly mutants for each of these receptors and found that serotonin receptor 5-HT2A is the sole molecular target for feeding inhibition by metitepine. These results highlight the conservation of molecular mechanisms controlling appetite and provide a method for unbiased whole-organism drug screens to identify novel drugs and molecular pathways modulating food intake. PMID:23817146

  9. 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

  10. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

    PubMed Central

    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance. PMID:25988960

  11. Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants.

    PubMed

    Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F M; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

    2015-01-01

    Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance. PMID:25988960

  12. Current status of drug screening and disease modelling in human pluripotent stem cells

    PubMed Central

    Rajamohan, Divya; Matsa, Elena; Kalra, Spandan; Crutchley, James; Patel, Asha; George, Vinoj; Denning, Chris

    2013-01-01

    The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost. PMID:22886688

  13. Screening α-glucosidase inhibitors from traditional Chinese drugs by capillary electrophoresis with electrophoretically mediated microanalysis.

    PubMed

    Guo, Li-Ping; Jiang, Ting-Fu; Lv, Zhi-Hua; Wang, Yuan-Hong

    2010-12-15

    In the present study, we report the study by a combination of electrophoretically mediated microanalysis method with a partial technique for screening alpha-glucosidase inhibitors from 21 traditional Chinese drugs. In the setup, substrates and enzymes were introduced into the capillary as distinct plugs, the electrophoretic conditions for enzyme reaction and separation of substrates and products were different in the composition and pH of the background electrolyte, which make more enzyme reactions possible. Part of the capillary was filled with the optimal buffer for the enzyme reaction, whereas the rest was filled with the background electrolyte optimal for the separation of substrates and products. With the optimal condition, the Michaelis-Menten constant and the inhibitive mechanism of acarbose were studied, which were in the same range as previous literature data. Furthermore, the inhibitory ratios of enzymatic activity (IRE) of 21 traditional Chinese drugs were determined. The classical method has superiorities over traditional assay methods, which not only minimizes the false-positive results but also simplifies the experimental processes. It could be used for screening inhibitors in natural extract. PMID:20719454

  14. Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z.

    PubMed

    Gosai, Sager J; Kwak, Joon Hyeok; Luke, Cliff J; Long, Olivia S; King, Dale E; Kovatch, Kevin J; Johnston, Paul A; Shun, Tong Ying; Lazo, John S; Perlmutter, David H; Silverman, Gary A; Pak, Stephen C

    2010-01-01

    The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms. PMID:21103396

  15. Results of drug screening from a producer's view.

    PubMed

    Adams, J B

    1994-07-01

    The dairy industry is faced with increasing governmental and public concern about the safety of the nation's milk supply. New regulations under the Grade A Pasteurized Milk Ordinance require that prescription drugs be properly labeled and that all tanker loads of milk be tested for beta-lactam antimicrobial residues. Concern over the use of animal drugs in an extralabel manner has prompted the National Milk Producers Federation and the American Veterinary Medical Association to develop a quality assurance program for on-farm residue prevention known as the Dairy Quality Assurance 10-Point Milk and Dairy Beef Residue Prevention Protocol. The program promotes the concept of Hazard Analysis Critical Control Points, applied to a pre-harvest farm environment. Screening limitations at point of milk receipt necessitates widespread adoption of the Dairy Quality Assurance protocol to address controlled use of all animal medications under a valid relationship among veterinarian, client, and animals, thus minimizing the potential for violative residues in the milk and meat supply. PMID:7929955

  16. 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.

  17. 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

  18. A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis

    PubMed Central

    Collins, Tony J.; Ylanko, Jarkko; Geng, Fei

    2015-01-01

    Abstract A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose–response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds. PMID:26422066

  19. A simple liposome assay for the screening of zinc ionophore activity of polyphenols.

    PubMed

    Clergeaud, Gael; Dabbagh-Bazarbachi, Husam; Ortiz, Mayreli; Fernández-Larrea, Juan B; O'Sullivan, Ciara K

    2016-04-15

    An efficient liposomal system for screening the zinc ionophore activity of a selected library consisting of the most relevant dietary polyphenols is presented. The zinc ionophore activity was demonstrated by exploring the use of zinc-specific fluorophore FluoZin-3 loaded liposomes as simple membrane tools that mimic the cell membrane. The zinc ionophore activity was demonstrated as the capacity of polyphenols to transport zinc cations across the liposome membrane and increase the zinc-specific fluorescence of the encapsulated fluorophore FluoZin-3. In addition, the zinc chelation strength of the polyphenols was also tested in a competition assay based on the fluorescence quenching of zinc-dependent fluorescence emitted by zinc-FluoZin-3 complex. Finally, the correlation between the chelation capacity and ionophore activity is demonstrated, thus underlining the sequestering or ionophoric activity that the phenolic compounds can display, thus, providing better knowledge of the importance of the structural conformation versus their biological activity. Furthermore, the assays developed can be used as tools for rapid, high-throughput screening of families of polyphenols towards different biometals. PMID:26617034

  20. A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

    PubMed

    Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W

    2015-11-01

    A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds. PMID:26422066

  1. Rapid Polymerase Chain Reaction-based Screening Assay for Bacterial Biothreat Agents

    PubMed Central

    Yang, Samuel; Rothman, Richard E.; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A.

    2013-01-01

    Objectives To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. Methods The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. Results The UniProbe detected the presence of all tested Eubacteria (31 / 31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. Conclusions A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents. PMID:18370996

  2. Automated assay for screening the enzymatic release of reducing sugars from micronized biomass

    PubMed Central

    2010-01-01

    Background To reduce the production cost of bioethanol obtained from fermentation of the sugars provided by degradation of lignocellulosic biomass (i.e., second generation bioethanol), it is necessary to screen for new enzymes endowed with more efficient biomass degrading properties. This demands the set-up of high-throughput screening methods. Several methods have been devised all using microplates in the industrial SBS format. Although this size reduction and standardization has greatly improved the screening process, the published methods comprise one or more manual steps that seriously decrease throughput. Therefore, we worked to devise a screening method devoid of any manual steps. Results We describe a fully automated assay for measuring the amount of reducing sugars released by biomass-degrading enzymes from wheat-straw and spruce. The method comprises two independent and automated steps. The first step is the making of "substrate plates". It consists of filling 96-well microplates with slurry suspensions of micronized substrate which are then stored frozen until use. The second step is an enzymatic activity assay. After thawing, the substrate plates are supplemented by the robot with cell-wall degrading enzymes where necessary, and the whole process from addition of enzymes to quantification of released sugars is autonomously performed by the robot. We describe how critical parameters (amount of substrate, amount of enzyme, incubation duration and temperature) were selected to fit with our specific use. The ability of this automated small-scale assay to discriminate among different enzymatic activities was validated using a set of commercial enzymes. Conclusions Using an automatic microplate sealer solved three main problems generally encountered during the set-up of methods for measuring the sugar-releasing activity of plant cell wall-degrading enzymes: throughput, automation, and evaporation losses. In its present set-up, the robot can autonomously

  3. Phenotypic Screening of Small-Molecule Inhibitors: Implications for Therapeutic Discovery and Drug Target Development in Traumatic Brain Injury.

    PubMed

    Al-Ali, Hassan; Lemmon, Vance P; Bixby, John L

    2016-01-01

    The inability of central nervous system (CNS) neurons to regenerate damaged axons and dendrites following traumatic brain injury (TBI) creates a substantial obstacle for functional recovery. Apoptotic cell death, deposition of scar tissue, and growth-repressive molecules produced by glia further complicate the problem and make it challenging for re-growing axons to extend across injury sites. To date, there are no approved drugs for the treatment of TBI, accentuating the need for relevant leads. Cell-based and organotypic bioassays can better mimic outcomes within the native CNS microenvironment than target-based screening methods and thus should speed the discovery of therapeutic agents that induce axon or dendrite regeneration. Additionally, when used to screen focused chemical libraries such as small-molecule protein kinase inhibitors, these assays can help elucidate molecular mechanisms involved in neurite outgrowth and regeneration as well as identify novel drug targets. Here, we describe a phenotypic cellular (high content) screening assay that utilizes brain-derived primary neurons for screening small-molecule chemical libraries. PMID:27604745

  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. Drug activity screening based on microsomes-hydrogel system in predicting metabolism induced antitumor effect of oroxylin A

    PubMed Central

    Yang, Huiying; Li, Jianfeng; Zheng, Yuanting; Zhou, Lu; Tong, Shanshan; Zhao, Bei; Cai, Weimin

    2016-01-01

    A novel microsomes-hydrogel added cell culture system (MHCCS) was employed in the antitumor activity screening of natural compounds, aiming to achieve drug screening with better in vivo correlation, higher initiative to explore the potential active metabolites, and investigation of the antitumor mechanism from the perspective of metabolism. MTT assay and cell apoptosis detection showed that test drug oroxylin A (OA) had enhanced cytotoxicity and wogonin (W) with reduced cytotoxicity on MCF-7 cell line upon MHCCS incubation. In vivo antitumor evaluations also demonstrated that OA induced higher tumor inhibition than W at the same dosage. To explore the reasons, nine major metabolites of OA were separated and collected through UPLC-Q-TOF and semi-preparative HPLC. Metabolites M318 exhibited higher cytotoxicity than OA and other metabolites by MTT assay. 1H NMR spectrums, HPLC and TOF MS/MS results revealed that OA was catalyzed into its active metabolite M318 via a ring-opening reaction. M318 induced significant cell apoptosis and S-phase arrest through affecting tumor survival related genes after mechanism study. In conclusion, our MHCCS could be a useful tool for drug activity screening from a perspective of metabolism. PMID:26905263

  6. 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

  7. Genome-wide screening of loci associated with drug resistance to 5-fluorouracil-based drugs.

    PubMed

    Ooyama, Akio; Okayama, Yoshihiro; Takechi, Teiji; Sugimoto, Yoshikazu; Oka, Toshinori; Fukushima, Masakazu

    2007-04-01

    Resistance to chemotherapeutic agents represents the chief cause of mortality in cancer patients with advanced disease. Chromosomal aberration and altered gene expression are the main genetic mechanisms of tumor chemoresistance. In this study, we have established an algorithm to calculate DNA copy number using the Affymetrix 10K array, and performed a genome-wide correlation analysis between DNA copy number and antitumor activity against 5-fluorouracil (5-FU)-based drugs (S-1, tegafur + uracil [UFT], 5'-DFUR and capecitabine) to screen for loci influencing drug resistance using 27 human cancer xenografts. A correlation analysis confirmed that the single nucleotide polymorphism (SNP) showing significant associations with drug sensitivity were concentrated in some cytogenetic regions (18p, 17p13.2, 17p12, 11q14.1, 11q11 and 11p11.12), and we identified some genes that have been indicated their relations to drug sensitivity. Among these regions, 18p11.32 at the location of the thymidylate synthase gene (TYMS) was strongly associated with resistance to 5-FU-based drugs. A change in copy number of the TYMS gene was reflected in the TYMS expression level, and showed a significant negative correlation with sensitivity against 5-FU-based drugs. These results suggest that amplification of the TYMS gene is associated with innate resistance, supporting the possibility that TYMS copy number might be a predictive marker of drug sensitivity to fluoropyrimidines. Further study is necessary to clarify the functional roles of other genes coded in significant cytogenetic regions. These promising data suggest that a comprehensive DNA copy number analysis might aid in the quest for optimal markers of drug response. PMID:17425594

  8. Generation of orientation tools for automated zebrafish screening assays using desktop 3D printing

    PubMed Central

    2014-01-01

    Background The zebrafish has been established as the main vertebrate model system for whole organism screening applications. However, the lack of consistent positioning of zebrafish embryos within wells of microtiter plates remains an obstacle for the comparative analysis of images acquired in automated screening assays. While technical solutions to the orientation problem exist, dissemination is often hindered by the lack of simple and inexpensive ways of distributing and duplicating tools. Results Here, we provide a cost effective method for the production of 96-well plate compatible zebrafish orientation tools using a desktop 3D printer. The printed tools enable the positioning and orientation of zebrafish embryos within cavities formed in agarose. Their applicability is demonstrated by acquiring lateral and dorsal views of zebrafish embryos arrayed within microtiter plates using an automated screening microscope. This enables the consistent visualization of morphological phenotypes and reporter gene expression patterns. Conclusions The designs are refined versions of previously demonstrated devices with added functionality and strongly reduced production costs. All corresponding 3D models are freely available and digital design can be easily shared electronically. In combination with the increasingly widespread usage of 3D printers, this provides access to the developed tools to a wide range of zebrafish users. Finally, the design files can serve as templates for other additive and subtractive fabrication methods. PMID:24886511

  9. 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

  10. 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

  11. A New Surface Plasmon Resonance Assay for In Vitro Screening of Mannose-Binding Lectin Inhibitors.

    PubMed

    Stravalaci, Matteo; De Blasio, Daiana; Orsini, Franca; Perego, Carlo; Palmioli, Alessandro; Goti, Giulio; Bernardi, Anna; De Simoni, Maria-Grazia; Gobbi, Marco

    2016-08-01

    Mannose-binding lectin (MBL) is a circulating protein that acts as a soluble pattern recognition molecule of the innate immunity. It binds to carbohydrate patterns on the surface of pathogens or of altered self-cells, with activation of the lectin pathway of the complement system. Recent evidence indicates that MBL contributes to the pathophysiology of ischemia-reperfusion injury and other conditions. Thus, MBL inhibitors offer promising therapeutic strategies, since they prevent the interaction of MBL with its target sugar arrays. We developed and characterized a novel assay based on surface plasmon resonance for in vitro screening of these compounds, which may be useful before the more expensive and time-consuming in vivo studies. The assay measures the inhibitor's ability to interfere with the binding of murine MBL-A or MBL-C, or of human recombinant MBL, to mannose residues immobilized on the sensor chip surface. We have applied the assay to measure the IC50 of synthetic glycodendrimers, two of them with neuroprotective properties in animal models of MBL-mediated injuries. PMID:26969323

  12. Assessment of estrogenic activity in Tunisian water and wastewater by E-screen assay.

    PubMed

    Limam, Atef; Talorete, Terence P N; Ali, Mourad Ben Sik; Kawano, Mitsuko; Jenhani, Amel Ben Rejeb; Abe, Yukuo; Ghrabi, Ahmed; Isoda, Hiroko

    2007-01-01

    Wastewater and surface water samples from three wastewater treatment plants (WWTPs) and three rivers in Tunisia were assayed for estrogenic activity using the E-screen assay and enzyme-linked immunosorbent assay (ELISA). Results showed that all the Tunisian raw wastewater samples as well as the Roriche river water sample induced a strong proliferative response in human MCF-7 breast cancer cells. Tunisian raw wastewater had an average 17beta-estradiol content of 2,705.4 pg/ml, whereas that of the Roriche river was 36.7 pg/ml, which is sufficient for inducing endocrine-mediated responses in aquatic organisms. Results further showed that the Mornag WWTP, which uses the activated-sludge treatment system, has a higher estrogen removal efficiency than the stabilization ponds of the Gammart and pilot WWTPs. This study, which is the first of such studies in Tunisia, and probably the first in the North African region, underscores the need to detect and monitor the estrogenic activity of water and wastewater, given the scarcity of water in Tunisia and the detrimental impact of endocrine-disrupting compounds on the physiology of both animals and humans. PMID:18382414

  13. Evaluation of a Fluorescence-Based Method for Antibabesial Drug Screening

    PubMed Central

    Guswanto, Azirwan; Sivakumar, Thillaiampalam; Rizk, Mohamed Abdo; Elsayed, Shimaa Abd Elsalam; Youssef, Mohamed Ahmed; ElSaid, ElSaid El Shirbini; Yokoyama, Naoaki

    2014-01-01

    In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become routine, and the effectiveness of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness-of-fit (r2) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC50s determined by the fluorescence-based method (408 nM and 8.13 μM, respectively) and microscopy (400.3 nM and 9.4 μM, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 μM) was similar to the recently described microscopy-based value (21.7 μM) for B. bovis. Additionally, the Z′ factor (0.80 to 0.90), signal-to-noise (S/N) ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay. PMID:24914124

  14. Evaluation of a fluorescence-based method for antibabesial drug screening.

    PubMed

    Guswanto, Azirwan; Sivakumar, Thillaiampalam; Rizk, Mohamed Abdo; Elsayed, Shimaa Abd Elsalam; Youssef, Mohamed Ahmed; ElSaid, ElSaid El Shirbini; Yokoyama, Naoaki; Igarashi, Ikuo

    2014-08-01

    In vitro evaluation of chemotherapeutic agents against Babesia and Theileria parasites has become routine, and the effectiveness of these chemicals is usually determined by comparing the parasitemia dynamics of untreated and treated parasites. Although microscopy is widely used to calculate parasitemia, several disadvantages are associated with this technique. The present study evaluated a fluorescence-based method using SYBR green I stain (SG I) to screen antibabesial agents in in vitro cultures of Babesia bovis. The linearity between relative fluorescence units (RFU) and parasitemia was found to be well correlated with a 0.9944 goodness-of-fit (r(2)) value. Subsequently, 50% inhibitory concentration (IC50) values were calculated for 3 antiprotozoan agents, diminazene aceturate, nimbolide, and gedunin, by this method. For diminazene aceturate and nimbolide, the IC(50)s determined by the fluorescence-based method (408 nM and 8.13 μM, respectively) and microscopy (400.3 nM and 9.4 μM, respectively) were in agreement. Furthermore, the IC50 of gedunin determined by the fluorescence-based method (19 μM) was similar to the recently described microscopy-based value (21.7 μM) for B. bovis. Additionally, the Z' factor (0.80 to 0.90), signal-to-noise (S/N) ratio (44.15 to 87.64), coefficient of variation at the maximum signal (%CVmax) (0.50 to 2.85), and coefficient of variation at the minimum signal (%CVmin) (1.23 to 2.21) calculated for the fluorescence method using diminazene aceturate were comparable to those previously determined in malaria research for this assay. These findings suggest that the fluorescence-based method might be useful for antibabesial drug screening and may have potential to be developed into a high-throughput screening (HTS) assay. PMID:24914124

  15. High-throughput screening of drug-binding dynamics to HERG improves early drug safety assessment.

    PubMed

    Di Veroli, Giovanni Y; Davies, Mark R; Zhang, Henggui; Abi-Gerges, Najah; Boyett, Mark R

    2013-01-01

    The use of computational models to predict drug-induced changes in the action potential (AP) is a promising approach to reduce drug safety attrition but requires a better representation of more complex drug-target interactions to improve the quantitative prediction. The blockade of the human ether-a-go-go-related gene (HERG) channel is a major concern for QT prolongation and Torsade de Pointes risk. We aim to develop quantitative in-silico AP predictions based on a new electrophysiological protocol (suitable for high-throughput HERG screening) and mathematical modeling of ionic currents. Electrophysiological recordings using the IonWorks device were made from HERG channels stably expressed in Chinese hamster ovary cells. A new protocol that delineates inhibition over time was applied to assess dofetilide, cisapride, and almokalant effects. Dynamic effects displayed distinct profiles for these drugs compared with concentration-effects curves. Binding kinetics to specific states were identified using a new HERG Markov model. The model was then modified to represent the canine rapid delayed rectifier K(+) current at 37°C and carry out AP predictions. Predictions were compared with a simpler model based on conductance reduction and were found to be much closer to experimental data. Improved sensitivity to concentration and pacing frequency variables was obtained when including binding kinetics. Our new electrophysiological protocol is suitable for high-throughput screening and is able to distinguish drug-binding kinetics. The association of this protocol with our modeling approach indicates that quantitative predictions of AP modulation can be obtained, which is a significant improvement compared with traditional conductance reduction methods. PMID:23103500

  16. Evaluation of novel assays for the detection of human papilloma virus in self-collected samples for cervical cancer screening.

    PubMed

    Chen, Q; Du, H; Zhang, R; Zhao, J H; Hu, Q C; Wang, C; Wang, G X; Tang, J L; Wu, R F

    2016-01-01

    The aim of this study was to evaluate the performance of three new high-risk human papillomavirus (HPV) assays for primary cervical cancer screening, by using self-collected samples, and to identify an HPV assay that could overcome the major obstacles faced during large-scale population-based screening. Two hundred and ten women showing abnormal cervical cytology (and referred for a colposcopy) were recruited in this study. Self-collected samples obtained from all women were tested with the Cobas, Seq, and BioPerfectus Multiplex Real Time HPV assays; simultaneously, clinician-collected samples (from the same women) were tested with the gold-standard Cobas HPV assay. The results of all the assays were consistent. The sensitivity, positive predictive value, and negative predictive value for cervical intraepithelial neoplasia 2+ (CIN2+) and CIN3+ were comparable between the self-collected samples tested with the three new assays and the clinician-collected samples tested with the Cobas HPV assay (P > 0.05). The single-genotype HPV load per sample did not differ significantly between the self- and clinician-collected samples (P = 0.195). In conclusion, the results of this study demonstrated the applicability of the three new HPV assays for primary cervical cancer screening based on self-collection. PMID:27420961

  17. Minimizing DILI risk in drug discovery - A screening tool for drug candidates.

    PubMed

    Schadt, S; Simon, S; Kustermann, S; Boess, F; McGinnis, C; Brink, A; Lieven, R; Fowler, S; Youdim, K; Ullah, M; Marschmann, M; Zihlmann, C; Siegrist, Y M; Cascais, A C; Di Lenarda, E; Durr, E; Schaub, N; Ang, X; Starke, V; Singer, T; Alvarez-Sanchez, R; Roth, A B; Schuler, F; Funk, C

    2015-12-25

    Drug-induced liver injury (DILI) is a leading cause of acute hepatic failure and a major reason for market withdrawal of drugs. Idiosyncratic DILI is multifactorial, with unclear dose-dependency and poor predictability since the underlying patient-related susceptibilities are not sufficiently understood. Because of these limitations, a pharmaceutical research option would be to reduce the compound-related risk factors in the drug-discovery process. Here we describe the development and validation of a methodology for the assessment of DILI risk of drug candidates. As a training set, 81 marketed or withdrawn compounds with differing DILI rates - according to the FDA categorization - were tested in a combination of assays covering different mechanisms and endpoints contributing to human DILI. These include the generation of reactive metabolites (CYP3A4 time-dependent inhibition and glutathione adduct formation), inhibition of the human bile salt export pump (BSEP), mitochondrial toxicity and cytotoxicity (fibroblasts and human hepatocytes). Different approaches for dose- and exposure-based calibrations were assessed and the same parameters applied to a test set of 39 different compounds. We achieved a similar performance to the training set with an overall accuracy of 79% correctly predicted, a sensitivity of 76% and a specificity of 82%. This test system may be applied in a prospective manner to reduce the risk of idiosyncratic DILI of drug candidates. PMID:26407524

  18. Screening American Indian Youth for Referral to Drug Abuse Prevention and Intervention Services

    ERIC Educational Resources Information Center

    Winters, Ken C.; Dewolfe, Jerome; Graham, Donald

    2006-01-01

    The development and psychometric properties of a brief screening tool for use with American Indian youth suspected of abusing substances is described. The Indian Health Service-Personal Experience Screening Questionnaire (IHS-PESQ) is a brief questionnaire that screens for drug abuse problem severity, response distortion tendencies, and…

  19. The 'BlueScreen HC' assay as a decision making test in the genotoxicity assessment of flavour and fragrance materials.

    PubMed

    Etter, Sylvain; Birrell, Louise; Cahill, Paul; Scott, Heather; Billinton, Nick; Walmsley, Richard M; Smith, Benjamin

    2015-10-01

    The genotoxicity of a library of 70 flavour and fragrance substances having a high proportion of in vivo and/or carcinogenicity test data has been assessed using the GADD45a-GLuc 'BlueScreen HC' genotoxicity assay, with and without exogenous metabolic activation. There are only limited genotoxicity and carcinogenicity study data for compounds in this applicability domain, but this study allowed the following conclusions: (i) The BlueScreen HC results are highly predictive of positive results from regulator-required in vitro genotoxicity assays for the test set of materials; the moderate negative predictivity of BlueScreen HC from the in vitro test set of material is mainly due to the high rate of false positive in regulatory in vitro mammalian tests. (ii) BlueScreen HC negative results are predictive of negative in vivo results and provide a specific prediction of in vivo genotoxicity assay results. (iii) In this applicability domain, which comprises a large proportion of relatively low molecular weight molecules, a 1mM testing limit maintains the sensitivity of the assay, and increases specificity. (iv) The predictive capacity and specificity to in vivo genotoxins and carcinogens, coupled to a microplate format with low compound requirement supports further investigation of the BlueScreen HC assay as a useful tool in prioritizing the assessment of new F&F materials and in filling data gaps on materials with no or limited regulatory test data for genotoxicity. PMID:26003925

  20. 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

  1. The University of Kansas High-Throughput Screening Laboratory. Part II: enabling collaborative drug-discovery partnerships through cutting-edge screening technology

    PubMed Central

    McDonald, Peter R; Roy, Anuradha; Chaguturu, Rathnam

    2011-01-01

    The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory. PMID:21806374

  2. The University of Kansas High-Throughput Screening Laboratory. Part II: enabling collaborative drug-discovery partnerships through cutting-edge screening technology.

    PubMed

    McDonald, Peter R; Roy, Anuradha; Chaguturu, Rathnam

    2011-07-01

    The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory. PMID:21806374

  3. Systematic Evaluation of Different Nucleic Acid Amplification Assays for Cytomegalovirus Detection: Feasibility of Blood Donor Screening.

    PubMed

    Vollmer, T; Knabbe, C; Dreier, J

    2015-10-01

    Acute primary cytomegalovirus (CMV) infections, which commonly occur asymptomatically among blood donors, represent a significant risk for serious morbidity in immunocompromised patients (a major group of transfusion recipients). We implemented a routine CMV pool screening procedure for plasma for the identification of CMV DNA-positive donors, and we evaluated the sensitivities and performance of different CMV DNA amplification systems. Minipools (MPs) of samples from 18,405 individual donors (54,451 donations) were screened for CMV DNA using the RealStar CMV PCR assay (Altona Diagnostic Technologies), with a minimum detection limit of 11.14 IU/ml. DNA was extracted with a high-volume protocol (4.8 ml, Chemagic Viral 5K kit; PerkinElmer) for blood donor pool screening (MP-nucleic acid testing [NAT]) and with the Nuclisens easyMAG system (0.5 ml; bioMérieux) for individual donation (ID)-NAT. In total, six CMV DNA-positive donors (0.03%) were identified by routine CMV screening, with DNA concentrations ranging from 4.35 × 10(2) to 4.30 × 10(3) IU/ml. Five donors already showed seroconversion and detectable IgA, IgM, and/or IgG antibody titers (IgA(+)/IgM(+)/IgG(-) or IgA(+)/IgM(+)/IgG(+)), and one donor showed no CMV-specific antibodies. Comparison of three commercial assays, i.e., the RealStar CMV PCR kit, the Sentosa SA CMV quantitative PCR kit (Vela Diagnostics), and the CMV R-gene PCR kit (bioMérieux), for MP-NAT and ID-NAT showed comparably good analytical sensitivities, ranging from 10.23 to 11.14 IU/ml (MP-NAT) or from 37.66 to 57.94 IU/ml (ID-NAT). The clinical relevance of transfusion-associated CMV infections requires further investigation, and the evaluated methods present powerful basic tools providing sensitive possibilities for viral testing. The application of CMV MP-NAT facilitated the identification of one donor with a window-phase donation during acute primary CMV infection. PMID:26202109

  4. Continuous colorimetric screening assays for the detection of specific L- or D-α-amino acid transaminases in enzyme libraries.

    PubMed

    Heuson, Egon; Petit, Jean-Louis; Debard, Adrien; Job, Aurélie; Charmantray, Franck; de Berardinis, Véronique; Gefflaut, Thierry

    2016-01-01

    In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of L- or D-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays' quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since L-CSA and D-CSA allowed the exclusive detection of L-TA and D-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable L- or D-homophenylalanine. PMID:26452497

  5. Pralatrexate Monitoring Using a Commercially Available Methotrexate Assay to Avoid Potential Drug Interactions.

    PubMed

    McPherson, Jordan P; Vrontikis, Alaina; Sedillo, Courtney; Halwani, Ahmad S; Gilreath, Jeffrey A

    2016-02-01

    Pralatrexate (PDX) is a folate antagonist structurally similar to methotrexate (MTX). Unlike MTX, it is currently not known whether PDX exhibits delayed clearance and heightened toxicity in the setting of fluid overload. A specific serum assay for PDX is not commercially available. To our knowledge, we report the first case using an MTX serum assay as a surrogate for PDX concentrations to avoid a potential drug-drug interaction with pralatrexate. We describe a 76-year-old man with refractory cutaneous T-cell lymphoma who began therapy with weekly PDX 15 mg/m(2) intravenous infusions on days 1, 8, and 15 of a 28-day cycle. He subsequently developed mucositis, a moderate right-sided pleural effusion, and peripheral edema over the next 5 weeks. Aggressive diuresis with furosemide was initiated, which was then withheld the day before his next PDX dose to avoid a potential drug-drug interaction between PDX and furosemide. His baseline MTX/PDX concentration (measured prior to administration of the cycle 2, week 2 PDX dose) was less than 0.20 μmol/L (i.e., undetectable). After PDX administration, his 1-hour peak MTX/PDX concentration increased to 0.58 μmol/L. Aggressive diuresis was withheld until his MTX/PDX concentration was undetectable, 43.5 hours later. PDX is more potent than MTX and displays similar pharmacokinetic properties. PDX concentrations using the serum MTX assay reflect lower values than those reported from PDX-specific assays in clinical studies. Because PDX is approved by the U.S. Food and Drug Administration for the treatment of uncommon malignancies, it is unlikely that a specific assay will be commercially developed. We propose that the MTX serum assay has merit for use in determining when to reinstate possible interacting drug therapies such as loop diuretics. PMID:26809959

  6. 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

  7. DNA intercalative potential of marketed drugs testing positive in in vitro cytogenetics assays.

    PubMed

    Snyder, Ronald D; Ewing, Douglas; Hendry, Lawrence B

    2006-10-10

    We have previously noted that the Physicians' Desk Reference (PDR) contains over 80 instances in which a drug elicited a positive genotoxic response in one or more in vitro assays, despite having no obvious structural features predictive of covalent drug/DNA interactive potential or known mechanistic basis. Furthermore, in most cases, these drugs were "missed" by computational genotoxicity-predicting models such as DEREK, MCASE and TOPKAT. We have previously reported the application of a V79 cell-based model and a 3D DNA docking model for predicting non-covalent chemical/DNA interactions. Those studies suggested that molecules that are very widely structurally diverse may be capable of intercalating into DNA. To determine whether such non-covalent drug/DNA interactions might be involved in unexpected drug genotoxicity, we evaluated, using both models where possible, 56 marketed pharmaceuticals, 40 of which were reported as being clastogenic in in vitro cytogenetics assays (chromosome aberrations/mouse lymphoma assay). As seen before, the two approaches showed good concordance (62%) and 26 of the 40 (65%) drugs exhibiting in vitro clastogenicity were predicted as intercalators by one or both methods. This finding provides support for the hypothesis that non-covalent DNA interaction may be a common mechanism of clastogenicity for many drugs having no obvious structural alerts for covalent DNA interaction. PMID:16857419

  8. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence–Quencher Probe as a Tool for Functional Antibody Screening

    PubMed Central

    Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D.; Hiraga, Kaori

    2015-01-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody–drug conjugates. Here we describe a novel activatable fluorescence–quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

  9. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening.

    PubMed

    Li, Yan; Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D; Hiraga, Kaori

    2015-08-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody-drug conjugates. Here we describe a novel activatable fluorescence-quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

  10. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay is a rapid, cheap, screening test for the in vitro anti-tuberculous activity of chalcones.

    PubMed

    Moodley, Suventha; Koorbanally, Neil A; Moodley, Thrineshen; Ramjugernath, Deresh; Pillay, Manormoney

    2014-09-01

    Rapid and reliable drug susceptibility testing facilitates replenishment of the TB drug pipeline in the fight against drug resistant Mycobacterium tuberculosis. This study compared the performance of the MTT and MABA assays on the anti-tuberculous activity of a set of chalcones. Twenty seven chalcones and chromenochalcones were screened against the laboratory strain M. tuberculosis H37Rv, using a microtitre plate MTT assay at 7 days. The MIC for 20 active compounds was subsequently determined using the MABA, MTT and the Macroscopic broth assays at 7, 14 and 21 days. No significant difference in the MICs, or increase in the MICs was observed over time between the MABA (p=0.209) and the MTT (p=0.207) assays, in contrast to the gold standard, the Macroscopic broth assay (p=0.000). The MICs (16 to >128μg/ml) were much higher than the currently used TB drugs. In conclusion, the MTT assay is a cost effective method (R0.06/well) for the rapid in vitro screening of chalcones against M. tuberculosis, producing reliable results in 8 days. The chalcone with a MIC of 16μg/mL shows promise as a potential lead compound and should be investigated further. PMID:24978593

  11. Animal models for screening anxiolytic-like drugs: a perspective

    PubMed Central

    Bourin, Michel

    2015-01-01

    Contemporary biological psychiatry uses experimental animal models to increase our understanding of affective disorder pathogenesis. Modern anxiolytic drug discovery mainly targets specific pathways and molecular determinants within a single phenotypic domain. However, greater understanding of the mechanisms of action is possible through animal models. Primarily developed with rats, animal models in anxiety have been adapted with mixed success for mice, easy-to-use mammals with better genetic possibilities than rats. In this review, we focus on the three most common animal models of anxiety in mice used in the screening of anxiolytics. Both conditioned and unconditioned models are described, in order to represent all types of animal models of anxiety. Behavioral studies require careful attention to variable parameters linked to environment, handling, or paradigms; this is also discussed. Finally, we focus on the consequences of re-exposure to the apparatus. Test-retest procedures can provide new answers, but should be intensively studied in order to revalidate the entire paradigm as an animal model of anxiety. PMID:26487810

  12. Multicenter evaluation of the nitrate reductase assay for drug resistance detection of Mycobacterium tuberculosis.

    PubMed

    Martin, Anandi; Montoro, Ernesto; Lemus, Dihadenys; Simboli, Norberto; Morcillo, Nora; Velasco, Maritza; Chauca, José; Barrera, Lucía; Ritacco, Viviana; Portaels, Françoise; Palomino, Juan Carlos

    2005-11-01

    The performance of the nitrate reductase assay was evaluated in a multicenter laboratory study to detect resistance of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin, isoniazid, ethambutol and streptomycin using a set of coded isolates. Compared with the gold standard proportion method on Löwenstein-Jensen medium, the assay was highly accurate in detecting resistance to rifampicin, isoniazid and ethambutol with an accuracy of 98%, 96.6% and 97.9%, respectively. For streptomycin, discrepant results were obtained with an overall accuracy of 85.3%. The assay proved easy to be implemented in countries with limited laboratory facilities. PMID:15893391

  13. 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

  14. 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

  15. Drug and bioactive molecule screening based on a bioelectrical impedance cell culture platform

    PubMed Central

    Ramasamy, Sakthivel; Bennet, Devasier; Kim, Sanghyo

    2014-01-01

    This review will present a brief discussion on the recent advancements of bioelectrical impedance cell-based biosensors, especially the electric cell-substrate impedance sensing (ECIS) system for screening of various bioactive molecules. The different technical integrations of various chip types, working principles, measurement systems, and applications for drug targeting of molecules in cells are highlighted in this paper. Screening of bioactive molecules based on electric cell-substrate impedance sensing is a trial-and-error process toward the development of therapeutically active agents for drug discovery and therapeutics. In general, bioactive molecule screening can be used to identify active molecular targets for various diseases and toxicity at the cellular level with nanoscale resolution. In the innovation and screening of new drugs or bioactive molecules, the activeness, the efficacy of the compound, and safety in biological systems are the main concerns on which determination of drug candidates is based. Further, drug discovery and screening of compounds are often performed in cell-based test systems in order to reduce costs and save time. Moreover, this system can provide more relevant results in in vivo studies, as well as high-throughput drug screening for various diseases during the early stages of drug discovery. Recently, MEMS technologies and integration with image detection techniques have been employed successfully. These new technologies and their possible ongoing transformations are addressed. Select reports are outlined, and not all the work that has been performed in the field of drug screening and development is covered. PMID:25525360

  16. Development and validation of cell-based assays for the detection of neutralizing antibodies to drug products: a practical approach.

    PubMed

    Jolicoeur, Pierre; Tacey, Richard L

    2012-12-01

    Neutralizing antibodies (NAbs) that bind to drug products and may diminish or eliminate the associated biological activity are an unintended and undesirable outcome of some drug products. Standard immunoassays can detect drug-specific antibodies but cannot distinguish NAbs, so cell-based assays are often preferred because they closely mimic the mechanism by which NAbs and drug products interact in vivo. Each cell-based NAb assay is unique and based on several factors, such as the drug product, study population and phase of development (preclinical or clinical). The type of NAb assay (direct or indirect) depends on the drug's mechanism of action. Key steps in assay development are: selecting a suitable cell line, choosing the proper cellular response (end point method), selection of proper controls and optimization of assay parameters. Once developed, the assay must be rigorously tested (validated) to ensure that it meets several important criteria and is fit for its intended purpose. PMID:23244285

  17. diskImageR: quantification of resistance and tolerance to antimicrobial drugs using disk diffusion assays.

    PubMed

    Gerstein, Aleeza C; Rosenberg, Alexander; Hecht, Inbal; Berman, Judith

    2016-07-01

    Microbial pathogens represent an increasing threat to human health. Although many infections can be successfully treated and cleared, drug resistance is a widespread problem. The existence of subpopulations of 'tolerant' cells (where a fraction of the population is able to grow above the population resistance level) may increase the rate of treatment failure; yet, existing methods to measure subpopulation effects are cumbersome. Here we describe diskImageR, a computational pipeline that analyses photographs of disk diffusion assays to determine the degree of drug susceptibility [the radius of inhibition, (RAD)], and two aspects of subpopulation growth [the fraction of growth (FoG) within the zone of inhibition, (ZOI), and the rate of change in growth from no drug to inhibitory drug concentrations, (SLOPE)]. diskImageR was used to examine the response of the human fungal pathogen Candida albicans to the antifungal drug fluconazole across different strain backgrounds and growth conditions. Disk diffusion assays performed under Clinical and Laboratory Standards Institute (CLSI) conditions led to more susceptibility and less tolerance than assays performed using rich medium conditions. We also used diskImageR to quantify the effects of three drugs in combination with fluconazole, finding that all three combinations affected tolerance, with the effect of one drug (doxycycline) being very strain dependent. The three drugs had different effects on susceptibility, with doxycycline generally having no effect, chloroquine generally increasing susceptibility and pyrvinium pamoate generally reducing susceptibility. The ability to simultaneously quantitate different aspects of microbial drug responses will facilitate the study of mechanisms of subpopulation responses in the presence of antimicrobial drugs. PMID:27126388

  18. The current status of time dependent CYP inhibition assay and in silico drug-drug interaction predictions.

    PubMed

    Yan, Zhengyin; Caldwell, Gary W

    2012-01-01

    Various CYP time-dependent inhibition (TDI) assays have been widely implemented in drug discovery and development which has led to great success in positively identifying compounds with mechanism-base inhibition liability. However, drug-drug interaction (DDI) predictions by various in-silico models utilizing kinetic parameters obtained from TDI assays have met with significant challenges including questionable kinetic data, over-simplified in-vitro models and unreliable mathematic algorithms. Although significant efforts have been made to standardize the TDI assay and refine mathematical models, recent evaluation studies have revealed that the kinetic parameters of TDI, the most important in-vitro data required by all DDI prediction models, are significantly impacted by a variety of experimental variables including microsomal protein concentration, metabolic stability, CYP-specific probes, and post-incubation time. This review attempts to provide medicinal chemists a brief overview on the current status of TDI assays, determination of kinetic parameters and in silico DDI predictions with emphasis on the complexity of the TDI kinetics and limitations of current in-vitro models and DDI prediction methodologies. PMID:22571791

  19. Electrochemical assay of α-glucosidase activity and the inhibitor screening in cell medium.

    PubMed

    Zhang, Juan; Liu, Ying; Wang, Xiaonan; Chen, Yangyang; Li, Genxi

    2015-12-15

    An electrochemical method is established in this work for the assay of α-glucosidase activity and the inhibitor screening through one-step displacement reaction, which can be directly used in cell medium. The displacement reaction can be achieved via strong binding of 4-aminophenyl-α-D-glucopyranoside (pAPG)/magnetic nanoparticles (MNPs) to pyrene boric acid (PBA) immobilized on the surface of graphite electrode (GE), compared to that of dopamine (DA)/sliver nanoparticles (AgNPs). Since α-glucosidase can specifically catalyze MNPs/pAPG into MNPs/pAP which has no binding capacity with PBA, the activity of both isolated and membrane bound enzyme can be well evaluated by using this proposed method. Meanwhile, signal amplification can be accomplished via the immobilization of DA at the outer layer of AgNPs, and the accuracy can be strengthened through magnetic separation. Moreover, this method can also be utilized for inhibitor screening not only in the medium containing the enzyme but also in cell medium. With good precision and accuracy, it may be extended to other proteases and their inhibitors as well. PMID:26201984

  20. Implementation of an interferon-gamma release assay to screen for tuberculosis in refugees and immigrants.

    PubMed

    Simpson, Terri; Tomaro, Julie; Jobb, Cynthia

    2013-08-01

    Despite increased use and accuracy of interferon-gamma release assays to detect latent tuberculosis infection (LTBI) in foreign-born arrivals in the United States, risk characteristics associated with positive results are not well characterized. We conducted a retrospective record review of 541 refugees and immigrants screened for LTBI with QuantiFERON(®)-TB Gold In-Tube (QFT-IT) at the Spokane Public Health Clinic from January 2, 2008, through June 5, 2009. Overall, 24 % of the arrivals had a positive QFT-IT, with the greatest frequency of positive results occurring in arrivals from Liberia (100 %) and Bhutan (39 %). More than the expected number of Burmese had indeterminate QFT-IT results. A positive QFT-IT was associated with age, race, ethnicity, and extent of TB burden in the country of origin. QFT-IT is useful to screen for LTBI in foreign-born arrivals, particularly middle-aged adults from high-burden countries. However, the QFT-IT may not yield meaningful results in groups with significant immunocompromise. PMID:23179470

  1. A Fluorescence-Based Assay for Proteinuria Screening in Larval Zebrafish (Danio rerio).

    PubMed

    Hanke, Nils; King, Benjamin L; Vaske, Bernhard; Haller, Hermann; Schiffer, Mario

    2015-10-01

    Analysis of genes compromising the glomerular filtration barrier in rodent models using transgenic or knockdown approaches is time- and resource-consuming and often leads to unsatisfactory results. Therefore, it would be beneficial to have a selection tool indicating that your gene of interest is in fact associated with proteinuria. Zebrafish (Danio rerio) is a rapid screening tool to study effects in glomerular filtration barrier integrity after genetic manipulation. We use either injection of high-molecular-weight dextrans or a transgenic fluorescent fish line [Tg(l-fabp:DBP:EGFP)] expressing a vitamin D-binding protein fused with eGFP for indirect detection of proteinuria. A loss of high-molecular-weight proteins from the circulation of the fish into the urine can be identified by monitoring fluorescence intensity in the zebrafish eye. Paired with an optimized analysis method, this assay provides an effective screening solution to detect filtration barrier damage with proteinuria before moving to a mammalian system. PMID:26125680

  2. Detection and prevalence of drug use in arrested drivers using the Dräger Drug Test 5000 and Affiniton DrugWipe oral fluid drug screening devices.

    PubMed

    Logan, Barry K; Mohr, Amanda L A; Talpins, Stephen K

    2014-09-01

    The use of oral fluid (OF) drug testing devices offers the ability to rapidly obtain a drug screening result at the time of a traffic stop. We describe an evaluation of two such devices, the Dräger Drug Test 5000 and the Affiniton DrugWipe, to detect drug use in a cohort of drivers arrested from an investigation of drug impaired driving (n = 92). Overall, 41% of these drivers were ultimately confirmed positive by mass spectrometry for the presence of one or more drugs. The most frequently detected drugs were cannabinoids (30%), benzodiazepines (11%) and cocaine (10%). Thirty-nine percent of drivers with blood alcohol concentrations >0.08 g/100 mL were found to be drug positive. Field test results obtained from OF samples were compared with collected OF and urine samples subsequently analyzed in the laboratory by gas or liquid chromatography-mass spectrometry. The Dräger Drug Test 5000 (DDT5000) and DrugWipe returned overall sensitivities of 51 and 53%, and positive predictive values of 93 and 63%, respectively. The most notable difference in performance was the DDT5000's better sensitivity in detecting marijuana use. Both devices failed to detect benzodiazepine use. Oral fluid proved to be a more effective confirmatory specimen, with more drugs being confirmed in OF than urine. PMID:24894458

  3. Non-invasive screening of cytochrome c oxidase deficiency in children using a dipstick immunocapture assay.

    PubMed

    Rodinová, M; Trefilová, E; Honzík, T; Tesařová, M; Zeman, J; Hansíková, H

    2014-01-01

    Cytochrome c oxidase (CIV) deficiency is among the most common childhood mitochondrial disorders. The diagnosis of this deficiency is complex, and muscle biopsy is used as the gold standard of diagnosis. Our aim was to minimize the patient burden and to test the use of a dipstick immunocapture assay (DIA) to determine the amount of CIV in non-invasively obtained buccal epithelial cells. Buccal smears were obtained from five children with Leigh syndrome including three children exhibiting a previously confirmed CIV deficiency in muscle and fibroblasts and two children who were clinical suspects for CIV deficiency; the smear samples were analysed using CI and CIV human protein quantity dipstick assay kits. Samples from five children of similar age and five adults were used as controls. Analysis of the controls demonstrated that only samples of buccal cells that were frozen for a maximum of 4 h after collection provide accurate results. All three patients with confirmed CIV deficiency due to mutations in the SURF1 gene exhibited significantly lower amounts of CIV than the similarly aged controls; significantly lower amounts were also observed in two new patients, for whom later molecular analysis also confirmed pathologic mutations in the SURF1 gene. We conclude that DIA is a simple, fast and sensitive method for the determination of CIV in buccal cells and is suitable for the screening of CIV deficiency in non-invasively obtained material from children who are suspected of having mitochondrial disease. PMID:25629267

  4. Development of a screening system for cystic fibrosis: meconium or blood spot trypsin assay or both?

    PubMed

    Pederzini, F; Faraguna, D; Giglio, L; Pedrotti, D; Perobelli, L; Mastella, G

    1990-10-01

    High blood trypsin levels during early days of life are found in newborns subsequently diagnosed to be affected by cystic fibrosis. The authors compared the validity of the traditional meconium test with the blood immunoreactive trypsin (IRT) assay, carried out in parallel on 113,302 neonates from three regions of North-eastern Italy. The meconium test showed a sensitivity of 57.7%. The sensitivity of the IRT test was higher (96.1%). It was possible to identify by IRT 10 out of 11 false negative CFs at the meconium test. A shortcoming of neonatal IRT, however, is its low specificity; 1.6% of the newborns had to be retested. A new screening policy was therefore proposed and carried out on 69,640 newborns: the Lactase test (LACT) on meconium was introduced as a complementary assay in IRT positive newborns. If LACT exceeded 2 U/g dry meconium, a confirmatory sweat test was immediately requested; if LACT test was negative and IRT exceeded 85 micrograms/l, IRT was repeated. Postneonatal retesting values above 25 micrograms/l required a sweat test. As a result, the estimated prevalence of CF was 1:4,352, the sensitivity was 93.3%; the specificity turned out to be 99.6%, considering all false positive newborns investigated with retesting and/or direct sweat test. PMID:2124772

  5. 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

  6. GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations

    SciTech Connect

    Cronn, M.T.; Miyada, C.G.; Fucini, R.V.

    1994-09-01

    GeneChip{sup {trademark}} assays are based on high density, carefully designed arrays of short oligonucleotide probes (13-16 bases) built directly on derivatized silica substrates. DNA target sequence analysis is achieved by hybridizing fluorescently labeled amplification products to these arrays. Fluorescent hybridization signals located within the probe array are translated into target sequence information using the known probe sequence at each array feature. The mutation screening assay for cystic fibrosis includes sets of oligonucleotide probes designed to detect numerous different mutations that have been described in 14 exons and one intron of the CFTR gene. Each mutation site is addressed by a sub-array of at least 40 probe sequences, half designed to detect the wild type gene sequence and half designed to detect the reported mutant sequence. Hybridization with homozygous mutant, homozygous wild type or heterozygous targets results in distinctive hybridization patterns within a sub-array, permitting specific discrimination of each mutation. The GeneChip probe arrays are very small (approximately 1 cm{sup 2}). There miniature size coupled with their high information content make GeneChip probe arrays a useful and practical means for providing CF mutation analysis in a clinical setting.

  7. Library screening by means of mass spectrometry (MS) binding assays-exemplarily demonstrated for a pseudostatic library addressing γ-aminobutyric acid (GABA) transporter 1 (GAT1).

    PubMed

    Sindelar, Miriam; Wanner, Klaus T

    2012-09-01

    In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ-aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine-3-carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays-the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1-{2-[2'-(1,1'-biphenyl-2-ylmethylidene)hydrazine]ethyl}piperidine-3-carboxylic acid and 1-(2-{2'-[1-(2-thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine-3-carboxylic acid, were found to be potent GAT1 ligands exhibiting pK(i) values of 6.186 ± 0.028 and 6.229 ± 0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including

  8. Establishment of a novel cell-based assay for screening small molecule antagonists of human interleukin-6 receptor

    PubMed Central

    He, Yang-yang; Yan, Yu; Zhang, Chang; Li, Peng-yuan; Wu, Ping; Du, Peng; Zeng, Da-di; Fang, Jian-song; Wang, Shuang; Du, Guan-hua

    2014-01-01

    Aim: Blockade of interleukin-6 (IL-6) or its receptor (IL-6R) is effective in preventing the progression of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. In the present study, we established a novel cell-based assay for identifying small molecule IL-6R antagonists. Methods: HEK293A cells were transfected with recombinant plasmids pTaglite-SNAP-IL6R and pABhFc-IL6 to obtain membrane-bound IL-6R and recombinant human IL-6 coupled with human Fc fragment (rhIL-6), respectively. A novel screening assay based on the interaction between IL-6R and rhIL-6 was established, optimized and validated. The stability of the assay was also assessed by calculating the Z′-factor. Results: RhIL-6 dose-dependently bound to IL-6R expressed at HEK293A cell surface. The IC50 value of the known antagonist ab47215 was 0.38±0.08 μg/mL, which was consistent with that obtained using the traditional method (0.36±0.14 μg/mL). The value of Z′-factor was 0.68, suggesting that the novel assay was stable for high throughput screening. A total of 474 compounds were screened using the novel screening assay, and 3 compounds exhibited antagonistic activities (IC50=8.73±0.28, 32.32±9.08, 57.83±4.24 μg/mL). Furthermore, the active compounds dose-dependently inhibited IL-6-induced proliferation of 7TD1 cells, and reduced IL-6-induced STAT3 phosphorylation in U937 cells. Conclusion: A novel cell-based screening assay for identifying small molecule IL-6R antagonists was established, which simplifies the procedures in traditional cellular ELISA screening and profiling and reduces the costs. PMID:25345743

  9. Cross-reactivity between Lyme and syphilis screening assays: Lyme disease does not cause false-positive syphilis screens.

    PubMed

    Patriquin, Glenn; LeBlanc, Jason; Heinstein, Charles; Roberts, Catherine; Lindsay, Robbin; Hatchette, Todd F

    2016-03-01

    Increased rates of Lyme disease and syphilis in the same geographic area prompted an assessment of screening test cross-reactivity. This study supports the previously described cross-reactivity of Lyme screening among syphilis-positive sera and reports evidence against the possibility of false-positive syphilis screening tests resulting from previous Borrelia burgdorferi infection. PMID:26707064

  10. Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine.

    PubMed

    Goodell, C K; Prickett, J; Kittawornrat, A; Johnson, J; Zhang, J; Wang, C; Zimmerman, J J

    2016-02-01

    Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti-IAV antibodies using homologous and heterologous haemagglutination-inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)-blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut-off of S/N ≤ 0.60, the sensitivity and specificity of the NP-blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post-inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost-effective approach for the detection and surveillance of IAV infections in swine populations. PMID:24571447

  11. Rapid facile solid-phase immunobead assay for screening ciguatoxic fish in the market place.

    PubMed

    Park, D L; Gamboa, P M; Goldsmith, C H

    1992-01-01

    The precision of the solid-phase immunobead assay (Ciguatect) to detect toxins associated with ciguatera poisoning have been evaluated through analysis of toxic and non-toxic fish obtained from fishing areas around the Hawaiian Islands. The Ciguatect test kit has been optimized for application to field/marketplace screening of ciguatoxic fish. Twelve parrot, surgeon, and amberjack fish fillet and fish extract test portions containing various concentrations of toxins were distributed to participating laboratories for analysis. The presence or absence of ciguatera-related toxins is determined by binding the toxins to a membrane attached to a plastic strip and exposing the toxin ladened membrane to a monoclonal antibody-colored latex bead complex which has a high specificity for ciguatera-related toxins. The intensity of the color on the membrane denotes the presence of the toxins in the fish or fish extract. Toxic components in the fish were confirmed by extraction, column purification, and toxicity testing using the brine shrimp (Artemia sp.) assay. Okadaic acid was used to standardize both the S-PIA and brine shrimp assays. For determination of ciguatoxin and related polyether toxins in parrot, surgeon, and amberjack fish fillets, the relative standard deviations for repeatability (RSDR) were 13.5, 9.0 and 4.3%, respectively, and the relative standard deviations for reproducibility (RSDR) were 44.4, 29.7 and 14.3%, respectively, for concentrations ranging from 1-4 ng/test strip. For determination of ciguatoxin and related polyether toxins in parrot, surgeon, and amberjack fish extracts, the RSDR were 5.8, 4.8, and 3.7%, respectively, and the RSDR were 11.9, 9.9, and 7.6%, respectively, for concentrations ranging from 3-5 ng/test strip.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1340354

  12. Toward antituberculosis drugs: in silico screening of synthetic compounds against Mycobacterium tuberculosisl,d-transpeptidase 2.

    PubMed

    Billones, Junie B; Carrillo, Maria Constancia O; Organo, Voltaire G; Macalino, Stephani Joy Y; Sy, Jamie Bernadette A; Emnacen, Inno A; Clavio, Nina Abigail B; Concepcion, Gisela P

    2016-01-01

    Mycobacterium tuberculosis (Mtb) the main causative agent of tuberculosis, is the main reason why this disease continues to be a global public health threat. It is therefore imperative to find a novel antitubercular drug target that is unique to the structural machinery or is essential to the growth and survival of the bacterium. One such target is the enzyme l,d-transpeptidase 2, also known as LdtMt2, a protein primarily responsible for the catalysis of 3→3 cross-linkages that make up the mycolyl-arabinogalactan-peptidoglycan complex of Mtb. In this study, structure-based pharmacophore screening, molecular docking, and in silico toxicity evaluations were employed in screening compounds from a database of synthetic compounds. Out of the 4.5 million database compounds, 18 structures were identified as high-scoring, high-binding hits with very satisfactory absorption, distribution, metabolism, excretion, and toxicity properties. Two out of the 18 compounds were further subjected to in vitro bioactivity assays, with one exhibiting a good inhibitory activity against the Mtb H37Ra strain. PMID:27042006

  13. Study on microvisualizing assay of delivered drug infiltration using 2-color optical coherence dosigraphy

    NASA Astrophysics Data System (ADS)

    Nakamichi, Yu; Saeki, Souichi; Saito, Takashi; Hiro, Takafumi; Matsuzaki, Masunori

    2009-02-01

    Recently, clinical treatments applying drug delivery system (DDS) have been being developed. However, it is quite difficult to in vivo diagnose spatiotemporal distribution of drug infiltration, so the validation study should be too insufficient to progress the DDS development. In this study, we propose a visualizing assay of DDS, namely 2-Color Optical Coherence Dosigraphy (2C-OCD). 2C-OCD is based on optical coherence tomography using two waveband "2-Color" light sources having different optical absorbance of drug. This can simultaneously provide microscale tomographic images of scatterer density and drug concentration. In order to evaluate the efficacy of this technique, this was applied to drug-diffusion phenomena in microchannel and lipidrich plaques of rabbit with drug administration, respectively. As a result of diffusion experiment, it was confirmed that 2C-OCD can visualize a cross-sectional map of drug concentration, with spatial resolution 5 micro m × 10 μm and accuracy plus-minus 13.0 μM. In ex vivo animal experiment, the enhancement of absorptivity could be observed inside lipidrich plaques, in which DDS drug could be therein uptaken by drug administration. The absorption maps corresponding to drug concentration were calculated, comparing with their histological images. Consequently, they had good coincidence with histological examinations, therefore, it was concluded that 2C-OCD could visualize drug infiltration in biological tissue with almost the same spatial resolution as OCT system.

  14. Screening and identification of proteins interacting with IL-24 by the yeast two-hybrid screen, Co-IP, and FRET assays.

    PubMed

    Hu, Hui; Wang, Tao; Chen, Jiaojiao; Yu, Fang; Liu, Huilin; Zuo, Zhenyu; Yang, Zhonghua; Fan, Handong

    2016-04-01

    Interleukin-24 (IL-24) is an ideal tumor-suppressor gene, but the mechanisms underlying its antitumor specificity remain to be elucidated. The best way to investigate these problems is to begin from the initiation of corresponding signaling cascades activated by IL-24 with screening and identifying those proteins that interacted with IL-24. With the aim of identifying these initial interactions, a yeast two-hybrid screening was performed by transforming AH109 cells containing PGBKT7-IL-24 with a liver cDNA plasmid library. These cells were then plated on synthetic nutrient medium (SD/-Trp/-Leu/-His) for the first screening and on quadruple dropout medium containing X-α-gal for the second screening. Positive colonies were further verified by repeating the MATE experiments, co-immunoprecipitation (Co-IP) analysis, and fluorescence resonance energy transfer (FRET) assays in vitro. Following the yeast two-hybrid screening, 15 genes were selected for sequencing, with two genes, HLA-C and NDUFA13, further verified using Co-IP assays and FRET assays. Both HLA-C and NDUFA13 were found to interact with IL-24. We found that HLA-C and NDUFA13 could interact with IL-24 and it may be involved in the signal induced by IL-24. Overall, this study contributes further insight into the cancer-specific apoptosis-inducing abilities of IL-24 to potentially enhance its therapeutic potential, and it also provides outlets for other biological functions of IL-24. PMID:26930462

  15. Development of a web-based tool for automated processing and cataloging of a unique combinatorial drug screen.

    PubMed

    Dalecki, Alex G; Wolschendorf, Frank

    2016-07-01

    Facing totally resistant bacteria, traditional drug discovery efforts have proven to be of limited use in replenishing our depleted arsenal of therapeutic antibiotics. Recently, the natural anti-bacterial properties of metal ions in synergy with metal-coordinating ligands have shown potential for generating new molecule candidates with potential therapeutic downstream applications. We recently developed a novel combinatorial screening approach to identify compounds with copper-dependent anti-bacterial properties. Through a parallel screening technique, the assay distinguishes between copper-dependent and independent activities against Mycobacterium tuberculosis with hits being defined as compounds with copper-dependent activities. These activities must then be linked to a compound master list to process and analyze the data and to identify the hit molecules, a labor intensive and mistake-prone analysis. Here, we describe a software program built to automate this analysis in order to streamline our workflow significantly. We conducted a small, 1440 compound screen against M. tuberculosis and used it as an example framework to build and optimize the software. Though specifically adapted to our own needs, it can be readily expanded for any small- to medium-throughput screening effort, parallel or conventional. Further, by virtue of the underlying Linux server, it can be easily adapted for chemoinformatic analysis of screens through packages such as OpenBabel. Overall, this setup represents an easy-to-use solution for streamlining processing and analysis of biological screening data, as well as offering a scaffold for ready functionality expansion. PMID:27117032

  16. The Drug Use Screening Inventory: School Adjustment Correlates of Substance Abuse.

    ERIC Educational Resources Information Center

    Tarter, Ralph E.; And Others

    1996-01-01

    The Drug Use Screening Inventory's (DUSI) sensitivity in detecting adolescents (N=706) who abuse drugs is demonstrated. Severity of drug involvement and psychiatric disturbance correlated with scores on the School Adjustment domain. Recommends additional research be conducted to determine the predictive validity and temporal stability of DUSI…

  17. Development of a voltammetric assay, using screen-printed electrodes, for clonazepam and its application to beverage and serum samples.

    PubMed

    Honeychurch, Kevin C; Brooks, Joshua; Hart, John P

    2016-01-15

    This paper describes the development of an electrochemical assay based on screen-printed carbon sensors for the determination of clonazepam in serum and in wine. The cyclic voltammetric behaviour of the drug was investigated and the effects of pH and scan rate on the peak current and peak potential determined. Two reduction peaks were recorded on the initial negative going scan, which were considered to result from the 2e(-), 2 H(+) reduction of the 4,5-azomethine and from the 4e(-), 4 H(+) reduction of the 7-NO2 to a hydroxylamine. On the return positive going scan an oxidation peak was seen, which was considered to result from the 2e(-), 2 H(+) oxidation (O1) of the hydroxylamine to the corresponding nitroso species. At pH 11 the solution of clonazepam was found to turn from clear to yellow in colour and the voltammetric signal of the O1 oxidation process was found to be adsorptive in nature, this was exploited in the development of an adsorptive stripping voltammetric assay. Experimental conditions were then optimised for the differential pulse adsorptive voltammetric measurement of clonazepam in wine and serum samples. It was shown that these analyses could be performed on only 100µL of sample which was deposited on the sensor surface. Mean recoveries of 79.53% (%CV=9.88%) and 88.22% (%CV=14.1%) were calculated for wine fortified with 3.16µg/mL and serum fortified with 12.6µg/mL. PMID:26592640

  18. High-throughput matrix screening identifies synergistic and antagonistic antimalarial drug combinations.

    PubMed

    Mott, Bryan T; Eastman, Richard T; Guha, Rajarshi; Sherlach, Katy S; Siriwardana, Amila; Shinn, Paul; McKnight, Crystal; Michael, Sam; Lacerda-Queiroz, Norinne; Patel, Paresma R; Khine, Pwint; Sun, Hongmao; Kasbekar, Monica; Aghdam, Nima; Fontaine, Shaun D; Liu, Dongbo; Mierzwa, Tim; Mathews-Griner, Lesley A; Ferrer, Marc; Renslo, Adam R; Inglese, James; Yuan, Jing; Roepe, Paul D; Su, Xin-Zhuan; Thomas, Craig J

    2015-01-01

    Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy. PMID:26403635

  19. High-throughput matrix screening identifies synergistic and antagonistic antimalarial drug combinations

    PubMed Central

    Mott, Bryan T.; Eastman, Richard T.; Guha, Rajarshi; Sherlach, Katy S.; Siriwardana, Amila; Shinn, Paul; McKnight, Crystal; Michael, Sam; Lacerda-Queiroz, Norinne; Patel, Paresma R.; Khine, Pwint; Sun, Hongmao; Kasbekar, Monica; Aghdam, Nima; Fontaine, Shaun D.; Liu, Dongbo; Mierzwa, Tim; Mathews-Griner, Lesley A.; Ferrer, Marc; Renslo, Adam R.; Inglese, James; Yuan, Jing; Roepe, Paul D.; Su, Xin-zhuan; Thomas, Craig J.

    2015-01-01

    Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs, and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators, and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy. PMID:26403635

  20. Identifying New Drug Targets for Potent Phospholipase D Inhibitors: Combining Sequence Alignment, Molecular Docking, and Enzyme Activity/Binding Assays.

    PubMed

    Djakpa, Helene; Kulkarni, Aditya; Barrows-Murphy, Scheneque; Miller, Greg; Zhou, Weihong; Cho, Hyejin; Török, Béla; Stieglitz, Kimberly

    2016-05-01

    Phospholipase D enzymes cleave phospholipid substrates generating choline and phosphatidic acid. Phospholipase D from Streptomyces chromofuscus is a non-HKD (histidine, lysine, and aspartic acid) phospholipase D as the enzyme is more similar to members of the diverse family of metallo-phosphodiesterase/phosphatase enzymes than phospholipase D enzymes with active site HKD repeats. A highly efficient library of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure was utilized to evaluate the inhibition of purified S. chromofuscus phospholipase D. The molecules exhibited inhibition of phospholipase D activity (IC50 ) in the nanomolar range with monomeric substrate diC4 PC and micromolar range with phospholipid micelles and vesicles. Binding studies with vesicle substrate and phospholipase D strongly indicate that these inhibitors directly block enzyme vesicle binding. Following these compelling results as a starting point, sequence searches and alignments with S. chromofuscus phospholipase D have identified potential new drug targets. Using AutoDock, inhibitors were docked into the enzymes selected from sequence searches and alignments (when 3D co-ordinates were available) and results analyzed to develop next-generation inhibitors for new targets. In vitro enzyme activity assays with several human phosphatases demonstrated that the predictive protocol was accurate. The strategy of combining sequence comparison, docking, and high-throughput screening assays has helped to identify new drug targets and provided some insight into how to make potential inhibitors more specific to desired targets. PMID:26691755

  1. A novel assay to assess the effectiveness of antiangiogenic drugs in human breast cancer.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many cytotoxic drugs maintain antiangiogenic properties, but there are no human, tumor-based assays to evaluate their antiangiogenic potential. We used a fibrin-thrombin clot-based angiogenesis model to evaluate the angiogenic response of human breast cancer to various cytotoxic agents commonly used...

  2. Detection of Resistance to Second-Line Antituberculosis Drugs by Use of the Genotype MTBDRsl Assay: a Multicenter Evaluation and Feasibility Study

    PubMed Central

    Ignatyeva, Olga; Kontsevaya, Irina; Kovalyov, Alexander; Balabanova, Yanina; Nikolayevskyy, Vladislav; Toit, Kadri; Dragan, Anda; Maxim, Daniela; Mironova, Svetlana; Kummik, Tiina; Muntean, Ionela; Koshkarova, Ekaterina

    2012-01-01

    The rate of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has been steadily increasing in countries of the former USSR. The availability of rapid and reliable methods for the detection of drug resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the Genotype MTBDRsl assay compared to that of phenotypic drug susceptibility testing (Becton Dickinson Bactec MGIT 960 system) with a test panel of 200 Mycobacterium tuberculosis isolates at four sites in Eastern Europe. The interpretability of the Genotype MTBDRsl assay was over 95%. The sensitivity for the detection of resistance to fluoroquinolones, ethambutol, amikacin, and capreomycin varied between 77.3% and 92.3%; however, it was much lower for kanamycin (42.7%). The sensitivity for the detection of XDR TB was 22.6%. The test specificity was over 82% for all drugs. The assay presents a good screening tool for the rapid detection of resistance to individual second-line drugs and can be recommended for use in countries with a high burden of MDR/XDR TB. The sensitivity for the detection of kanamycin resistance needs improvement. PMID:22378910

  3. New drug susceptibility test for Mycobacterium tuberculosis using the hybridization protection assay.

    PubMed Central

    Miyamoto, J; Koga, H; Kohno, S; Tashiro, T; Hara, K

    1996-01-01

    We developed a novel method for early detection of drug-resistant strains of Mycobacterium tuberculosis by using the hybridization protection assay (HPA). The number of viable bacteria during the incubation period correlated well with the number of relative light units measured by the HPA. In addition, the relative light unit values of susceptible strains on the first, third and fifth days of incubation were significantly different from those of resistant strains for both isoniazid and rifampin. Our results suggest that after isolation of the organism from clinical specimens, drug-resistant strains of M. tuberculosis are accurately detected by the HPA even after 1 day of incubation with the drug. PMID:8727932

  4. Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome1,2,3

    PubMed Central

    Dinday, Matthew T.

    2015-01-01

    Abstract Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate salient phenotypes associated with DS, including seizures, early fatality, and resistance to antiepileptic drugs. To discover new drug candidates for the treatment of DS, we screened a chemical library of ∼1000 compounds and identified 4 compounds that rescued the behavioral seizure component, including 1 compound (dimethadione) that suppressed associated electrographic seizure activity. Fenfluramine, but not huperzine A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were also identified. We conclude that the combination of behavioral and electrophysiological assays provide a convenient, sensitive, and rapid basis for phenotype-based drug screening in zebrafish mimicking a genetic form of epilepsy. PMID:26465006

  5. Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome(1,2,3).

    PubMed

    Dinday, Matthew T; Baraban, Scott C

    2015-01-01

    Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate salient phenotypes associated with DS, including seizures, early fatality, and resistance to antiepileptic drugs. To discover new drug candidates for the treatment of DS, we screened a chemical library of ∼1000 compounds and identified 4 compounds that rescued the behavioral seizure component, including 1 compound (dimethadione) that suppressed associated electrographic seizure activity. Fenfluramine, but not huperzine A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were also identified. We conclude that the combination of behavioral and electrophysiological assays provide a convenient, sensitive, and rapid basis for phenotype-based drug screening in zebrafish mimicking a genetic form of epilepsy. PMID:26465006

  6. Network-based in silico drug efficacy screening

    PubMed Central

    Guney, Emre; Menche, Jörg; Vidal, Marc; Barábasi, Albert-László

    2016-01-01

    The increasing cost of drug development together with a significant drop in the number of new drug approvals raises the need for innovative approaches for target identification and efficacy prediction. Here, we take advantage of our increasing understanding of the network-based origins of diseases to introduce a drug-disease proximity measure that quantifies the interplay between drugs targets and diseases. By correcting for the known biases of the interactome, proximity helps us uncover the therapeutic effect of drugs, as well as to distinguish palliative from effective treatments. Our analysis of 238 drugs used in 78 diseases indicates that the therapeutic effect of drugs is localized in a small network neighborhood of the disease genes and highlights efficacy issues for drugs used in Parkinson and several inflammatory disorders. Finally, network-based proximity allows us to predict novel drug-disease associations that offer unprecedented opportunities for drug repurposing and the detection of adverse effects. PMID:26831545

  7. Raman micro spectroscopy for in vitro drug screening: subcellular localisation and interactions of doxorubicin.

    PubMed

    Farhane, Z; Bonnier, F; Casey, A; Byrne, H J

    2015-06-21

    Vibrational spectroscopy, including Raman micro spectroscopy, has been widely used over the last few years to explore potential biomedical applications. Indeed, Raman micro spectroscopy has been demonstrated to be a powerful non-invasive tool in cancer diagnosis and monitoring. In confocal microscopic mode, the technique is also a molecularly specific analytical tool with optical resolution which has potential applications in subcellular analysis of biochemical processes, and therefore as an in vitro screening tool of the efficacy and mode of action of, for example, chemotherapeutic agents. In order to demonstrate and explore the potential in this field, established, model chemotherapeutic agents can be valuable. In this study paper, Raman micro spectroscopy coupled with confocal microscopy were used for the localization and tracking of the commercially available drug, doxorubicin (DOX), in the intracellular environment of the lung cancer cell line, A549. Cytotoxicity assays were employed to establish clinically relevant drug doses for 24 h exposure, and Confocal Laser Scanning Fluorescence Microscopy was conducted in parallel with Raman micro spectroscopy profiling to confirm the drug internalisation and localisation. Multivariate statistical analysis, consisting of PCA (principal components analysis) was used to highlight doxorubicin interaction with cancer cells and spectral variations due to its effects before and after DOX spectral features subtraction from nuclear and nucleolar spectra, were compared to non-exposed control spectra. Results show that Raman micro spectroscopy is not only able to detect doxorubicin inside cells and profile its specific subcellular localisation, but, it is also capable of elucidating the local biomolecular changes elicited by the drug, differentiating the responses in different sub cellular regions. Further analysis clearly demonstrates the early apoptotic effect in the nuclear regions and the initial responses of cells to this

  8. In vitro screening of clinical drugs identifies sensitizers of oncolytic viral therapy in glioblastoma stem-like cells.

    PubMed

    Berghauser Pont, L M E; Balvers, R K; Kloezeman, J J; Nowicki, M O; van den Bossche, W; Kremer, A; Wakimoto, H; van den Hoogen, B G; Leenstra, S; Dirven, C M F; Chiocca, E A; Lawler, S E; Lamfers, M L M

    2015-12-01

    Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV. PMID:26196249

  9. Developing highER-throughput zebrafish screens for in-vivo CNS drug discovery.

    PubMed

    Stewart, Adam Michael; Gerlai, Robert; Kalueff, Allan V

    2015-01-01

    The high prevalence of brain disorders and the lack of their efficient treatments necessitate improved in-vivo pre-clinical models and tests. The zebrafish (Danio rerio), a vertebrate species with high genetic and physiological homology to humans, is an excellent organism for innovative central nervous system (CNS) drug discovery and small molecule screening. Here, we outline new strategies for developing higher-throughput zebrafish screens to test neuroactive drugs and predict their pharmacological mechanisms. With the growing application of automated 3D phenotyping, machine learning algorithms, movement pattern- and behavior recognition, and multi-animal video-tracking, zebrafish screens are expected to markedly improve CNS drug discovery. PMID:25729356

  10. Developing highER-throughput zebrafish screens for in-vivo CNS drug discovery

    PubMed Central

    Stewart, Adam Michael; Gerlai, Robert; Kalueff, Allan V.

    2015-01-01

    The high prevalence of brain disorders and the lack of their efficient treatments necessitate improved in-vivo pre-clinical models and tests. The zebrafish (Danio rerio), a vertebrate species with high genetic and physiological homology to humans, is an excellent organism for innovative central nervous system (CNS) drug discovery and small molecule screening. Here, we outline new strategies for developing higher-throughput zebrafish screens to test neuroactive drugs and predict their pharmacological mechanisms. With the growing application of automated 3D phenotyping, machine learning algorithms, movement pattern- and behavior recognition, and multi-animal video-tracking, zebrafish screens are expected to markedly improve CNS drug discovery. PMID:25729356

  11. Identification of novel EZH2 inhibitors through pharmacophore-based virtual screening and biological assays.

    PubMed

    Wu, Yunlong; Hu, Junchi; Ding, Hong; Chen, Limin; Zhang, Yuanyuan; Liu, Rongfeng; Xu, Pan; Du, Daohai; Lu, Wenchao; Liu, Jingqiu; Liu, Yan; Liu, Yu-Chih; Lu, Junyan; Zhang, Jin; Yao, Zhiyi; Luo, Cheng

    2016-08-01

    Polycomb repressive complex 2 (PRC2) acts as a primary writer for di- and tri-methylation of histone H3 at lysine 27. This protein plays an essential role in silencing gene expression. Enhancer of zeste 2 (EZH2), the catalytic subunit of PRC2, is considered as a promising therapeutic target for cancer. GSK126, a specific inhibitor of EZH2, is undergoing phase I trials for hypermethylation-related cancers. In addition, many derivatives of GSK126 are also commonly used in laboratory investigations. However, studies on the mechanism and drug development of EZH2 are limited by the absence of structural diversity of these inhibitors because they share similar SAM-like scaffolds. In this study, we generated a pharmacophore model based on reported EZH2 inhibitors and performed in silico screenings. Experimental validations led to the identification of two novel EZH2 inhibitors, DCE_42 and DCE_254, with IC50 values of 23 and 11μM, respectively. They also displayed significant anti-proliferation activity against lymphoma cell lines. Thus, we discovered potent EZH2 inhibitors with novel scaffold using combined in silico screening and experimental study. Results from this study can also guide further development of novel specific EZH2 inhibitors. PMID:27289323

  12. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants.

    PubMed Central

    Soto, A M; Sonnenschein, C; Chung, K L; Fernandez, M F; Olea, N; Serrano, F O

    1995-01-01

    Estrogens are defined by their ability to induce the proliferation of cells of the female genital tract. The wide chemical diversity of estrogenic compounds precludes an accurate prediction of estrogenic activity on the basis of chemical structure. Rodent bioassays are not suited for the large-scale screening of chemicals before their release into the environment because of their cost, complexity, and ethical concerns. The E-SCREEN assay was developed to assess the estrogenicity of environmental chemicals using the proliferative effect of estrogens on their target cells as an end point. This quantitative assay compares the cell number achieved by similar inocula of MCF-7 cells in the absence of estrogens (negative control) and in the presence of 17 beta-estradiol (positive control) and a range of concentrations of chemicals suspected to be estrogenic. Among the compounds tested, several "new" estrogens were found; alkylphenols, phthalates, some PCB congeners and hydroxylated PCBs, and the insecticides dieldrin, endosulfan, and toxaphene were estrogenic by the E-SCREEN assay. In addition, these compounds competed with estradiol for binding to the estrogen receptor and increased the levels of progesterone receptor and pS2 in MCF-7 cells, as expected from estrogen mimics. Recombinant human growth factors (bFGF, EGF, IGF-1) and insulin did not increase in cell yields. The aims of the work summarized in this paper were a) to validate the E-SCREEN assay; b) to screen a variety of chemicals present in the environment to identify those that may be causing reproductive effects in wildlife and humans; c) to assess whether environmental estrogens may act cumulatively; and finally d) to discuss the reliability of this and other assays to screen chemicals for their estrogenicity before they are released into the environment. PMID:8593856

  13. Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

    PubMed Central

    Paape, Daniel; Bell, Andrew S.; Heal, William P.; Hutton, Jennie A.; Leatherbarrow, Robin J.; Tate, Edward W.; Smith, Deborah F.

    2014-01-01

    We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors. PMID:25522361

  14. Microbioassay system for antiallergic drug screening using suspension cells retaining in a poly(dimethylsiloxane) microfluidic device.

    PubMed

    Tokuyama, Takahito; Fujii, Shin-Ichiro; Sato, Kiichi; Abo, Mitsuru; Okubo, Akira

    2005-05-15

    This article describes an antiallergic drug-screening system by the detection of histamine released from mast cells (suspension cells) on a multilayer microchip. In this study, the elastmeric material, poly(dimethylsiloxane) (PDMS), was employed to fabricate microchannels and microchambers. The microchip consists of two sections: a histamine-releasing one, which has a cell chamber, and a histamine-derivatizing one. Both were laminated to one microchip. Rat peritoneal mast cells were retained in the cell chamber (1.2 microL) with a filtering system using a cellulose nitrate membrane. This filtering system could easily retain suspension cells without cell damage. Mast cells were viable for a sufficient time to conduct the assay on the cell chamber. The cells were stimulated with a chemical release compound 48/80 (C48/80), and then histamine flowed into the lower layer, where it was derivatized to the fluorescent molecules with o-phthalaldehyde and its fluorescence was detected on the microchip. This flow system could detect the time course of the histamine release, and this microchip system required only 20 min for the assay. By this integrated system, 51 pmol of histamine released from 500 cells was detected, and the number of cells required for the assay was reduced to 1% compared with conventional bulk systems. By comparing the released histamine levels with and without drugs, their effect could be evaluated. The inhibition ratio of C48/80 induced-histamine release using an antiallergic drug, disodium cromoglicate (DSCG), was related to the concentration of DSCG. This flow system was applicable for antiallergy drug screening by rapid measurement of the inhibition of histamine release from a very small amount of mast cells. PMID:15889923

  15. Understanding the Supersensitive Anti-Drug Antibody Assay: Unexpected High Anti-Drug Antibody Incidence and Its Clinical Relevance

    PubMed Central

    2016-01-01

    Numbers of biotherapeutic products in development have increased over past decade. Despite providing significant benefits to patients with unmet needs, almost all protein-based biotherapeutics could induce unwanted immunogenicity, which result in a loss of efficacy and/or increase the risk of adverse reactions, such as infusion reactions, anaphylaxis, and even life-threatening response to endogenous proteins. Recognizing these possibilities, regulatory agencies request that immunogenicity be assessed as part of the approval process for biotherapeutics. Great efforts have been made to reduce drug immunogenicity through protein engineering. Accordingly the immunogenicity incidence has been reduced from around 80% in murine derived products to 0–10% in fully human products. However, recent improvements in immunogenicity assays have led to unexpectedly high immunogenicity rates, even in fully human products, leading to new challenges in assessing immunogenicity and its clinical relevance. These new immunogenicity assays are becoming supersensitive and able to detect more of anti-drug antibodies (ADA) than with earlier assays. This paper intends to review and discuss our understanding of the supersensitive ADA assay and the unexpected high ADA incidence and its potential clinical relevance. PMID:27340678

  16. Validation of the Drug Abuse Screening Test (DAST-10): A study on illicit drug use among Chinese pregnant women

    PubMed Central

    Lam, Lap Po; Leung, Wing Cheong; Ip, Patrick; Chow, Chun Bong; Chan, Mei Fung; Ng, Judy Wai Ying; Sing, Chu; Lam, Ying Hoo; Mak, Wing Lai Tony; Chow, Kam Ming; Chin, Robert Kien Howe

    2015-01-01

    We assessed the Chinese version of the Drug Abuse Screening Test (DAST-10) for identifying illicit drug use during pregnancy among Chinese population. Chinese pregnant women attending their first antenatal visit or their first unbooked visit to the maternity ward were recruited during a 4-month study period in 2011. The participants completed self-administered questionnaires on demographic information, a single question on illicit drug use during pregnancy and the DAST-10. Urine samples screened positive by the urine Point-of-Care Test were confirmed by gas chromatography-mass spectrometry. DAST-10 performance was compared with three different gold standards: urinalysis, self-reported drug use, and evidence of drug use by urinalysis or self-report. 1214 Chinese pregnant women participated in the study and 1085 complete DAST-10 forms were collected. Women who had used illicit drugs had significantly different DAST-10 scores than those who had not. The sensitivity of DAST-10 for identify illicit drug use in pregnant women ranged from 79.2% to 33.3% and specificity ranged from 67.7% to 99.7% using cut-off scores from ≥1 to ≥3. The ~80% sensitivity of DAST-10 using a cut-off score of ≥1 should be sufficient for screening of illicit drug use in Chinese pregnant women, but validation tests for drug use are needed. PMID:26091290

  17. Validation of the Drug Abuse Screening Test (DAST-10): A study on illicit drug use among Chinese pregnant women.

    PubMed

    Lam, Lap Po; Leung, Wing Cheong; Ip, Patrick; Chow, Chun Bong; Chan, Mei Fung; Ng, Judy Wai Ying; Sing, Chu; Lam, Ying Hoo; Mak, Wing Lai Tony; Chow, Kam Ming; Chin, Robert Kien Howe

    2015-01-01

    We assessed the Chinese version of the Drug Abuse Screening Test (DAST-10) for identifying illicit drug use during pregnancy among Chinese population. Chinese pregnant women attending their first antenatal visit or their first unbooked visit to the maternity ward were recruited during a 4-month study period in 2011. The participants completed self-administered questionnaires on demographic information, a single question on illicit drug use during pregnancy and the DAST-10. Urine samples screened positive by the urine Point-of-Care Test were confirmed by gas chromatography-mass spectrometry. DAST-10 performance was compared with three different gold standards: urinalysis, self-reported drug use, and evidence of drug use by urinalysis or self-report. 1214 Chinese pregnant women participated in the study and 1085 complete DAST-10 forms were collected. Women who had used illicit drugs had significantly different DAST-10 scores than those who had not. The sensitivity of DAST-10 for identify illicit drug use in pregnant women ranged from 79.2% to 33.3% and specificity ranged from 67.7% to 99.7% using cut-off scores from ≥ 1 to ≥ 3. The ~ 80% sensitivity of DAST-10 using a cut-off score of ≥ 1 should be sufficient for screening of illicit drug use in Chinese pregnant women, but validation tests for drug use are needed. PMID:26091290

  18. Data quality in drug discovery: the role of analytical performance in ligand binding assays.

    PubMed

    Wätzig, Hermann; Oltmann-Norden, Imke; Steinicke, Franziska; Alhazmi, Hassan A; Nachbar, Markus; El-Hady, Deia Abd; Albishri, Hassan M; Baumann, Knut; Exner, Thomas; Böckler, Frank M; El Deeb, Sami

    2015-09-01

    Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development. PMID:26070362

  19. AlphaScreen HTS and Live Cell Bioluminescence Resonance Energy Transfer (BRET) Assays for Identification of Tau–Fyn SH3 Interaction Inhibitors for Alzheimer’s Disease

    PubMed Central

    Cochran, J. Nicholas; Diggs, Pauleatha V.; Nebane, N. Miranda; Rasmussen, Lynn; White, E. Lucile; Bostwick, Robert; Maddry, Joseph A.; Suto, Mark J.; Roberson, Erik D.

    2014-01-01

    Alzheimer’s Disease (AD) is the most common neurodegenerative disease and with Americans’ increasing longevity it is becoming an epidemic. There are currently no effective treatments for this disorder. Abnormalities of Tau track more closely with cognitive decline than the most studied therapeutic target in AD, amyloid-beta, but the optimal strategy for targeting Tau has not yet been identified. Based on considerable preclinical data from AD models, we hypothesize that interactions between Tau and the Src-family tyrosine kinase, Fyn, are pathogenic in AD. Genetically reducing either Tau or Fyn is protective in AD mouse models, and a dominant negative fragment of Tau that alters Fyn localization is also protective. Here, we describe a new AlphaScreen assay and a live-cell BRET assay using a novel BRET pair for quantifying the Tau–Fyn interaction. We used these assays to map the binding site on Tau for Fyn to the 5th and 6th PXXP motifs, to show that AD-associated phosphorylation at MARK sites increase the affinity of the Tau–Fyn interaction, and to identify Tau–Fyn interaction inhibitors by HTS. This screen has identified a variety of chemically tractable hits, suggesting that the Tau–Fyn interaction may represent a good drug target for AD. PMID:25156556

  20. Whole Blood Interferon-Gamma Assay for Baseline Tuberculosis Screening among Japanese Healthcare Students

    PubMed Central

    Hotta, Katsuyuki; Ogura, Toshio; Nishii, Kenji; Kodani, Tsuyoshi; Onishi, Masaru; Shimizu, Yukito; Kanehiro, Arihiko; Kiura, Katsuyuki; Tanimoto, Mitsune; Tobe, Kazuo

    2007-01-01

    Background The whole blood interferon-gamma assay (QuantiFERON-TB-2G; QFT) has not been fully evaluated as a baseline tuberculosis screening test in Japanese healthcare students commencing clinical contact. The aim of this study was to compare the results from the QFT with those from the tuberculin skin test (TST) in a population deemed to be at a low risk for infection with Mycobacterium tuberculosis. Methodology/Principal Findings Healthcare students recruited at Okayama University received both the TST and the QFT to assess the level of agreement between these two tests. The interleukin-10 levels before and after exposure to M tuberculosis-specific antigens (early-secreted antigenic target 6-kDa protein [ESAT-6] and culture filtrate protein 10 [CFP-10]) were also measured. Of the 536 healthcare students, most of whom had been vaccinated with bacillus-Calmette-Guérin (BCG), 207 (56%) were enrolled in this study. The agreement between the QFT and the TST results was poor, with positive result rates of 1.4% vs. 27.5%, respectively. A multivariate analysis also revealed that the induration diameter of the TST was not affected by the interferon-gamma concentration after exposure to either of the antigens but was influenced by the number of BCG needle scars (p = 0.046). The whole blood interleukin-10 assay revealed that after antigen exposure, the median increases in interleukin-10 concentration was higher in the subgroup with the small increase in interferon-gamma concentration than in the subgroup with the large increase in interferon-gamma concentration (0.3 vs. 0 pg/mL; p = 0.004). Conclusions/Significance As a baseline screening test for low-risk Japanese healthcare students at their course entry, QFT yielded quite discordant results, compared with the TST, probably because of the low specificity of the TST results in the BCG-vaccinated population. We also found, for the first time, that the change in the interleukin-10 level after exposure to specific

  1. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    EPA Science Inventory

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  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. 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...

  4. Unique Nanoparticle Optical Properties Confound Fluorescent Based Assays Widely Employed in Their In Vitro Toxicity Screening and Ranking

    EPA Science Inventory

    Nanoparticles (NPs) are novel materials having at least one dimension less than 100 nm and display unique physicochemical properties due to their nanoscale size. An emphasis has been placed on developing high throughput screening (HTS) assays to characterize and rank the toxiciti...

  5. Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

    PubMed Central

    Testa, Maria Pia; Alvarado, Omar; Wournell, Andrea; Lee, Jonathan; Guilford, Frederick T.; Henriksen, Steven H.; Phillips, Tom R.

    2011-01-01

    An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of the Central Nervous System (CNS). Astrocytes help maintain a homeostatic environment for neurons as well as protecting neurons from Reactive Oxygen Species (ROS). CM-H2DCFDA is a cell-permeable indicator for the presence of ROS. CM-H2DCFDA enters the cell as a non-fluorescent compound, and becomes fluorescent after cellular esterases remove the acetate groups, and the compound is oxidized. The number of cells, measured by flow cytometry, that are found to be green fluorescing is an indication of the number of cells that are in an oxidative state. CM-H2DCFDA is susceptible to oxidation by a large number of different ROS. This lack of specificity, regarding which ROS can oxidize CM-H2DCFDA, makes this compound a valuable regent for use in the early stages of a pathogenesis investigation, as this assay can be used to screen for an oxidative cellular environment regardless of which oxygen radical or combination of ROS are responsible for the cellular conditions. Once it has been established that ROS are present by oxidation of CM-H2DCFDA, then additional experiments can be performed to determine which ROS or combination of ROSs are involved in the particular pathogenesis process. The results of this study demonstrate that with the addition of hydrogen peroxide an increase in CM-H2DCFDA fluoresce was detected relative to the saline controls, indicating that this assay is a valuable test for detecting an oxidative environment within G355-5 cells, a feline astrocyte cell line. PMID:21775965

  6. CARS based label-free assay for assessment of drugs by monitoring lipid droplets in tumour cells.

    PubMed

    Steuwe, Christian; Patel, Imran I; Ul-Hasan, Mahmud; Schreiner, Alexander; Boren, Joan; Brindle, Kevin M; Reichelt, Stefanie; Mahajan, Sumeet

    2014-11-01

    Coherent anti-Stokes Raman scattering (CARS) is becoming an established tool for label-free multi-photon imaging based on molecule specific vibrations in the sample. The technique has proven to be particularly useful for imaging lipids, which are abundant in cells and tissues, including cytoplasmic lipid droplets (LD), which are recognized as dynamic organelles involved in many cellular functions. The increase in the number of lipid droplets in cells undergoing cell proliferation is a common feature in many neoplastic processes [1] and an increase in LD number also appears to be an early marker of drug-induced cell stress and subsequent apoptosis [3]. In this paper, a CARS-based label-free method is presented to monitor the increase in LD content in HCT116 colon tumour cells treated with the chemotherapeutic drugs Etoposide, Camptothecin and the protein kinase inhibitor Staurosporine. Using CARS, LDs can easily be distinguished from other cell components without the application of fluorescent dyes and provides a label-free non-invasive drug screening assay that could be used not only with cells and tissues ex vivo but potentially also in vivo. PMID:24343869

  7. 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

  8. A Microplate Format Assay for Real-Time Screening for New Aldolases that Accept Aryl-Substituted Acceptor Substrates.

    PubMed

    Ma, Huan; Enugala, Thilak Reddy; Widersten, Mikael

    2015-12-01

    Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose 6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been reported to be a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with bulky acceptor substrates of this type is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an aldehyde reductase produced in vitro by directed evolution; it reduces the aldehyde product formed after cleavage of the aldol by FSA. The assay is suitable both for steady-state enzyme kinetics and for real-time activity screening in a 96-well format. PMID:26449620

  9. Automation of cell-based drug absorption assays in 96-well format using permeable support systems.

    PubMed

    Larson, Brad; Banks, Peter; Sherman, Hilary; Rothenberg, Mark

    2012-06-01

    Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics. PMID:22357561

  10. Model for high-throughput screening of drug immunotoxicity--study of the anti-microbial G1 over peritoneal macrophages using flow cytometry.

    PubMed

    Tenorio-Borroto, Esvieta; Peñuelas-Rivas, Claudia G; Vásquez-Chagoyán, Juan C; Castañedo, Nilo; Prado-Prado, Francisco J; García-Mera, Xerardo; González-Díaz, Humberto

    2014-01-24

    Quantitative Structure-Activity (mt-QSAR) techniques may become an important tool for prediction of cytotoxicity and High-throughput Screening (HTS) of drugs to rationalize drug discovery process. In this work, we train and validate by the first time mt-QSAR model using TOPS-MODE approach to calculate drug molecular descriptors and Linear Discriminant Analysis (LDA) function. This model correctly classifies 8258 out of 9000 (Accuracy = 91.76%) multiplexing assay endpoints of 7903 drugs (including both train and validation series). Each endpoint correspond to one out of 1418 assays, 36 molecular and cellular targets, 46 standard type measures, in two possible organisms (human and mouse). After that, we determined experimentally, by the first time, the values of EC50 = 21.58 μg/mL and Cytotoxicity = 23.6% for the anti-microbial/anti-parasite drug G1 over Balb/C mouse peritoneal macrophages using flow cytometry. In addition, the model predicts for G1 only 7 positive endpoints out 1251 cytotoxicity assays (0.56% of probability of cytotoxicity in multiple assays). The results obtained complement the toxicological studies of this important drug. This work adds a new tool to the existing pool of few methods useful for multi-target HTS of ChEMBL and other libraries of compounds towards drug discovery. PMID:24445280

  11. Dictyostelium discoideum Ax2 as an Assay System for Screening of Pharmacological Chaperones for Phenylketonuria Mutations.

    PubMed

    Kim, Yu-Min; Yang, Yun Gyeong; Kim, Hye-Lim; Park, Young Shik

    2015-06-01

    In this study, we developed an assay system for missense mutations in human phenylalanine hydroxylases (hPAHs). To demonstrate the reliability of the system, eight mutant proteins (F39L, K42I, L48S, I65T, R252Q, L255V, S349L, and R408W) were expressed in a mutant strain (pah(-)) of Dictyostelium discoideum Ax2 disrupted in the indigenous gene encoding PAH. The transformed pah- cells grown in FM minimal medium were measured for growth rate and PAH activity to reveal a positive correlation between them. The protein level of hPAH was also determined by western blotting to show the impact of each mutation on protein stability and catalytic activity. The result was highly compatible with the previous ones obtained from other expression systems, suggesting that Dictyostelium is a dependable alternative to other expression systems. Furthermore, we found that both the protein level and activity of S349L and R408W, which were impaired severely in protein stability, were rescued in HL5 nutrient medium. Although the responsible component(s) remains unidentified, this unexpected finding showed an important advantage of our expression system for studying unstable proteins. As an economic and stable cell-based expression system, our development will contribute to mass-screening of pharmacological chaperones for missense PAH mutations as well as to the in-depth characterization of individual mutations. PMID:25563416

  12. 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

  13. Expedient screening for HIV-1 protease inhibitors using a simplified immunochromatographic assay.

    PubMed

    Kitidee, Kuntida; Khamaikawin, Wannisa; Thongkum, Weeraya; Tawon, Yardpiroon; Cressey, Tim R; Jevprasesphant, Rachaneekorn; Kasinrerk, Watchara; Tayapiwatana, Chatchai

    2016-05-15

    A colloidal gold-based immunochromatographic (IC) strip test was developed and validated for the detection of HIV-1 protease (HIV-PR) activity and inhibitory effect of HIV-PR inhibitors (PIs). It is a unique 'two-step' process requiring the combination of proteolysis of HIV-PR and an immunochromatographic reaction. Monoclonal antibodies to the free C-terminus of HIV matrix protein (HIV-MA) conjugated to gold particles and a monoclonal antibody against intact and cleaved forms of the HIV-MA are immobilized on the 'Test'-line of the IC strip. Using lopinavir, a potent HIV protease inhibitor, the IC-strip was optimized to detect inhibitory activity against HIV-protease. At a lopinavir concentration of 1000ng/mL (its suggested minimum effective concentration), a HIV-PRH6 concentration of 6mg/mL and incubation period of 60min were the optimal conditions. A preliminary comparison between a validated high-performance liquid chromatography assay and the IC-strip to semi-quantify HIV protease inhibitor concentrations (lopinavir and atazanavir) demonstrated good agreement. This simplified method is suitable for the rapid screening of novel protease inhibitors for future therapeutic use. Moreover, the IC strip could also be optimized to semi-quantify PIs concentrations in plasma samples. PMID:26490422

  14. Tuberculin Skin Tests versus Interferon-Gamma Release Assays in Tuberculosis Screening among Immigrant Visa Applicants

    PubMed Central

    Chuke, Stella O.; Yen, Nguyen Thi Ngoc; Laserson, Kayla F.; Phuoc, Nguyen Huu; Trinh, Nguyen An; Nhung, Duong Thi Cam; Mai, Vo Thi Chi; Qui, An Dang; Hai, Hoang Hoa; Loan, Le Thien Huong; Jones, Warren G.; Whitworth, William C.; Shah, J. Jina; Painter, John A.; Mazurek, Gerald H.; Maloney, Susan A.

    2014-01-01

    Objective. Use of tuberculin skin tests (TSTs) and interferon gamma release assays (IGRAs) as part of tuberculosis (TB) screening among immigrants from high TB-burden countries has not been fully evaluated. Methods. Prevalence of Mycobacterium tuberculosis infection (MTBI) based on TST, or the QuantiFERON-TB Gold test (QFT-G), was determined among immigrant applicants in Vietnam bound for the United States (US); factors associated with test results and discordance were assessed; predictive values of TST and QFT-G for identifying chest radiographs (CXRs) consistent with TB were calculated. Results. Of 1,246 immigrant visa applicants studied, 57.9% were TST positive, 28.3% were QFT-G positive, and test agreement was 59.4%. Increasing age was associated with positive TST results, positive QFT-G results, TST-positive but QFT-G-negative discordance, and abnormal CXRs consistent with TB. Positive predictive values of TST and QFT-G for an abnormal CXR were 25.9% and 25.6%, respectively. Conclusion. The estimated prevalence of MTBI among US-bound visa applicants in Vietnam based on TST was twice that based on QFT-G, and 14 times higher than a TST-based estimate of MTBI prevalence reported for the general US population in 2000. QFT-G was not better than TST at predicting abnormal CXRs consistent with TB. PMID:24738031

  15. Development a monoclonal antibody-based enzyme-linked immunosorbent assay for screening carotenoids in eggs.

    PubMed

    Peng, Dapeng; Liao, Feng; Pan, Yuanhu; Chen, Dongmei; Liu, Zhenli; Wang, Yulian; Yuan, Zonghui

    2016-07-01

    In this study, a monoclonal antibody (mAb) with broad-specificity against several carotenoid analogs with equal or similar efficacy was prepared. The obtained mAb C11, with the IgG1 isotype, showed cross-reactivity (CR) with canthaxanthin (100%), β-ionone acid (140.4%), β-carotene (92.9%), capsanthin (90.1%), β-apo-8'-carotenal (92.7%), and xanthophyll (95.8%). Using the mAb C11, a highly sensitive and inexpensive indirect competitive enzyme linked immunosorbent assay (ic-ELISA) was developed with a simple sample preparation procedure for the simultaneous detection of these carotenoid compounds in eggs. The limit of detection of the various carotenoids ranged from 1.31mgkg(-1) to 1.48mgkg(-1). Recoveries from egg yolks spiked with the above carotenoids ranged from 91.8% to 113.3%, with coefficients of variation (CVs) of less than 14.8%. These results suggest that the developed ic-ELISA is a sensitive, specific, accurate, and inexpensive method that is suitable for the screening of carotenoid residues in routine monitoring. PMID:26920278

  16. A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

    PubMed Central

    Mendoza, Oscar; Gueddouda, Nassima Meriem; Boulé, Jean-Baptiste; Bourdoncle, Anne; Mergny, Jean-Louis

    2015-01-01

    Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5′ to 3′ DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures. PMID:25765657

  17. New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass

    PubMed Central

    2013-01-01

    Background Fungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies. Results Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast. Conclusions The high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for

  18. Development of a Fluorescence-based Trypanosoma cruzi CYP51 Inhibition Assay for Effective Compound Triaging in Drug Discovery Programmes for Chagas Disease.

    PubMed

    Riley, Jennifer; Brand, Stephen; Voice, Michael; Caballero, Ivan; Calvo, David; Read, Kevin D

    2015-09-01

    Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is a life threatening global health problem with only two drugs available for treatment (benznidazole and nifurtimox), both having variable efficacy in the chronic stage of the disease and high rates of adverse drug reactions. Inhibitors of sterol 14α-demethylase (CYP51) have proven effective against T. cruzi in vitro and in vivo in animal models of Chagas disease. Consequently two azole inhibitors of CYP51 (posaconazole and ravuconazole) have recently entered clinical development by the Drugs for Neglected Diseases initiative. Further new drug treatments for this disease are however still urgently required, particularly having a different mode of action to CYP51 in order to balance the overall risk in the drug discovery portfolio. This need has now been further strengthened by the very recent reports of treatment failure in the clinic for both posaconazole and ravuconazole. To this end and to prevent enrichment of drug candidates against a single target, there is a clear need for a robust high throughput assay for CYP51 inhibition in order to evaluate compounds active against T. cruzi arising from phenotypic screens. A high throughput fluorescence based functional assay using recombinantly expressed T. cruzi CYP51 (Tulahuen strain) is presented here that meets this requirement. This assay has proved valuable in prioritising medicinal chemistry resource on only those T. cruzi active series arising from a phenotypic screening campaign where it is clear that the predominant mode of action is likely not via inhibition of CYP51. PMID:26394211

  19. Use of the in Vivo Hollow Fiber Assay in Natural Products Anticancer Drug Discovery#

    PubMed Central

    Mi, Qiuwen; Pezzuto, John M.; Farnsworth, Norman R.; Wani, Mansukh C.; Kinghorn, A. Douglas; Swanson, Steven M.

    2009-01-01

    The in vivo hollow fiber assay was developed at the National Cancer Institute (NCI) to help bridge the gap between in vitro cell-based assays and human tumor models propagated in immunodeficient mice. The goal was to develop an intermediate assay that could help predict which compounds found active in the 60-cell line panel would be active in a subsequent xenograft system. This was necessary due to the high cost of the traditional xenograft assay in terms of number of animals required, time for assay completion, and financial commitment necessary. To address this problem, investigators of the NCI Developmental Therapeutics Program designed a method of propagating human cancer cells in inert hollow fibers with pores small enough to retain the cancer cells but large enough to permit entry of potential chemotherapeutic drugs, including large proteins and other important substances. Fibers containing proliferating cancer cells are transplanted into the peritoneum or under the skin, the host mice are treated with a test agent and the fibers are subsequently retrieved for analysis of viable cell mass. The assay has been successful in helping investigators from around the world, including our own research group, prioritize compounds active in vitro for further testing in the traditional xenograft system. PMID:19161316

  20. Detection and measurement of surface contamination by multiple antineoplastic drugs using multiplex bead assay

    PubMed Central

    Smith, Jerome P; Sammons, Deborah L; Robertson, Shirley A; Pretty, Jack; Debord, D Gayle; Connor, Thomas H; Snawder, John

    2015-01-01

    Objectives Contamination of workplace surfaces by antineoplastic drugs presents an exposure risk for healthcare workers. Traditional instrumental methods to detect contamination such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) are sensitive and accurate but expensive. Since immunochemical methods may be cheaper and faster than instrumental methods, we wanted to explore their use for routine drug residue detection for preventing worker exposure. Methods In this study we examined the feasibility of using fluorescence covalent microbead immunosorbent assay (FCMIA) for simultaneous detection and semi-quantitative measurement of three antineoplastic drugs (5-fluorouracil, paclitaxel, and doxorubicin). The concentration ranges for the assay were 0–1000 ng/ml for 5-fluorouracil, 0–100 ng/ml for paclitaxel, and 0–2 ng/ml for doxorubicin. The surface sampling technique involved wiping a loaded surface with a swab wetted with wash buffer, extracting the swab in storage/blocking buffer, and measuring drugs in the extract using FCMIA. Results There was no significant cross reactivity between these drugs at the ranges studied indicated by a lack of response in the assay to cross analytes. The limit of detection (LOD) for 5-fluorouracil on the surface studied was 0.93 ng/cm2 with a limit of quantitation (LOQ) of 2.8 ng/cm2, the LOD for paclitaxel was 0.57 ng/cm2 with an LOQ of 2.06 ng/cm2, and the LOD for doxorubicin was 0.0036 ng/cm2 with an LOQ of 0.013 ng/cm2. Conclusion The use of FCMIA with a simple sampling technique has potential for low cost simultaneous detection and semi-quantitative measurement of surface contamination from multiple antineoplastic drugs. PMID:25293722

  1. Identification of Multiple Cryptococcal Fungicidal Drug Targets by Combined Gene Dosing and Drug Affinity Responsive Target Stability Screening

    PubMed Central

    Park, Yoon-Dong; Sun, Wei; Salas, Antonio; Antia, Avan; Carvajal, Cindy; Wang, Amy; Xu, Xin; Meng, Zhaojin; Zhou, Ming; Tawa, Gregory J.; Dehdashti, Jean; Zheng, Wei; Henderson, Christina M.; Zelazny, Adrian M.

    2016-01-01

    ABSTRACT Cryptococcus neoformans is a pathogenic fungus that is responsible for up to half a million cases of meningitis globally, especially in immunocompromised individuals. Common fungistatic drugs, such as fluconazole, are less toxic for patients but have low efficacy for initial therapy of the disease. Effective therapy against the disease is provided by the fungicidal drug amphotericin B; however, due to its high toxicity and the difficulty in administering its intravenous formulation, it is imperative to find new therapies targeting the fungus. The antiparasitic drug bithionol has been recently identified as having potent fungicidal activity. In this study, we used a combined gene dosing and drug affinity responsive target stability (GD-DARTS) screen as well as protein modeling to identify a common drug binding site of bithionol within multiple NAD-dependent dehydrogenase drug targets. This combination genetic and proteomic method thus provides a powerful method for identifying novel fungicidal drug targets for further development. PMID:27486194

  2. Urine Toxicology Screen in Multiple Sleep Latency Test: The Correlation of Positive Tetrahydrocannabinol, Drug Negative Patients, and Narcolepsy

    PubMed Central

    Dzodzomenyo, Samuel; Stolfi, Adrienne; Splaingard, Deborah; Earley, Elizabeth; Onadeko, Oluwole; Splaingard, Mark

    2015-01-01

    Objective: Drugs can influence results of multiple sleep latency tests (MSLT). We sought to identify the effect of marijuana on MSLT results in pediatric patients evaluated for excessive daytime sleepiness (EDS). Methods: This is a retrospective study of urine drug screens performed the morning before MSLT in 383 patients < 21 years old referred for EDS. MSLT results were divided into those with (1) (−) urine drug screens, (2) urine drug screens (+) for tetrahydrocannabinol (THC) alone or THC plus other drugs, and (3) urine drug screens (+) for drugs other than THC. Groups were compared with Fisher exact tests or one-way ANOVA. Results: 38 (10%) urine drug tests were (+): 14 for THC and 24 for other drugs. Forty-three percent of patients with drug screen (+) for THC had MSLT findings consistent with narcolepsy, 0% consistent with idiopathic hypersomnia, 29% other, and 29% normal. This was statistically different from those with (−) screens (24% narcolepsy, 20% idiopathic hypersomnia, 6% other, 50% normal), and those (+) for drugs other than THC (17% narcolepsy, 33% idiopathic hypersomnia, 4% other, 46% normal (p = 0.01). Six percent (6/93) of patients with MSLT findings consistent with narcolepsy were drug screen (+) for THC; 71% of patients with drug screen (+) for THC had multiple sleep onset REM periods (SOREMS). There were no (+) urine drug screens in patients < 13 years old. Conclusion: Many pediatric patients with (+) urine drug screens for THC met MSLT criteria for narcolepsy or had multiple SOREMs. Drug screening is important in interpreting MSLT findings for children ≥ 13 years. Citation: Dzodzomenyo S, Stolfi A, Splaingard D, Earley E, Onadeko O, Splaingard M. Urine toxicology screen in multiple sleep latency test: the correlation of positive tetrahydrocannabinol, drug negative patients, and narcolepsy. J Clin Sleep Med 2015;11(2):93–99. PMID:25348245

  3. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

    PubMed

    Wu, Yuanzhong; Kang, Tiebang

    2016-01-01

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability. PMID:27357860

  4. Protein-based multiplex assays: mock presubmissions to the US Food and Drug Administration.

    PubMed

    Regnier, Fred E; Skates, Steven J; Mesri, Mehdi; Rodriguez, Henry; Tezak, Zivana; Kondratovich, Marina V; Alterman, Michail A; Levin, Joshua D; Roscoe, Donna; Reilly, Eugene; Callaghan, James; Kelm, Kellie; Brown, David; Philip, Reena; Carr, Steven A; Liebler, Daniel C; Fisher, Susan J; Tempst, Paul; Hiltke, Tara; Kessler, Larry G; Kinsinger, Christopher R; Ransohoff, David F; Mansfield, Elizabeth; Anderson, N Leigh

    2010-02-01

    As a part of ongoing efforts of the NCI-FDA Interagency Oncology Task Force subcommittee on molecular diagnostics, members of the Clinical Proteomic Technology Assessment for Cancer program of the National Cancer Institute have submitted 2 protein-based multiplex assay descriptions to the Office of In Vitro Diagnostic Device Evaluation and Safety, US Food and Drug Administration. The objective was to evaluate the analytical measurement criteria and studies needed to validate protein-based multiplex assays. Each submission described a different protein-based platform: a multiplex immunoaffinity mass spectrometry platform for protein quantification, and an immunological array platform quantifying glycoprotein isoforms. Submissions provided a mutually beneficial way for members of the proteomics and regulatory communities to identify the analytical issues that the field should address when developing protein-based multiplex clinical assays. PMID:20007858

  5. Off-Target Effects of Psychoactive Drugs Revealed by Genome-Wide Assays in Yeast

    PubMed Central

    Ericson, Elke; Gebbia, Marinella; Heisler, Lawrence E.; Wildenhain, Jan; Tyers, Mike; Giaever, Guri; Nislow, Corey

    2008-01-01

    To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac) interfered with establishment of cell polarity, cyproheptadine (Periactin) targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil) interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril) had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol) and pimozide (Orap). Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes. PMID:18688276

  6. 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

  7. High-Throughput Screening of Ototoxic and Otoprotective Pharmacological Drugs

    ERIC Educational Resources Information Center

    Kalinec, Federico

    2005-01-01

    Drug ototoxicity research has relied traditionally on animal models for the discovery and development of therapeutic interventions. More than 50 years of research, however, has delivered few--if any--successful clinical strategies for preventing or ameliorating the ototoxic effects of common pharmacological drugs such as aminoglycoside…

  8. Hydration of nail plate: a novel screening model for transungual drug permeation enhancers.

    PubMed

    Chouhan, P; Saini, T R

    2012-10-15

    Drug delivery by topical route for the treatment of onychomycosis, a nail fungal infection, is challenging due to the unique barrier properties of the nail plate which imparts high resistance to the passage of antifungal drugs. Permeation enhancers are used in transungual formulations to improve the drug flux across the nail plate. Selection of the effective permeation enhancer among the available large pool of permeation enhancers is a difficult task. Screening the large number of permeation enhancers using conventional Franz diffusion cells is laborious and expensive. The objective of present study was to evolve a simple, accurate and rapid method for screening of transungual drug permeation enhancers based on the principle of hydration of nail plate. The permeation enhancer which affects the structural or physicochemical properties of nail plate would also affect their hydration capacity. Two screening procedures namely primary and secondary screenings were evolved wherein hydration and uptake of ciclopirox olamine by nail plates were measured. Hydration enhancement factor, HEF(24) and drug uptake enhancement factor, UEF(24) were determined for screening of 23 typical permeation enhancers. The Pearson's correlation coefficient between HEF(24) and UEF(24) was determined. A good agreement between the HEF(24) and UEF(24) data proved the validity of the proposed nail plate hydration model as a screening technique for permeation enhancers. PMID:22705091

  9. SCIENTIFIC AND TECHNOLOGICAL SUPPORT ON IN VITRO ASSAYS FOR THE AGENCY'S ENDOCRINE DISRUPTOR SCREENING PROGRAM

    EPA Science Inventory

    In response to the 1996 legislative mandate for an endocrine screening and testing program, we are helping develop, standardize and validate relatively sensitive, robust and relatively simple methods for in vitro screening of chemicals that affect estrogen, and androgen function ...

  10. DISIS: Prediction of Drug Response through an Iterative Sure Independence Screening

    PubMed Central

    Zhang, Naiqian; Wang, Jun; Wang, Haiyun; Zheng, Xiaoqi

    2015-01-01

    Prediction of drug response based on genomic alterations is an important task in the research of personalized medicine. Current elastic net model utilized a sure independence screening to select relevant genomic features with drug response, but it may neglect the combination effect of some marginally weak features. In this work, we applied an iterative sure independence screening scheme to select drug response relevant features from the Cancer Cell Line Encyclopedia (CCLE) dataset. For each drug in CCLE, we selected up to 40 features including gene expressions, mutation and copy number alterations of cancer-related genes, and some of them are significantly strong features but showing weak marginal correlation with drug response vector. Lasso regression based on the selected features showed that our prediction accuracies are higher than those by elastic net regression for most drugs. PMID:25794193

  11. Recent trends and future prospects in computational GPCR drug discovery: from virtual screening to polypharmacology.

    PubMed

    Carrieri, Antonio; Pérez-Nueno, Violeta I; Lentini, Giovanni; Ritchie, David W

    2013-01-01

    Extending virtual screening approaches to deal with multi-target drug design and polypharmacology is an increasingly important aspect in drug design. In light of this, the concept of accessible chemical space and its exploration should be reviewed. The great advantages of re-using drugs with safe pharmacological profiles with favourable pharmacokinetic properties highlights drug repositioning as a valid alternative to rational drug design, massive drug development efforts, and high-throughput screening, especially when supported by in silico techniques. Here, we discuss some of the advantages of multi-target approaches, and we review some significant examples of their application in the last decade to that well known class of pharmaceutical targets, the G-protein coupled receptors. PMID:23651484

  12. Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development

    PubMed Central

    2013-01-01

    Background With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs. Calpain inhibitor, ALLN, is proposed to inhibit parasite proliferation by suppressing haemoglobin degradation. This provides Plasmodium calpain as a potential target for drug development. Pf-calpain, a cysteine protease of Plasmodium falciparum, belongs to calpain-7 family, which is an atypical calpain not harboring Ca2+-binding regulatory motifs. In this present study, in order to establish the screening system for Pf-calpain specific inhibitors, the active form of Pf-calpain was first identified. Methods Recombinant Pf-calpain including catalytic subdomain IIa (rPfcal-IIa) was heterologously expressed and purified. Enzymatic activity was determined by both fluorogenic substrate assay and gelatin zymography. Molecular homology modeling was carried out to address the activation mode of Pf-calpain in the aspect of structural moiety. Results Based on the measurement of enzymatic activity and protease inhibitor assay, it was found that the active form of Pf-calpain only contains the catalytic subdomain IIa, suggesting that Pf-calpain may function as a monomeric form. The sequence prediction indicates that the catalytic subdomain IIa contains all amino acid residues necessary for catalytic triad (Cys-His-Asn) formation. Molecular modeling suggests that the Pf-calpain subdomain IIa makes an active site, holding the catalytic triad residues in their appropriate orientation for catalysis. The mutation analysis further supports that those amino acid residues are functional and have enzymatic activity. Conclusion The identified active form of Pf-calpain could be utilized to establish high-throughput screening system for Pf-calpain inhibitors. Due to its unique monomeric structural property, Pf-calpain could be served as a novel anti-malarial drug target, which has a high specificity for malaria parasite. In addition, the monomeric

  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. 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

  15. 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

  16. 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

  17. Development of a rapid multiplexed assay for the direct screening of antimicrobial residues in raw milk.

    PubMed

    McGrath, Terry F; McClintock, Laura; Dunn, John S; Husar, Gregory M; Lochhead, Michael J; Sarver, Ronald W; Klein, Frank E; Rice, Jennifer A; Campbell, Katrina; Elliott, Christopher T

    2015-06-01

    Antimicrobial residues found to be present in milk can have both health and economic impacts. For these reasons, the widespread routine testing of milk is required. Due to delays with sample handling and test scheduling, laboratory-based tests are not always suited for making decisions about raw material intake and product release, especially when samples require shipping to a central testing facility. Therefore, rapid on-site screening tests that can produce results within a matter of minutes are required to facilitate rapid intake and product release processes. Such tests must be simple for use by non-technical staff. There is increasing momentum towards the development and implementation of multiplexing tests that can detect a range of important antimicrobial residues simultaneously. A simple in situ multiplexed planar waveguide device that can simultaneously detect chloramphenicol, streptomycin and desfuroylceftiofur in raw dairy milk, without sample preparation, has been developed. Samples are simply mixed with antibody prior to an aliquot being passed through the detection cartridge for 5 min before reading on a field-deployable portable instrument. Multiplexed calibration curves were produced in both buffer and raw milk. Buffer curves, for chloramphenicol, streptomycin and desfuroylceftiofur, showed linear ranges (inhibitory concentration (IC)20-IC80) of 0.1-0.9, 3-129 and 12-26 ng/ml, whilst linear range in milk was 0.13-0.74, 11-376 and 2-12 ng/ml, respectively, thus meeting European legislated concentration requirements for both chloramphenicol and streptomycin, in milk, without the need for any sample preparation. Desfuroylceftiofur-contaminated samples require only simple sample dilution to bring positive samples within the range of quantification. Assay repeatability and reproducibility were lower than 12 coefficient of variation (%CV), whilst blank raw milk samples (n = 9) showed repeatability ranging between 4.2 and 8.1%CV when measured on all

  18. Validation of a semiconductor next-generation sequencing assay for the clinical genetic screening of CFTR

    PubMed Central

    Trujillano, Daniel; Weiss, Maximilian E R; Köster, Julia; Papachristos, Efstathios B; Werber, Martin; Kandaswamy, Krishna Kumar; Marais, Anett; Eichler, Sabrina; Creed, Jenny; Baysal, Erol; Jaber, Iqbal Yousuf; Mehaney, Dina Ahmed; Farra, Chantal; Rolfs, Arndt

    2015-01-01

    Genetic testing for cystic fibrosis and CFTR-related disorders mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the CFTR gene. We have explored a more efficient genetic screening strategy based on next-generation sequencing (NGS) of the CFTR gene. We validated this approach in a cohort of 177 patients with previously known CFTR mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq™ CFTR panel. The DNA libraries were pooled, barcoded, and sequenced using an Ion Torrent PGM sequencer. The combination of different robust bioinformatics tools allowed us to detect previously known pathogenic mutations and polymorphisms in the 177 samples, without detecting spurious pathogenic calls. In summary, the assay achieves a sensitivity of 94.45% (95% CI: 92% to 96.9%), with a specificity of detecting nonvariant sites from the CFTR reference sequence of 100% (95% CI: 100% to 100%), a positive predictive value of 100% (95% CI: 100% to 100%), and a negative predictive value of 99.99% (95% CI: 99.99% to 100%). In addition, we describe the observed allelic frequencies of 94 unique definitely and likely pathogenic, uncertain, and neutral CFTR variants, some of them not previously annotated in the public databases. Strikingly, a seven exon spanning deletion as well as several more technically challenging variants such as pathogenic poly-thymidine-guanine and poly-thymidine (poly-TG-T) tracts were also detected. Targeted NGS is ready to substitute classical molecular methods to perform genetic testing on the CFTR gene. PMID:26436105

  19. Application of a fish DNA damage assay as a biological toxicity screening tool for metal plating wastewater

    SciTech Connect

    Choi, K.; Zong, M.; Meier, P.G.

    2000-01-01

    The utility of a fish DNA damage assay as a rapid monitoring tool was investigated. Metal plating wastewater was chosen as a sample because it contains various genotoxic metal species. Fish DNA damage assay results were compared to data generated from the conventional whole effluent toxicity (WET) test procedure. The Microtox{reg_sign} assay (Azur Environmental, Carlsbad, CA, USA) using Vibrio fischeri was also employed. Eleven samples from two metal plating companies were collected for this evaluation. For the fish DNA damage assay, 7-d-old fathead minnow larvae, Pimephales promelas, were utilized. They were exposed to a series of dilutions at 20 C for 2 h. Whole effluent toxicity tests conducted in this study included two acute toxicity tests with Daphnia magna and fathead minnows and two chronic toxicity tests with Ceriodaphnia dubia and fathead minnows. The fish DNA damage assay showed good correlations with both the acute and chronic WET test results, especially with those obtained with fathead minnows. The kappa values, an index of agreement, between the fish DNA damage assay and WET tests were shown to be acceptable. These findings imply that this novel fish DNA damage assay has use as an expedient toxicity screening procedure since it produces comparable results to those of the acute and chronic fathead minnow toxicity tests.

  20. Stem cells: a model for screening, discovery and development of drugs

    PubMed Central

    Kitambi, Satish Srinivas; Chandrasekar, Gayathri

    2011-01-01

    The identification of normal and cancerous stem cells and the recent advances made in isolation and culture of stem cells have rapidly gained attention in the field of drug discovery and regenerative medicine. The prospect of performing screens aimed at proliferation, directed differentiation, and toxicity and efficacy studies using stem cells offers a reliable platform for the drug discovery process. Advances made in the generation of induced pluripotent stem cells from normal or diseased tissue serves as a platform to perform drug screens aimed at developing cell-based therapies against conditions like Parkinson’s disease and diabetes. This review discusses the application of stem cells and cancer stem cells in drug screening and their role in complementing, reducing, and replacing animal testing. In addition to this, target identification and major advances in the field of personalized medicine using induced pluripotent cells are also discussed. PMID:24198530

  1. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane.

    PubMed

    Ziegler, Hanne L; Staerk, Dan; Christensen, Jette; Hviid, Lars; Hägerstrand, Henry; Jaroszewski, Jerzy W

    2002-05-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 +/- 0.5 microM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay. PMID:11959580

  2. In Vitro Plasmodium falciparum Drug Sensitivity Assay: Inhibition of Parasite Growth by Incorporation of Stomatocytogenic Amphiphiles into the Erythrocyte Membrane

    PubMed Central

    Ziegler, Hanne L.; Stærk, Dan; Christensen, Jette; Hviid, Lars; Hägerstrand, Henry; Jaroszewski, Jerzy W.

    2002-01-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 ± 0.5 μM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, l