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Primary and secondary drug screening assays for Friedreich ataxia.  


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

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



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

PubMed Central

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 suitable as primary screen in drug discovery efforts, which ultimately aim for a broad-spectrum antischistosomal drug as a larger number of S. mansoni NTS can be generated.



Use of the BRET 7TM receptor/beta-arrestin assay in drug discovery and screening.  


To perform functional cell-based screening assays on seven-transmembrane (7TM) receptors, also known as G-protein coupled receptors, at least three distinct assays are currently needed to screen for G(alphas), G(alphai/0) or G(alphaq/11) signaling receptors. Therefore, there has long been a desire for a universal screening assay that could be used to screen all 7TM receptors independent of their signaling pathway. The receptor/beta-arrestin interaction is common to virtually all 7TM receptors. Therefore, an assay based on this interaction should achieve just that. Bioluminescence resonance energy transfer technology can be used to measure the receptor/beta-arrestin interaction in living cells but due to various technical and biological reasons, the use of the technology for compound screening has been limited. The recent development of beta-arrestin mutants that significantly improve the assay signal, in combination with new improved instrumentation, has transformed bioluminescence resonance energy transfer technology from being a highly specialized research tool in molecular pharmacology to a more drug screening-friendly technique that is useful in an industrial setting. PMID:15137906

Heding, Anders



Formalization, Annotation and Analysis of Diverse Drug and Probe Screening Assay Datasets Using the BioAssay Ontology (BAO)  

PubMed Central

Huge amounts of high-throughput screening (HTS) data for probe and drug development projects are being generated in the pharmaceutical industry and more recently in the public sector. The resulting experimental datasets are increasingly being disseminated via publically accessible repositories. However, existing repositories lack sufficient metadata to describe the experiments and are often difficult to navigate by non-experts. The lack of standardized descriptions and semantics of biological assays and screening results hinder targeted data retrieval, integration, aggregation, and analyses across different HTS datasets, for example to infer mechanisms of action of small molecule perturbagens. To address these limitations, we created the BioAssay Ontology (BAO). BAO has been developed with a focus on data integration and analysis enabling the classification of assays and screening results by concepts that relate to format, assay design, technology, target, and endpoint. Previously, we reported on the higher-level design of BAO and on the semantic querying capabilities offered by the ontology-indexed triple store of HTS data. Here, we report on our detailed design, annotation pipeline, substantially enlarged annotation knowledgebase, and analysis results. We used BAO to annotate assays from the largest public HTS data repository, PubChem, and demonstrate its utility to categorize and analyze diverse HTS results from numerous experiments. BAO is publically available from the NCBO BioPortal at BAO provides controlled terminology and uniform scope to report probe and drug discovery screening assays and results. BAO leverages description logic to formalize the domain knowledge and facilitate the semantic integration with diverse other resources. As a consequence, BAO offers the potential to infer new knowledge from a corpus of assay results, for example molecular mechanisms of action of perturbagens.

Vempati, Uma D.; Przydzial, Magdalena J.; Chung, Caty; Abeyruwan, Saminda; Mir, Ahsan; Sakurai, Kunie; Visser, Ubbo; Lemmon, Vance P.; Schurer, Stephan C.



Bioluminescence screening in vitro (Bio-Siv) assays for high-volume antimycobacterial drug discovery.  

PubMed Central

Bioluminescence-based assays to indicate antimicrobial susceptibility have been developed and validated for recombinant strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, and Mycobacterium intracellulare expressing an integrated eukaryotic luciferase gene. MICs determined with these bioluminescence assays for several antimycobacterial agents, including isoniazid, ethambutol, rifampin, amikacin, streptomycin, ciprofloxacin, and clarithromycin, compared favorably with traditional BACTEC methods and visual estimations of the inhibitory end point. Assay methodology has been optimized for the analysis of large numbers of novel compounds and is simple, inexpensive, and labor efficient. The availability of these four recombinant mycobacteria has permitted a strategy for drug discovery employing the nonpathogenic BCG strain for mass screening purposes with subsequent confirmation of activity against the pathogenic mycobacteria. Furthermore, evidence suggests that the BCG-based screen may allow the direct identification of bactericidal agents.

Arain, T M; Resconi, A E; Hickey, M J; Stover, C K



Assay development and high throughput antiviral drug screening against Bluetongue virus  

PubMed Central

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 ?M, 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.

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



Optimization of the Caco-2 permeability assay to screen drug compounds for intestinal absorption and efflux.  


In vitro permeability assays are a valuable tool for scientists during lead compound optimization. As a majority of discovery projects are focused on the development of orally bioavailable drugs, correlation of in vitro permeability data to in vivo absorption results is critical for understanding the structural-physicochemical relationship (SPR) of drugs exhibiting low levels of absorption. For more than a decade, the Caco-2 screening assay has remained a popular, in vitro system to test compounds for both intestinal permeability and efflux liability. Despite advances in artificial membrane technology and in silico modeling systems, drug compounds still benefit from testing in cell-based epithelial monolayer assays for lead optimization. This chapter provides technical information for performing and optimizing the Caco-2 assay. In addition, techniques are discussed for dealing with some of the most pressing issues surrounding in vitro permeability assays (i.e., low aqueous solubility of test compounds and low postassay recovery). Insights are offered to help researchers avoid common pitfalls in the interpretation of in vitro permeability data, which can often lead to the perception of misleading results for correlation to in vivo data. PMID:21874449

Press, Barry



Fluorescence-based high-throughput screening assay for drug interactions with UGT1A6.  


The increasing awareness and the rising importance of UDP-glucuronosyltransferases (UGTs) in the pharmacokinetics of drugs have evoked a need to develop more powerful tools for studying the role of UGTs in the metabolism of drug candidates. To this end, we have developed a fluorescent high-throughput screening assay for screening potential inhibitors and/or substrates for recombinant human UGTs-here, for the UGT1A6. The assay is based on the increase in fluorescence intensity when 1-naphthol is glucuronidated. The formation of the highly fluorescent product, 1-naphthylglucuronide, is followed at excitation wavelengths of 295 and 300 nm with fixed emission (335 nm) in real time directly from the reaction mixture. A probe concentration of 5 ?M with 2.5 ?g of total protein in phosphate buffer at pH 7.4 with 5% dimethyl sulfoxide resulted in optimal linearity and acceptable signal separation (signal-to-base, 3.0) for the probe reaction. The interactions of test compounds with the enzyme are detected as lower rate of 1-naphthylglucuronide formation and thus lower rate of fluorescence increase. The success of the assay was first demonstrated with the known UGT1A6 substrates 4-hydroxyindole and scopoletin (Z' factor ?0.5) and later with nonsteroidal anti-inflammatory drugs and salicylate derivatives. Diclofenac, 5-methylsalicylic acid, 5-bromosalicylic acid, 5-chlorosalicylic acid, and 5-fluorosalicylic acid decreased the probe glucuronidation rate at 500??M by >50%. Further, the results gained with the high-throughput screening assay correlated well with the results obtained, in parallel, with the reference high-performance liquid chromatography method. PMID:21438674

Soikkeli, Anne; Kurkela, Mika; Hirvonen, Jouni; Yliperttula, Marjo; Finel, Moshe



A semi-automated neutral red based chemosensitivity assay for drug screening  

Microsoft Academic Search

A semi-automated colorometric chemosensitivity assay was developed. The assay utilizes the vital stain neutral red for the rapid screening of potential anticancer agents using solid tumor cell lines. The cell lines used in this assay and presented in this report are CX-1 colon adenocarcinoma and A549 lung carcinoma. The assay, performed in 96 well tissue culture plates, allows for short

Paul F. Cavanaugh Jr; Patricia S. Moskwa; William H. Donish; Paula J. Pera; Debra Richardson; Angelo P. Andrese



A Generally Applicable, High-Throughput Screening–Compatible Assay to Identify, Evaluate, and Optimize Antimicrobial Agents for Drug Therapy  

Microsoft Academic Search

Efficacy and tolerability are the key criteria for a successful medication in the clinic. Therefore, a new test method to obtain selective and active lead molecules has been developed. Recently, this novel screening strategy enabled a breakthrough in drug discovery in the field of herpes viruses. Here the authors report that this assay is a generally applicable screening test, which

Gerald Kleymann; Hans-Otto Werling



Assessment and Continued Validation of the Malaria SYBR Green I-Based Fluorescence Assay for Use in Malaria Drug Screening  

Microsoft Academic Search

Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability

Jacob D. Johnson; Richard A. Dennull; Lucia Gerena; Miriam Lopez-Sanchez; Norma E. Roncal; Norman C. Waters



Drug-induced liver steatosis and phospholipidosis: cell-based assays for early screening of drug candidates.  


The liver plays a key role in fat metabolism, and excessive lipid accumulation in liver cells is characterised by a large spectrum of lesions, e.g., steatosis and phospholipidosis. Steatosis is increased lipid accumulation, mainly as triglycerides, in the liver, while phospholipidosis is a lysosomal storage disorder characterised by intracellular accumulation of phospholipids. These alterations can be induced by several factors, including exposure to certain drugs. Drug-induced steatosis is often reversible, and prolonged exposure to certain drugs can cause macrovacuolar steatosis, a benign hepatic lesion, that can evolve into steatohepatitis and cirrhosis in some patients. Some drugs may acutely induce microvesicular steatosis which, despite having a good short-term prognosis, can lead to chronic lipid peroxidation and to the development of steatohepatitis lesions with time. Over 50 marketed drugs have been reported to induce phospholipidosis in different tissues, including the liver. Although drug-induced phospholipidosis is often reversible and there is no definitive evidence for its toxicological implications, it is considered an adverse side finding by regulatory agencies. As developing new drugs is a complex, lengthy and expensive process that aims to identify pharmacologically active, low-toxicity drug candidates among closely related compounds, it could be advantageous to determine which drugs are able to induce lipid metabolic disorders in early developmental stages. To this end, in vitro predictive screening assays, particularly cell-based approaches in which many drug candidates are evaluated, have been developed to identify and rule out compounds with a strong liver steatosis and/or phospholipidosis-inducing potential. PMID:22746303

Donato, M Teresa; Gómez-Lechón, M José



Screening for Drugs of Abuse with the Roche ONTRAK Assays. (Reannouncement with New Availability Information).  

National Technical Information Service (NTIS)

Manual, qualitative latex agglutination inhibition screening tests (ONTRAK, Rocehe) for amphetamines, barbiturates, cocaine, marijuana, morphine, and phencyclidine were evaluated. The assays are inexpensive, rapid, and easy to perform, requiring little tr...

D. A. Armbruster J. M. Krolak



Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug  

PubMed Central

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known anti-leishmanial drugs, i. e. amphotericin B and glucantime®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.

Varela, Ruben Eduardo M; Munoz, Diana Lorena; Robledo, Sara M.; Kolli, Bala K.; Dutta, Sujoy; Chang, Kwang Poo; Muskus, Carlos



Screening for phospholipidosis induced by central nervous drugs: comparing the predictivity of an in vitro assay to high throughput in silico assays.  


Drug-induced phospholipidosis is a side effect for which drug candidates can be screened in the drug discovery phase. The numerous in silico models that have been developed as a first line of screening are based on the characteristic physicochemical properties of phospholipidosis-inducing drugs, e.g. high logP and pK(b) values. However, applying these models on a predominantly high lipophilic, basic CNS chemistry results in a high false positive rate and consequently in a wrong classification of a large number of valuable drug candidates. Here, we tested 33 CNS-compounds (24 in vivo negative and 9 in vivo positive phospholipidosis-inducers) in our in house developed in vitro phospholipidosis screening assay (Mesens et al., 2009) and compared its predictivity with the outcome of three different, well established in silico prediction models. Our in vitro assay demonstrates an increased specificity of 79% over the in silico models (29%). Moreover, by considering the proposed plasma concentration at the efficacious dose we can show a clear correlation between the in vitro and in vivo occurrence of phospholipidosis, improving the specificity of prediction to 96%. Through its high predictive value, the in vitro low throughput assay is thus preferred above high throughput in silico assays, characterized by a high false positive rate. PMID:20430096

Mesens, Natalie; Steemans, Margino; Hansen, Erik; Verheyen, Geert R; Van Goethem, Freddy; Van Gompel, Jaques



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


Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA2'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, GpppA2'OMe-RNA74, by ligation of two RNA fragments. Then, we use GpppA2'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

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



Improved toxicogenomic screening for drug-induced phospholipidosis using a multiplexed quantitative gene expression ArrayPlate assay.  


We previously showed that a toxicogenomics analysis of drug-induced phospholipidosis enabled the identification of 12 specific gene markers and the establishment of an in vitro real-time PCR screening assay for the assessment of the phospholipidosis-inducing potential of compounds. The purpose of this study was to transfer our PCR-based assay into a 96-well microplate-based multiple mRNAs measuring assay (ArrayPlate assay) in order to increase throughput. Specifically, we determined the expression of the 12 marker genes using real-time PCR and ArrayPlate in human hepatoma HepG2 cells that were treated for 24h with each of amiodarone and 80 proprietary compounds. The following three performance criteria were satisfied in the ArrayPlate analysis: 1. Sensitivity-the expression of mRNA for all target genes was detected at quantifiable levels. 2. Repeatability-signal intensities and fold change values of each marker gene were highly repeatable. 3. Correlation-fold change values and their average values, which were used as indices of phospholipidosis induction potential, showed apparent correlation between the ArrayPlate and real-time PCR assays. Thus, the in vitro screening assay for compound-induced phospholipidosis should be transferable from a PCR-based assay to the higher-throughput ArrayPlate-based method. PMID:16919414

Sawada, Hiroshi; Taniguchi, Keiko; Takami, Kenji



Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential.  


Cryopreserved human hepatocytes were extensively characterized in our laboratory. The post-thaw viability, measured via dye exclusion, ranged from 55 to 83%, for hepatocytes cryopreserved from 17 donors. Post-thaw viability and yield (viable cells per vial) were found to be stable up to the longest storage duration evaluated of 120 days. Drug-metabolizing enzyme activities of the cryopreserved hepatocytes (mean of ten donors) as percentages of the freshly isolated cells were: 97%, for cytochrome P450 isoform (CYP) 1A2, 78% for CYP2A6, 96% for CYP2C9. 86% for CYP2Cl9, 90% for CYP2D6, 164% for CYP3A4, 76% for UDP-glucuronidase, and 88% for umbelliferone sulfotransferase. Known species-differences in 7-ethoxycoumarin (7-EC) metabolism were reproduced by cryopreserved hepatocytes from human, rat, rabbit, dog, and monkey, illustrating the utility of cryopreserved hepatocytes from multiple animal species in the evaluation of species-differences in drug metabolism. Higher throughput screening (HTS) assays were developed using cryopreserved human hepatocytes for hepatotoxicity, metabolic stability, and inhibitory drug-drug interactions. Dose-dependent cytotoxicity, measured using MTT metabolism as an endpoint, was observed for the known hepatotoxic chemicals tamoxifen, clozapine, cadmium chloride, diclofenac, amiodarone, tranylcypromine, precocene II, but not for 2-thiouracil. Cell density- and time-dependent metabolism of 7-EC and dextromethorphan were observed in the HTS assay for metabolic stability. Known CYP isoform-specific inhibitors were evaluated in the HTS assay for inhibitory drug-drug interactions. Furafylline, sulfaphenazole, quinidine, and ketoconazole were found to be specific inhibitors of CYP1A2, CYP2C9, CYP2D6, and CYP3A4, respectively. Tranylcypromine and diethyldithiocarbamate were found to be less specific, with inhibitory effects towards several CYP isoforms, including CYP2A6, CYP2C9, CYP2C19, and CYP2E1. These results suggest that cryopreserved human hepatocytes represent a useful experimental tool for the evaluation of drug metabolism, toxicity, and inhibitory drug-drug interaction potential. PMID:10418968

Li, A P; Lu, C; Brent, J A; Pham, C; Fackett, A; Ruegg, C E; Silber, P M



Cell-based bioluminescence screening assays.  


Drug screening is an essential and widely used technique for drug discovery in various biomedical fields notably in oncology. Here we describe a functional screening assay based on the bioluminescence detection of a secreted luciferase for monitoring cell viability of cancer cells in a high-throughput format. This assay allows the screening of large libraries comprising thousands of compounds and the identification of potential anticancer molecules in a rapid, facile, and cost-effective manner. PMID:24166378

Amante, Romain J; Badr, Christian E



A Novel High Throughput Assay for Anthelmintic Drug Screening and Resistance Diagnosis by Real-Time Monitoring of Parasite Motility  

Microsoft Academic Search

BackgroundHelminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed

Michael J. Smout; Andrew C. Kotze; James S. McCarthy; Alex Loukas



Current status of targets and assays for anti-HIV drug screening  

Microsoft Academic Search

HIV\\/AIDS is one of the most serious public health challenges globally. Despite the great efforts that are being devoted to\\u000a prevent, treat and to better understand the disease, it is one of the main causes of morbidity and mortality worldwide. Currently,\\u000a there are 30 drugs or combinations of drugs approved by FDA. Because of the side-effects, price and drug resistance,

Ren-rong Tian; Qing-jiao Liao; Xu-lin Chen



Development and Application of a GFP-FRET Intracellular Caspase Assay for Drug Screening  

Microsoft Academic Search

Apoptosis is a crucial biological process, and activation of caspase endoproteases is essential for proper regulation and execution of apoptosis. Because caspases also appear to be central players in several pathological states, there is a practical need within the biopharmaceutical research community for facile, noninvasive cellular assays for the discovery of compounds that modulate caspase activity. Tandem molecules of green

Jay Jones; Roger Heim; Eric Hare; Jeffrey Stack; Brian A. Pollok



Bioluminescent Assays for High-Throughput Screening  

Microsoft Academic Search

In the development of high throughput screening (HTS) as a central paradigm of drug discovery, fluorescence has generally been adopted as the favored methodology. Nevertheless, luminescence has maintained a prominent position among certain assay formats, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a wider range of applications due to their sensitivity, broad linearity,

Frank Fan; Keith V. Wood



Development and validation of a chemiluminescent immunodetection assay amenable to high throughput screening of antiviral drugs for Nipah and Hendra virus  

Microsoft Academic Search

There are currently no antiviral drugs approved for the highly lethal Biosafety Level 4 pathogens Nipah and Hendra virus. A number of researchers are developing surrogate assays amenable to Biosafety Level 2 biocontainment but ultimately, the development of a high throughput screening method for directly quantifying these viruses in a Biosafety Level 4 environment will be critical for final evaluation

Mohamad Aljofan; Matteo Porotto; Anne Moscona; Bruce A. Mungall



Bioluminescence and chemiluminescence in drug screening  

Microsoft Academic Search

Drug screening, that is, the evaluation of the biological activity of candidate drug molecules, is a key step in the drug discovery and development process. In recent years, high-throughput screening assays have become indispensable for early stage drug discovery because of the developments in synthesis technologies, such as combinatorial chemistry and automated synthesis, and the discovery of an increasing number

Aldo Roda; Massimo Guardigli; Patrizia Pasini; Mara Mirasoli



A 96-well flow cytometric screening assay for detecting in vitro phospholipidosis-induction in the drug discovery phase.  


Drug-induced phospholipidosis is caused by lysosomal accumulation of the drug, resulting in the disturbance of phospholipid degradation and a consequent excessive phospholipid accumulation. Depending on the type and number of tissues affected, phospholipidosis occurrence in test animals can raise safety issues, which may be critical for the risk assessment. Safety profiling of potential phospholipidosis-inducing drugs in the drug discovery phase can predict these late obstructions of drug development. For this purpose, a flow cytometric assay based on the difference in fluorescent phospholipid accumulation in a human monocyte cell line was initially established. Modifying the assay studying degradation of the fluorescent phospholipids instead of accumulation drastically improved sensitivity. By testing various phospholipidosis-inducers and negative compounds, it was found that the assay could detect the occurrence of phospholipidosis by a 2-fold difference in fluorescence compared to control cells, demonstrating the superior sensitivity of the novel approach. Implementation of a higher throughput 96-well flow cytometric set up did not affect the sensitivity of detection or the reproducibility of the assay. Based on an extended test set of reference compounds a profiling approach was introduced, by which we show we can rank our drug candidates according to their phospholipidosis-inducing potential. PMID:19101623

Natalie, Mesens; Margino, Steemans; Erik, Hansen; Annelieke, Peters; Geert, Verheyen; Philippe, Vanparys



Selection of drugs to test the specificity of the Tg.AC assay by screening for induction of the gadd153 promoter in vitro.  


Short-term assays for carcinogenicity testing of chemicals that use transgenic mice designed to have altered expression of genes mechanistically relevant to carcinogenesis are attractive alternatives to two-year dosing studies in rodents. The models that have been the received the greatest level of performance evaluation include p53(+/-), rasH2, Xpa/p53(+/-), and Tg.AC mice. For use of these models in a regulatory setting to evaluate the carcinogenic potential of pharmaceuticals, it is important to establish an assurance of assay specificity and positive predictivity based on studies using drugs with a wide spectrum of pharmacologic activity. For this purpose, 99 noncarcinogenic drugs were prioritized based on their activity in an in vitro induction assay correlative with a positive response in the Tg.AC assay (induction of the gadd153 promoter in HepG2 cells). Activities in two assays less predictive of Tg.AC activity (induction of c-fos and zeta-globin gene promoters) were also measured. Nine percent of the screened drugs induced the gadd153 promoter by at least fourfold. Several criteria were used to select candidates for subsequent in vivo testing in the Tg.AC assay: (1) sufficient drug solubility in appropriate skin paint vehicles to elicit systemic toxicity, (2) the level of induction of the gadd153 promoter by the drug, (3) the in vitro potency of the drug, and (4) the cost of the drug required for a 6-month study. Based on these criteria, amiloride, dipyridamole, and pyrimethamine were selected from 99 rodent noncarcinogens in a drug database for testing the specificity of the Tg.AC assay. PMID:12730611

Thompson, Karol L; Sistare, Frank D



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

PubMed Central

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.

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



Hydrogel-based diffusion chip with Electric Cell-substrate Impedance Sensing (ECIS) integration for cell viability assay and drug toxicity screening.  


In this study, we have provided a novel analytical integration between hydrogel-based cell chip and Electric Cell-substrate Impedance Sensing (ECIS) technique to apply to a high-throughput, real-time cell viability assay and drug screening. For simulating the drug diffusion model, we have developed a hydrogel-based tissue-mimicking structure with microfluidic channel, without unwanted flow, to generate a gradient concentration with long-term stability. Along the gradient line, four individual micro-electrodes were installed to record the impedance signal changes, which result from the cell viability under drug effects. By watching for cellular impedance changes, we successfully estimated the cytotoxicity of the treatment corresponding to the various concentration values of stimuli, generated by the diffusion process along the channel. Reliable IC50 values and time-dose relationships were also achieved. With the feature of real-time monitoring capability, the advantages of non-invasion, label-free detection, time saving and simple manipulation, our integrative device has become a promising high throughput cell-based on-chip platform for cell viability assay and drug screening. PMID:23911660

Tran, Trong Binh; Cho, Sungbo; Min, Junhong



Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference  

PubMed Central

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [125I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.

Ansbro, Megan R.; Shukla, Suneet; Ambudkar, Suresh V.; Yuspa, Stuart H.; Li, Luowei



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


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

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



Development of an assay for Complex I/Complex III of the respiratory chain using solid supported membranes and its application in mitochondrial toxicity screening in drug discovery.  


Membrane-bound transporter proteins are involved in cell signal transduction and metabolism as well as influencing key pharmacological properties such as drug bioavailability. The functional activity of transporters that belong to the group of electrically active membrane proteins can be directly monitored using the solid-supported membrane-based SURFE(2)R™ technology (SURFace Electrogenic Event Reader; Scientific Devices Heidelberg GmbH, Heidelberg, Germany). The method makes use of membrane fragments or vesicles containing transport proteins adsorbed onto solid-supported membrane-covered electrodes and allows the direct measurement of their activity. This technology has been used to develop a robust screening compatible assay for Complex I/Complex III, key components of the respiratory chain in 96-well microtiter plates. The assay was screened against 1,000 compounds from the ComGenex Lead-like small molecule library to ascertain whether mitochondrial liabilities might be an underlying, although undesirable feature of typical commercial screening libraries. Some 105 hits (compounds exhibiting >50% inhibition of Complex I/Complex III activity at 10??M) were identified and their activities were subsequently confirmed in duplicate, yielding a confirmation rate of 68%. Analysis of the confirmed hits also provided evidence of structure-activity relationships and two compounds from one structural class were further evaluated in dose-response experiments. This study provides evidence that profiling of compounds for potential mitochondrial liabilities, even at an early stage of drug discovery, may be a necessary additional quality filter that should be considered during the compound screening and profiling cascade. PMID:21133681

Preissl, Sebastian; Bick, Inga; Obrdlik, Petr; Diekert, Kerstin; Gul, Sheraz; Gribbon, Philip



Fluorescence Polarization Assays in Small Molecule Screening  

PubMed Central

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.

Lea, Wendy A.; Simeonov, Anton



Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a Microculture Tetrazolium Assay1  

Microsoft Academic Search

For the past 30 years strategies for the prcclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro\\/in vivo screening for selective

Michael C. Alley; Dominic A. Scudiere; Anne Monks; Miriam L. Hursey; Maciej J. Czerwinski; Donald L. Fine; Betty J. Abbott; Joseph G. Mayo; Robert H. Shoemaker; Michael R. Boyd



Development of a high-throughput antiviral assay for screening inhibitors of chikungunya virus and generation of drug-resistant mutations in cultured cells.  


Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that has already infected millions of people in recent large-scale epidemics in Africa, the islands of the Indian Ocean, South and Southeast Asia, and northern Italy. The infection is still ongoing in many countries, such as India. Although the fatal rate is approximately 0.1% in the La Réunion outbreak, it causes painful arthritis-like symptoms that can last for months or even years. Currently, neither vaccine nor approved antiviral therapy exists to protect humans from chikungunya infection. Therefore, there is an urgent unmet medical need for the development of antiviral drugs for pre-exposure prophylaxis and/or treatment of chikungunya infections. In this chapter, we describe a fully validated ATP/luminescence assay that is effective for high-throughput screening of CHIKV inhibitors. Protocols for growing CHIKV stocks and generating drug-resistant viral variants for modes of action studies of compounds are also described. PMID:23821286

Gong, Edwin Yunhao; Bonfanti, Jean-François; Ivens, Tania; Van der Auwera, Marijke; Van Kerckhove, Barbara; Kraus, Guenter



Signify® ER Drug Screen Test evaluation: comparison to Triage® Drug of Abuse Panel plus tricyclic antidepressants  

Microsoft Academic Search

Signify® ER Drug Screen Test (Signify ER) and Triage® Drug of Abuse Panel plus TCA (Triage DOA Panel) rapid drug screening devices were compared at four laboratories. Both assay systems are point of care immunoassays, measuring phencyclidine, barbiturates, amphetamine, cocaine metabolite, methamphetamine, tricyclic antidepressants, opiates, marijuana metabolite, and benzodiazepines in human urine. The performance of these two assay systems, including

Jane Ellen Phillips; Stuart Bogema; Paul Fu; Wieslaw Furmaga; Alan H. B Wu; Vlasta Zic; Catherine Hammett-Stabler



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


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

Marquet, Pierre



Solid-state electrochemical assay of heme-binding molecules for screening of drugs with antimalarial potential.  


The interaction between heme and ligands is the basis for a variety of tests aimed at the discovery of antiplasmodial molecules. Two electrochemical methods for the screening of molecules with potential antimalarial activity through heme-binding mechanism are described. The first method is applicable to lipophilic environment, by using solution phase electrochemistry in DMSO solutions of Fe(III)-heme plus the tested compounds at carbon electrodes. This method provides well-defined voltammetric signals, characteristic of the heme-ligand (L) interaction. The second method involves aqueous media at biological pH and the use of voltammetry of immobilized particles, by means of microparticulate films of the tested compounds immersed into Fe(III)-heme solutions with no need of prior incubation. These methodologies are applied to the testing of heme-binding activity in macromolecular level systems like hemoglobin, or much more complex mixtures like total blood, erythrocytes, or hemolyzed samples. PMID:23506072

Doménech-Carbó, Antonio; Maciuk, Alexandre; Figadère, Bruno; Poupon, Erwan; Cebrián-Torrejón, Gerardo



Screening of Antifungal Azole Drugs and Agrochemicals with an Adapted alamarBlue-Based Assay Demonstrates Antibacterial Activity of Croconazole against Mycobacterium ulcerans  

PubMed Central

An alamarBlue-based growth inhibition assay has been adapted for the thermosensitive and slow-growing pathogen Mycobacterium ulcerans. The standardized test procedure enables medium-throughput screening of preselected compound libraries. Testing of a set of 48 azoles with known antifungal activity led to the identification of an imidazole antifungal displaying an inhibitory dose (ID) of 9 ?M for M. ulcerans.

Roltgen, Katharina; Witschel, Matthias; Pluschke, Gerd



A high content screening assay for identifying lysosomotropic compounds.  


Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable. Hence, we developed a 96-well format, high content screening assay that measures lysosomotropism and cytotoxicity by quantitative image analysis. Forty drugs, including antidepressants, antipsychotics, antiarrhythmics and anticancer agents, were tested for their effects on lysosomotropism and cytotoxicity in H9c2 cells. The assay correctly identified drugs known to cause lysosomotropism and revealed novel information showing that the anticancer drugs, gefitinib, lapatinib, and dasatinib, caused lysosomotropism. Although structurally and pharmacologically diverse, drugs that were lysosomotropic shared certain physicochemical properties, possessing a ClogP>2 and a basic pKa between 6.5 and 11. In contrast, drugs which did not lie in this physicochemical property space were not lysosomotropic. The assay is a robust, rapid screen that can be used to identify lysosomotropic, as well as, cytotoxic compounds, and can be positioned within a screening paradigm to understand the role of lysosomotropism as a contributor to drug-induced toxicity. PMID:21184822

Nadanaciva, Sashi; Lu, Shuyan; Gebhard, David F; Jessen, Bart A; Pennie, William D; Will, Yvonne



Design and Implementation of High Throughput Screening Assays  

Microsoft Academic Search

High throughput screening (HTS) is at the core of the drug discovery process, and so it is critical to design and implement\\u000a HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics.\\u000a This requires careful analysis of many variables, starting with the choice of assay target and ending with the discovery of\\u000a lead

Ricardo Macarrón; Robert P. Hertzberg



Schistosomiasis Drug Screening.  

National Technical Information Service (NTIS)

From a total of 5,000 WR compounds which have been screened so far, 75 have been found to produce oogram changes in laboratory animals infected with Schistosoma mansoni. The antischistosomal activity of quinine and isomers, thiosinamine, diphenylsulfones,...

J. Pellegrino



Screening of antifungal azole drugs and agrochemicals with an adapted alamarBlue-based assay demonstrates antibacterial activity of croconazole against Mycobacterium ulcerans.  


An alamarBlue-based growth inhibition assay has been adapted for the thermosensitive and slow-growing pathogen Mycobacterium ulcerans. The standardized test procedure enables medium-throughput screening of preselected compound libraries. Testing of a set of 48 azoles with known antifungal activity led to the identification of an imidazole antifungal displaying an inhibitory dose (ID) of 9 ?M for M. ulcerans. PMID:23006761

Scherr, Nicole; Röltgen, Katharina; Witschel, Matthias; Pluschke, Gerd



Non-Invasive Screening Techniques for Drugs of Abuse.  

National Technical Information Service (NTIS)

A review of current drug of abuse screening methods available in Canada in 1982 is presented. These methods include classical thin-layer chromatography (TLC), the commercially available Toxi-Lab assay technique (EMIT), and radioimmunoassay (RIA - Aburscre...

L. J. McBurney



Urine drug screening in the medical setting  

Microsoft Academic Search

Background: The term drug screen is a misnomer since it implies screening for all drugs, which is not possible. Current practice is to limit the testing to the examination of serum for several drugs such as ethanol, acetaminophen, salicylate, and of urine for several specific drugs or classes of drugs. In the emergency setting the screen should be performed in

Catherine A Hammett-Stabler; Amadeo J Pesce; Donald J Cannon



A novel Leishmania major amastigote assay in 96-well format for rapid drug screening and its use for discovery and evaluation of a new class of leishmanicidal quinolinium salts.  


In most laboratories, the screening for leishmanicidal compounds is carried out with Leishmania promastigotes or axenic amastigotes. However, the best approach to identify leishmanicidal compounds is the use of amastigotes residing in macrophages. Reporter gene-based assays are relatively new tools in the search for drugs against eucaryotic protozoa, permitting the development of faster, more automated assays. In this paper, we report on the establishment of a rapid screening assay in a 96-well format. A luciferase-transgenic (Luc-tg) Leishmania major strain was generated and used to infect bone marrow-derived macrophages (BMDM). Amastigote-infected BMDM were treated with different compound concentrations. Cells were lysed with a luciferin-containing buffer, and the resulting luminescence was measured to determine the half-maximal inhibitory concentration (IC50). To validate this new amastigote screening assay, a library of a new class of quinolinium salts was synthesized and tested for leishmanicidal activity. Some of the quinolinium salts showed very promising activities, with IC50s against intracellular amastigotes (IC50 < 1 ?g/ml) and selectivity indices (SI > 20) that match the criteria of World Health Organization (WHO) for hits. Compound 21c (IC50 = 0.03 ?g/ml; SI = 358) could become a new lead structure for the development of improved chemotherapeutic drugs against L. major. In summary, we describe the establishment of a new 96-well format assay with Luc-transgenic L. major for the rapid screening of compounds for leishmanicidal activity against intracellular amastigotes and its application to the identification of a new class of quinolinium salts with most promising leishmanicidal activity. PMID:23587955

Bringmann, Gerhard; Thomale, Katja; Bischof, Sebastian; Schneider, Christoph; Schultheis, Martina; Schwarz, Tobias; Moll, Heidrun; Schurigt, Uta




Technology Transfer Automated Retrieval System (TEKTRAN)

Certain fungi that are commonly found in commodities such as corn and wheat produce toxins, known as mycotoxins. Monitoring of mycotoxins is routinely done to minimize their occurrence in the human and animal food supplies. This article describes recent advances in screening assays and focuses on ...


Screening models for antipsychotic drugs  

Microsoft Academic Search

\\u000a Development of effective screening strategies for improved antipsychotic drugs (APDs) rely primarily on the actual hypotheses\\u000a for mechanism of action of these compounds. In the early times of the dopamine (DA) hypothesis of schizophrenia animal models\\u000a were designed to detect compounds which blocked the behavioural effects of high doses of dopaminergic drugs (e.g. amphetamine-,\\u000a apomorphine-and methylphenidate-induced stereotyped behaviour). Whenin vitroreceptor

Jorn Arnt


A cell-based assay for screening spindle checkpoint inhibitors.  


In eukaryotes, the spindle checkpoint acts as a surveillance mechanism that ensures faithful chromosome segregation. The spindle checkpoint prevents premature separation of sister chromatids and the onset of anaphase until every chromosome is properly attached to the mitotic spindle. Tumorigenesis might result from generation of aneuploidy by dysfunction of the spindle checkpoint. Differences of the checkpoint system in normal cells versus tumor cells might provide a new opportunity in cancer drug development; therefore, efforts to identify the spindle checkpoint inhibitors have been fostered. Based on spindle checkpoint inhibitors being able to induce cells to exit mitotic arrest caused by microtubule drug treatment, we developed a cell-based assay to screen compounds that were potential spindle checkpoint inhibitors. This assay was validated with a known spindle checkpoint inhibitor and was easy to adapt to a large-scale screening. It also had the advantages of being high in sensitivity and low in cost. PMID:22352901

Wu, Zhen Hua; Hu, Long Yu; Xu, Da Qian; Li, Xiaotong



A Cell-Based Assay for Screening Spindle Checkpoint Inhibitors  

PubMed Central

Abstract In eukaryotes, the spindle checkpoint acts as a surveillance mechanism that ensures faithful chromosome segregation. The spindle checkpoint prevents premature separation of sister chromatids and the onset of anaphase until every chromosome is properly attached to the mitotic spindle. Tumorigenesis might result from generation of aneuploidy by dysfunction of the spindle checkpoint. Differences of the checkpoint system in normal cells versus tumor cells might provide a new opportunity in cancer drug development; therefore, efforts to identify the spindle checkpoint inhibitors have been fostered. Based on spindle checkpoint inhibitors being able to induce cells to exit mitotic arrest caused by microtubule drug treatment, we developed a cell-based assay to screen compounds that were potential spindle checkpoint inhibitors. This assay was validated with a known spindle checkpoint inhibitor and was easy to adapt to a large-scale screening. It also had the advantages of being high in sensitivity and low in cost.

Wu, Zhen Hua; Hu, Long Yu; Xu, Da Qian



Zebrafish assays as early safety pharmacology screens: Paradigm shift or red herring?  

Microsoft Academic Search

The recent flurry of interest in the potential use of the zebrafish (Danio rerio) in Drug Discovery has also led to the development of a range of assays purported to be useful as early screens in safety pharmacology. The purpose of this commentary is to take stock of the available zebrafish assays in the context of alternative mammalian cell-based assays,

William S. Redfern; Gareth Waldron; Matthew J. Winter; Paul Butler; Mark Holbrook; Rob Wallis; Jean-Pierre Valentin



In vitro screening for drug-induced depression and/or suicidal adverse effects: a new toxicogenomic assay based on CE-SSCP analysis of HTR2C mRNA editing in SH-SY5Y cells.  


Many drugs in clinical trials, or already on the market, can have psychiatric side effects, independently of their therapeutic indication (e.g., Acomplia, Taranabant, and Roaccutane). There is currently no in vitro or in vivo approved test for the detection/prediction of such adverse effects, and the Food and Drugs Administration (FDA) can only issue general alerts on specific therapeutic classes. The development of a screening assay is therefore of real interest. The anti-viral and anti-tumor action of human interferon-alpha (hIFN?) is associated with a variety of neuropsychiatric side effects, including major depression, suicidal ideation and suicide. RNA editing of the serotonin 2C receptor (HTR2C) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification, the regulation of which is altered in depressed suicide victims. In this study, we show that in the SH-SY5Y neuroblastoma cell line, hIFN? specifically activates the ADAR1a isoform and thereby modifies the HTR2C mRNA editing profile. As this hIFN?-induced altered profile partly overlaps with that observed in the brain of depressed suicide victims, we investigated whether it could be used as a signature to identify drugs with depression and/or suicidal side effects. By means of the Biocortech proprietary screening assay, which allows the relative quantification of all the edited HTR2C isoforms in a sample, we blind-tested the effect of 50 marketed molecules on HTR2C mRNA editing in SH-SY5Y cells and identified 17 compounds with an IFN-like editing profile. This new toxicogenomic assay can identify compounds with potential psychiatric adverse events with a positive predictive value of 90 %. PMID:22528247

Cavarec, Laurent; Vincent, Laurent; Le Borgne, Claudia; Plusquellec, Camille; Ollivier, Nathalie; Normandie-Levi, Priscilla; Allemand, Frédéric; Salvetat, Nicolas; Mathieu-Dupas, Eve; Molina, Franck; Weissmann, Dinah; Pujol, Jean-François



A fluorescent screening assay for identifying modulators of GIRK channels.  


G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue(1,2). GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate. GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders(3). However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations(3). Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry(4) or radiotracer analysis(5), the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure. PMID:22706581

Vazquez, Maribel; Dunn, Charity A; Walsh, Kenneth B



Virtual screening and in vitro assay of potential drug like inhibitors from spices against glutathione-S-transferase of filarial nematodes.  


Glutathione-S-transferase(s) (GST) enzyme from Brugia malayi has been exploited as a target in lymphatic filariasis therapeutics. An active GST is a homodimer of a 208 residue long monomer consisting of two domains, a smaller ?/? domain and a larger ? domain. The components of the glutathione (GSH) system, mainly GST enzymes, are critical antioxidant and detoxification system responsible for the long-term existence of filarial worms in mammalian host; hence they are major chemotherapeutic targets in filarial species. In the present study, 58 phytochemicals from 10 plants, predicted and reported to have potential nematicidal activity and ADMET satisfaction, have been docked to GST enzyme of B. malayi to assess their binding affinity and consequently their inhibitory activity. A comparative study has been made with commonly employed chemotherapeutic GST inhibitors such as cibacron-blue, butylated hydroxyanisole, hexyl glutathione and ethacrynic acid. In vitro effects of potential drug like compound from in silico results have been done for validation of docking studies. In vitro assay revealed efficacy in GST inhibition in the following compounds: linalool (97.50%), alpha-pinene (90.00%), strychnine (87.49%), vanillin (84.99%), piperine (79.99%), isoeugenol (62.49%), curcumin (57.49%), beta-caryophyllene (39.50%), cinnamic acid (27.49%), capsaicin (19.99%), citronellol (19.99%) and geraniol (17.49%). An online database ( ) has been developed, which will serve as a useful repository of information on GST inhibitors for future development of drugs against filarial nematodes. These findings thus suggest that the above phytochemicals could be potentially developed as lead molecules for targeting GST of lymphatic filarial parasites. PMID:21523552

Azeez, Shamina; Babu, Rosana O; Aykkal, Riju; Narayanan, Reena



Clinical implications of urinary drug screens  

Microsoft Academic Search

Urine drug screens are routinely used in the substance abuse treatment setting along with other clinical settings when illicit drug use is suspected. With the importance placed on the results of urine drug screens, clinicians should be aware of the strengths and weaknesses of the tests involved when interpreting the results. Such interpretation is quite complicated and depends on a

Michael F. Barber



Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds  

Microsoft Academic Search

Drugs currently available for leishmaniasis treatment often show parasite resistance, highly toxic side effects and prohibitive costs commonly incompatible with patients from the tropical endemic countries. In this sense, there is an urgent need for new drugs as a treatment solution for this neglected disease. Here we show the development and implementation of an automated high-throughput viability screening assay for

Jair L. Siqueira-Neto; Ok-Ryul Song; Hyunrim Oh; Jeong-Hun Sohn; Gyongseon Yang; Jiyoun Nam; Jiyeon Jang; Jonathan Cechetto; Chang Bok Lee; Seunghyun Moon; Auguste Genovesio; Eric Chatelain; Thierry Christophe; Lucio H. Freitas-Junior



Screening for Drugs of Abuse Using the Abbott ADx. (Reannouncement with New Availability Information).  

National Technical Information Service (NTIS)

We evaluated the Abbott ADx analyzer for abused substances for six different drug classes and compared it to the Abbott TDx analyzer for screening urines for drugs of abuse. The sensitivities of five of the assays amphetamines, barbiturates, benzodiazepin...

D. T. Green D. A. Armbruster




EPA Science Inventory

An in vitro fish liver tissue assay has been developed to test for estrogenic and anti-estrogenic effects of xenobiotics. The assay is part of a broader effort to provide multi-level screening of chemicals to enhance......


A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening  

Microsoft Academic Search

Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect\\u000a reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel\\u000a non-radioactive assay based on the principle of pyrosequencing coupled-enzyme system to monitor the activity of HIV-RT by\\u000a indirectly measuring the release of pyrophosphate (PPi),

Chang Zhang; Yang Wu; Yuna Sun; Chuan Hong; Kehui Xiang; Yu Guo; Mark Bartlam; Zhiyong Lou



Alternative Statistical Parameter for High-Throughput Screening Assay Quality Assessment  

Microsoft Academic Search

High-throughput screening is an essential process in drug discovery. The ability to identify true active compounds depends on the high quality of assays and proper analysis of data. The Z factor, presented by Zhang et al. in 1999, provides an easy and useful summary of assay quality and has been a widely accepted standard. However, as data analysis has undergone

Yunxia Sui; Zhijin Wu



Chemical Genetics: Drug Screens in Zebrafish  

Microsoft Academic Search

High throughput chemical genetic screens for compounds with specific biological activity in a whole organism are feasible using zebrafish embryos. At least two medium to large scale drug screens have been carried out to date, leading to the identification of compounds that disturb zebrafish development. Chemical genetics using zebrafish embryos may become an important step in the discovery of drugs

Jeroen den Hertog



Safety Pharmacology Screening: Practical Problems in Drug Development  

Microsoft Academic Search

Undesired pharmacologic activities of novel drugs or biologies may limit development of a therapeutic prior to the characterization of any toxicologic effects. In rodent species, general pharmacology assays have traditionally been used to screen new agents for pharmacologic effects on the central and peripheral nervous systems, the autonomic nervous system and smooth muscles, the respiratory and cardiovascular systems, the digestive

Lawrence I. Mortin; Christopher J. Horvath; Michael S. Wyand



Comparison of alamar blue and MTT assays for high through-put screening.  


The performance of alamar blue and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell viability assays in a high through-put format were compared. A total of 117 drugs chosen for their wide range of therapeutic areas were screened at 10 microM using both assays in human hepatoma cell line HepG2. Except for terfenadine and astemizole, which performed consistently in both assays, the alamar blue assay was slightly more sensitive than the MTT assay for most compounds. The MTT assay was less sensitive detecting an effect for daunorubicin and trifluoperazine. Seven drugs, astemizole, daunorubicin, ellipticine, fluphenazine, terfenadine, thioridazine and trifluoperazine, had percent viability results of 55% or less in the alamar blue assay at the single point screen. These were re-tested in both assays for reconfirmation of cytotoxicity and determination of the EC50 values. Except for daunorubicin, the EC50 values were comparable in both assays. Based on these results and the Z'-factor assessment of assay quality, both assays provided useful information to identify in vitro cytotoxic drugs at early stages of drug candidate selection. However, careful interpretation of data is warranted due to the possibility of false positive or negative results caused by inducers and/or inhibitors of metabolic enzymes that are responsible for transformation of cell toxicity end points, as we demonstrated using dicumarol. PMID:15251189

Hamid, R; Rotshteyn, Y; Rabadi, L; Parikh, R; Bullock, P



Adolescents and Drug Abuse: Clinical Use of Urine Drug Screening  

Microsoft Academic Search

Urine screening for clinical diagnostic purposes is used to answer the question of whether adolescents are continuing drug abuse. The purpose of this study is to examine the use of urine screening as a procedure to assess and monitor adolescents in an outpatient program who are suspected of continued drag abuse. To systematically evaluate the procedure, 296 adolescent urine screens

William H. James; David D. Moore



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

Microsoft Academic Search

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

Pirjo Lillsunde; Leena Michelson; Tarja Forsström; Taimi Korte; Eija Schultz; Kari Ariniemi; Maria Portman; Marja-Liisa Sihvonen; Timo Seppälä



Screening assay for the identification of deoxyhypusine synthase inhibitors.  


The 1st step in the posttranslational hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine] modification of eukaryotic translation initiation factor 5A (eIF5A) is catalyzed by deoxyhypusine synthase (DHS). The eIF5A intermediate is subsequently hydroxylated by deoxyhypusine hydroxylase (DHH), thereby converting the eIF5A precursor into a biologically active protein. Depletion of eIF5A causes inhibition of cell growth, and the identification of eIF5A as a cofactor of the HIV Rev protein turns this host protein and therefore DHS into an interesting target for drugs against abnormal cell growth and/or HIV replication. The authors developed a 96-well format DHS assay applicable for the screening of DHS inhibitors. Using this assay, they demonstrate DHS inhibition by AXD455 (Semapimod, CNI-1493). This assay represents a powerful tool for the identification of new DHS inhibitors with potency against cancer and HIV. PMID:15296643

Sommer, Marc-Nicola; Bevec, Dorian; Klebl, Bert; Flicke, Birgit; Hölscher, Kerstin; Freudenreich, Tatjana; Hauber, Ilona; Hauber, Joachim; Mett, Helmut



Lifecodes LifeScreen: A Luminex 100 Screening Assay for the ...  

Center for Biologics Evaluation and Research (CBER)

... Lifecodes LifeScreen: A Luminex 100 Screening Assay for the Detection of IgG Antibodies to HLA Class I and Class II Molecules of Human Origin. ... More results from


In vitro solubility assays in drug discovery.  


The solubility of a compound depends on its structure and solution conditions. Structure determines the lipophilicity, hydrogen bonding, molecular volume, crystal energy and ionizability, which determine solubility. Solution conditions are affected by pH, co-solvents, additives, ionic strength, time and temperature. Many drug discovery experiments are conducted under "kinetic" solubility conditions. In drug discovery, solubility has a major impact on bioassays, formulation for in vivo dosing, and intestinal absorption. A good goal for the solubility of drug discovery compounds is >60 ug/mL. Equilibrium solubility assays can be conducted in moderate throughput, by incubating excess solid with buffer and agitating for several days, prior to filtration and HPLC quantitation. Kinetic solubility assays are performed in high throughput with shorter incubation times and high throughput analyses using plate readers. The most frequently used of these are the nephelometric assay and direct UV assay, which begin by adding a small volume of DMSO stock solution of each test compound to buffer. In nephelometry, this solution is serially diluted across a microtitre plate and undissolved particles are detected via light scattering. In direct UV, undissolved particles are separated by filtration, after which the dissolved material is quantitated using UV absorption. Equilibrium solubility is useful for preformulation. Kinetic solubility is useful for rapid compound assessment, guiding optimization via structure modification, and diagnosing bioassays. It is often useful to customize solubility experiments using conditions that answer specific research questions of drug discovery teams, such as compound selection and vehicle development for pharmacology and PK studies. PMID:18991584

Kerns, Edward H; Di, Li; Carter, Guy T



In vitro assays and biomarkers for drug-induced phospholipidosis.  


Drug-induced phospholipidosis is the cytoplasmic accumulation of phospholipids as a result of xenobiotic exposure. This accumulation results in a unique histological effect in cells noted as electron-dense lamellar inclusions or whorls in the cytoplasm when observed with transmission electron microscopy. Electron microscopy has been the widely accepted standard for classification of the phospholipidosis effect. Molecules that have been prone to induce such an effect are made up of a lipophilic region and a positively charged region. Phospholipidosis has most commonly been associated with drugs that are cationic, amphiphilic drugs and can occur in a variety of tissues. Although phospholipidosis is not considered adverse in isolation, depending on the tissue affected or the occasional circumstance of concurrent toxicity, phospholipidosis can be perplexing if identified in early drug development. In most circumstances, characterisation of the effect with in vivo studies allows for determination of exposure and the magnitude of the effect. On occasion in drug development, there may be an interest to screen early stage compounds to minimise phospholipidosis. In such circumstances, in silico and in vitro assays can be employed in a strategy with in vivo assessments. In addition, there may be an interest to monitor for the potential development of phospholipidosis in longer-term animal studies. In such cases, biomarker approaches could be used. The challenge in the overall assessment of phospholipidosis remains the question of the possible relevance to any toxicity, and, therefore, any screening approach, while assessing the potential to induce phospholipidosis, must be considered in relation to prediction of findings in vivo. The status of current assays and biomarkers is presented with strategies for screening. PMID:17014389

Monteith, David K; Morgan, Ryan E; Halstead, Bartley



Establishing Assay Cutoffs for HLA Antibody Screening of Apheresis Donors  

PubMed Central

BACKGROUND TRALI is the leading cause of transfusion-related deaths. Donor HLA antibodies have been implicated in TRALI cases. Blood centers are implementing TRALI risk reduction strategies based on HLA antibody screening of some subpopulations of ever-pregnant apheresis platelet donors. However, if screening assay cutoffs are too sensitive, donation loss may adversely impact blood availability. STUDY DESIGN Pregnancy history and HLA antibody screening and single antigen bead (SAB) data from blood donors in the REDS-II Leukocyte Antibody Prevalence Study (LAPS) were evaluated for correlations between assay screening values, HLA antibody titer, and number of HLA antigen specificities. The probabilities of matching a cognate antigen in a recipient were calculated and examined in association with total number of specificities observed and screening values. The relative impact of imposing various screening assay cutoffs or pregnancy stratification was examined in relation to detection of HLA antibody reactive donations and loss of donors and donations. RESULTS We provide evidence that higher HLA Ab screening assay values are associated with maintaining higher screening signals upon dilution and an increased breadth of specificities compared with lower screening values; the latter correlated with an increased risk of a cognate antigen match in potential recipients. Depending upon the TRALI risk reduction strategy used, the potential loss of donations ranged between 0.9 and 6.0%. CONCLUSION This analysis should enable blood centers to decide upon a TRALI risk reduction strategy for apheresis platelets that is consistent with how much donation loss the blood center can tolerate.

Carrick, Danielle M.; Norris, Philip J.; Endres, Robert O.; Pandey, Suchitra; Kleinman, Steven H.; Wright, David; Sun, Yu; Busch, Michael P.



Assay System for Screening Protease Inhibitors.  

National Technical Information Service (NTIS)

Compositions and methods for identifying agents that inhibit protease activity are provided. In particular, polynucleotides, recombinant expression vectors, and host cells are provided that may be used in a bacterial cell-based assay for identifying agent...

T. J. Cheng C. C. Kan



Improved benzodiazepine radioreceptor assay using the MultiScreen® Assay System  

Microsoft Academic Search

In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time. The filtration in this method was performed by using the MultiScreen® Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom, which allows both the incubation and the filtration of the specimen in the same plate.

M. J. Janssen; K. Ensing; R. A. de Zeeuw



[Comparison of four drug interaction screening programs].  


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

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



Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds  

PubMed Central

Drugs currently available for leishmaniasis treatment often show parasite resistance, highly toxic side effects and prohibitive costs commonly incompatible with patients from the tropical endemic countries. In this sense, there is an urgent need for new drugs as a treatment solution for this neglected disease. Here we show the development and implementation of an automated high-throughput viability screening assay for the discovery of new drugs against Leishmania. Assay validation was done with Leishmania promastigote forms, including the screening of 4,000 compounds with known pharmacological properties. In an attempt to find new compounds with leishmanicidal properties, 26,500 structurally diverse chemical compounds were screened. A cut-off of 70% growth inhibition in the primary screening led to the identification of 567 active compounds. Cellular toxicity and selectivity were responsible for the exclusion of 78% of the pre-selected compounds. The activity of the remaining 124 compounds was confirmed against the intramacrophagic amastigote form of the parasite. In vitro microsomal stability and cytochrome P450 (CYP) inhibition of the two most active compounds from this screening effort were assessed to obtain preliminary information on their metabolism in the host. The HTS approach employed here resulted in the discovery of two new antileishmanial compounds, bringing promising candidates to the leishmaniasis drug discovery pipeline.

Sohn, Jeong-Hun; Yang, Gyongseon; Nam, Jiyoun; Jang, Jiyeon; Cechetto, Jonathan; Lee, Chang Bok; Moon, Seunghyun; Genovesio, Auguste; Chatelain, Eric; Christophe, Thierry; Freitas-Junior, Lucio H.




PubMed Central

Pooling in high-throughput screening (HTS) refers to the act of testing mixtures of compounds in a primary screen to accurately identify hits for secondary screening. The gain in compression of the number of compounds to be further screened by pooling can also be extended to achieve much-needed error tolerance in HTS. Despite the success of pooling in other biological experiments, pooling in high-throughput drug screening has been a controversial and often marginalized paradigm. At first appearance, pooling appears to promise gains from reduced effort, or possibly could create more problems than solutions. However, this article demonstrates that pooling is a practical and necessary part of HTS: discussions include the rationale for pooling compounds in HTS, a unifying view of pooling design theory, a review of past attempts at pooling and their success, and recent advances in the field.




Immunoassays for drug screening in urine  

Microsoft Academic Search

Immunoassays are presently used worldwide for the rapid screening of drugs. Despite the fact that they are a highly valuable\\u000a tool for the testing of legal and illicit drugs, there is a real risk of false-positive and false-negative findings and many\\u000a pitfalls must be taken into account when these tests are used in an uncritical manner and without valid confirmation

Harald Schütz; Alexandre Paine; Freidoon Erdmann; Günter Weiler; Marcel A. Verhoff



Rapid screening assay for calcium bioavailability studies  

SciTech Connect

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.

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



[Application of zebrafish models in drug screening].  


Due to its small size, fast external development, transparent embryos, and amenability to genetic analysis, zebrafish has become an ideal vertebrate animal model. In addition to studies in genetics and developmental biology, zebrafish has also been widely used in human disease modeling and drug screening. As a small whole-organism model, zebrafish can be used to comprehensively test and evaluate the activity and side effect of a compound at the same time, fulfilling high content screening. Recently, new zebrafish disease models and screening technologies have been developed. A number of active compounds were identified and most of them have similar functions in mammal models. One compound prostaglandin E2 has been subjected to clinical trial to test if it can promote the growth of umbilical cord blood units after transplantation. Another compound leflunomide has also been approved in clinical trial to cure melanoma in combination with vemurafenib. These findings demonstrate that zebrafish model is appropriate for drug screening. This review summarizes the unique features of zebrafish model and the recent progresses of zebrafish based drug screening. PMID:23017455

Xin, Sheng-Chang; Zhao, Yan-Qiu; Li, Song; Lin, Shuo; Zhong, Han-Bing



Screening of antipsychotic drugs in animal models  

Microsoft Academic Search

Behavioral models of antipsychotic drug (APD) action in the rat are widely used for the screening and developing APDs. Valid models are not only required to be selective and specific for APDs, but also to be able to dissociate between typical and atypical APDs. In recent years, newer models have been developed that are claimed to model processes impaired in

Ina Weiner; Inna Gaisler; Daniela Schiller; Amit Green; Lee Zuckerman; Daphna Joel



Continuous colorimetric assay for acetylcholinesterase and inhibitor screening with gold nanoparticles.  


We report herein a new colorimetric assay method for acetylcholinesterase (AChE) activity and its inhibitor screening by making use of the following facts: (1) the aggregation of gold nanoparticles (Au-NPs) results in the red-shift of the plasmon absorption due to interparticle plasmon interactions and (2) AChE can catalyze the hydrolysis of acetylthiocholine into thiocholine which can induce the aggregation of Au-NPs. With this convenient method, the activity of AChE with a concentration as low as 0.6 mU/mL can be assayed. Moreover, this assay method is also useful for screening inhibitors of AChE. Given its simplicity and easy-operation, this method may extend to high-throughput screening of AChE inhibitors and relevant drug discovery. PMID:19154124

Wang, Ming; Gu, Xinggui; Zhang, Guanxin; Zhang, Deqing; Zhu, Daoben



Membrane vesicle ABC transporter assays for drug safety assessment.  


The use of plasma membrane vesicles that overexpress the bile salt export pump (BSEP) or multidrug resistance-associated protein 2, 3, or 4 (MRP2-4) with an in vitro vacuum filtration system offers a rapid and reliable means for screening drug candidates for their effects on transporter function in hepatocytes and thus their potential for causing drug-induced liver injury (DILI). Comparison of transporter activity in the presence and absence of ATP allows for determination of a specific assay window for each transporter. This window is used to determine the degree to which each test compound inhibits transporter activity. This assay battery is helpful for prioritizing and rank-ordering compounds within a chemical series with respect to each other and in the context of known inhibitors of transporter activity and/or liver injury. This model can be used to influence the drug development process at an early stage and provide rapid feedback regarding the selection of compounds for advancement to in vivo safety evaluations. A detailed protocol for the high-throughput assessment of ABC transporter function is provided, including specific recommendations for curve-fitting to help ensure consistent results. PMID:23169270

van Staden, Carlo J; Morgan, Ryan E; Ramachandran, Bharath; Chen, Yuan; Lee, Paul H; Hamadeh, Hisham K



Diced electrophoresis gel assay for screening enzymes with specified activities.  


We have established the diced electrophoresis gel (DEG) assay as a proteome-wide screening tool to identify enzymes with activities of interest using turnover-based fluorescent substrates. The method utilizes the combination of native polyacrylamide gel electrophoresis (PAGE) with a multiwell-plate-based fluorometric assay to find protein spots with the specified activity. By developing fluorescent substrates that mimic the structure of neutrophil chemoattractants, we could identify enzymes involved in metabolic inactivation of the chemoattractants. PMID:23581642

Komatsu, Toru; Hanaoka, Kenjiro; Adibekian, Alexander; Yoshioka, Kentaro; Terai, Takuya; Ueno, Tasuku; Kawaguchi, Mitsuyasu; Cravatt, Benjamin F; Nagano, Tetsuo



Detecting drug promiscuity using Gaussian ensemble screening.  


Polypharmacology describes the binding of a ligand to multiple protein targets (a promiscuous ligand) or multiple diverse ligands binding to a given target (a promiscuous target). Pharmaceutical companies are discovering increasing numbers of both promiscuous drugs and drug targets. Hence, polypharmacology is now recognized as an important aspect of drug design. Here, we describe a new and fast way to predict polypharmacological relationships between drug classes quantitatively, which we call Gaussian Ensemble Screening (GES). This approach represents a cluster of molecules with similar spherical harmonic surface shapes as a Gaussian distribution with respect to a selected center molecule. Calculating the Gaussian overlap between pairs of such clusters allows the similarity between drug classes to be calculated analytically without requiring thousands of bootstrap comparisons, as in current promiscuity prediction approaches. We find that such cluster similarity scores also follow a Gaussian distribution. Hence, a cluster similarity score may be transformed into a probability value, or "p-value", in order to quantify the relationships between drug classes. We present results obtained when using the GES approach to predict relationships between drug classes in a subset of the MDL Drug Data Report (MDDR) database. Our results indicate that GES is a useful way to study polypharmacology relationships, and it could provide a novel way to propose new targets for drug repositioning. PMID:22747187

Pérez-Nueno, Violeta I; Venkatraman, Vishwesh; Mavridis, Lazaros; Ritchie, David W



Respirometric acute toxicity screening assay using Daphnia magna  

Microsoft Academic Search

Oxygen consumption rate is a universal and sensitive biomarker of viability and metabolic status of aerobic organisms. We applied the optical oxygen sensing method to monitor the respiration of individual Daphnia magna (D. magna) and to develop a simple, automated screening assay for the assessment of acute toxicity of large numbers of chemical and environmental samples. D. magna were exposed

Alice Zitova; Maud Cross; Robert Hernan; John Davenport; Dmitri B. Papkovsky



Multiplexing Fluorescence Polarization Assays to Increase Information Content Per Screen: Applications for Screening Steroid Hormone Receptors  

Microsoft Academic Search

As the push to reduce cost per well in high-throughput screening reaches the practical limitations of liquid handling, future cost savings will likely arise from an increase in information content per well. One strategy to increase information content is to perform discreet assays against multiple targets in a single well. In such assays, reagent usage and liquid handling steps do

Paul Blommel; George T. Hanson; Kurt W. Vogel



Fluorescence resonance energy transfer assay for high-throughput screening of ADAMTS1 inhibitors.  


A disintegrin and metalloprotease with thrombospondin type I motifs-1 (ADAMTS1) plays a crucial role in inflammatory joint diseases and its inhibitors are potential candidates for anti-arthritis drugs. For the purposes of drug discovery, we reported the development and validation of fluorescence resonance energy transfer (FRET) assay for high-throughput screening (HTS) of the ADAMTS1 inhibitors. A FRET substrate was designed for a quantitative assay of ADAMTS1 activity and enzyme kinetics studies. The assay was developed into a 50-µL, 384-well assay format for high throughput screening of ADAMTS1 inhibitors with an overall Z' factor of 0.89. ADAMTS1 inhibitors were screened against a diverse library of 40,960 total compounds with the established HTS system. Four structurally related hits, naturally occurring compounds, kuwanon P, kuwanon X, albafuran C and mulberrofuran J, extracted from the Chinese herb Morus alba L., were identified for further investigation. The results suggest that this FRET assay is an excellent tool, not only for measurement of ADAMTS1 activity but also for discovery of novel ADAMTS1 inhibitors with HTS. PMID:22186957

Peng, Jianhao; Gong, Lili; Si, Kun; Bai, Xiaoyu; Du, Guanhua



Development and application of a screening assay for glycoside phosphorylases.  


Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They require only a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme's substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen a residue E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity toward alkyl beta-glucosides and phenyl beta-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity. PMID:20188057

De Groeve, M R M; Tran, G H; Van Hoorebeke, A; Stout, J; Desmet, T; Savvides, S N; Soetaert, W




Microsoft Academic Search

Two overpressured layer chromatography (OPLC) systems were developed for the screening of toxicologically relevant basic drugs in forensic and clinical contexts. The OPLC1 system was trichloroethylene - methylethylketone - n-butanol - acetic acid - water 17+8+25+6+4 and the OPLC2 system was butyl acetate - ethanol - tripropylamine - water 85+9.25+5+0.75 with presaturation. Both systems were tested on high performance silica

I. Ojanperä; K. Goebel; E. Vuori



Structural ensemble in computational drug screening.  


Importance of the field: Structure-based in silico drug screening is now widely used in drug development projects. Structure-based in silico drug screening is generally performed using a protein-compound docking program and docking scoring function. Many docking programs have been developed over the last 2 decades, but their prediction accuracy remains insufficient. Areas covered in this review: This review highlights the recent progress of the post-processing of protein-compound complexes after docking. What the reader will gain: These methods utilize ensembles of docking poses of compounds to improve the prediction accuracy for the ligand-docking pose and screening results. While the individual docking poses are not reliable, the free energy surface or the most probable docking pose can be estimated from the ensemble of docking poses. Take home message: The protein-compound docking program provides an arbitral rather than a canonical ensemble of docking poses. When the ensemble of docking poses satisfies the canonical ensemble, we can discuss how these post-docking analysis methods work and fail. Thus, improvements to the docking software will be needed in order to generate well-defined ensembles of docking poses. PMID:20465522

Fukunishi, Yoshifumi



An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.  


The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell(maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation. PMID:21553282

Casalino, Laura; Magnani, Dario; De Falco, Sandro; Filosa, Stefania; Minchiotti, Gabriella; Patriarca, Eduardo J; De Cesare, Dario



Spheroid-based drug screen: considerations and practical approach.  


Although used in academic research for several decades, 3D culture models have long been regarded expensive, cumbersome and unnecessary in drug development processes. Technical advances, coupled with recent observations showing that gene expression in 3D is much closer to clinical expression profiles than those seen in 2D, have renewed attention and generated hope in the feasibility of maturing organotypic 3D systems to therapy test platforms with greater power to predict clinical efficacies. Here we describe a standardized setup for reproducible, easy-handling culture, treatment and routine analysis of multicellular spheroids, the classical 3D culture system resembling many aspects of the pathophysiological situation in human tumor tissue. We discuss essential conceptual and practical considerations for an adequate establishment and use of spheroid-based drug screening platforms and also provide a list of human carcinoma cell lines, partly on the basis of the NCI-DTP 60-cell line screen, that produce treatable spheroids under identical culture conditions. In contrast to many other settings with which to achieve similar results, the protocol is particularly useful to be integrated into standardized large-scale drug test routines as it requires a minimum number of defined spheroids and a limited amount of drug. The estimated time to run the complete screening protocol described herein--including spheroid initiation, drug treatment and determination of the analytical end points (spheroid integrity, and cell survival through the acid phosphatase assay)--is about 170 h. Monitoring of spheroid growth kinetics to determine growth delay and regrowth, respectively, after drug treatment requires long-term culturing (> or =14 d). PMID:19214182

Friedrich, Juergen; Seidel, Claudia; Ebner, Reinhard; Kunz-Schughart, Leoni A



A Real-Time Screening Assay for GIRK1\\/4 Channel Blockers  

Microsoft Academic Search

The cardiac acetylcholine-activated K+ channel (IK,Ach) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K+ (GIRK) channel superfamily and is composed of the GIRK1\\/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers

Kenneth B. Walsh



Assessment of a combination screening assay for celiac disease  

Microsoft Academic Search

Purpose  A serological screening assay for celiac disease (CD), designed to simultaneously detect IgA and IgG anti-tissue transglutaminase\\u000a (a-tTG) and IgA and IgG deamidated gliadin peptide antibodies (a-DGP), was recently developed. In this study, we establish\\u000a the performance of this assay.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  We enrolled 41 CD patients and 18 CD patients on gluten-free diets. The diagnosis of CD was based on histological

Brunetta Porcelli; Fabio Ferretti; Carla Vindigni; Carlo Scapellaato; Lucia Terzuoli


Mass screening for coeliac disease using antihuman transglutaminase antibody assay  

Microsoft Academic Search

Aims: To determine coeliac disease prevalence by an anti-transglutaminase antibody assay in a large paediatric population; to evaluate acceptance of the screening programme, dietary compliance, and long term health effects.Methods: Cross-sectional survey of 3188 schoolchildren (aged 6–12) and prospective follow up of diagnosed cases. Main outcome measures were: prevalence of coeliac disease defined by intestinal biopsy or positivity to both

A Tommasini; V Kiren; V Baldas; D Santon; C Trevisiol; I Berti; E Neri; T Gerarduzzi; I Bruno; A Lenhardt; E Zamuner; A Spano?; S Crovella; S Martellossi; G Torre; D Sblattero; R Marzari; A Bradbury; G Tamburlini; A Ventura



Emergency physicians perceptions of drug screens at their own hospitals.  


Previous studies evaluated the prudent use and potential over use of drug screens in clinical decision making. However, the percentage of emergency physicians who can correctly identify which drugs are found on their hospital's basic drug screens has not been established. Results of physician closed-ended questionnaires were compared to the results of a telephone survey with each physician's individual hospital laboratory. Eighty-one (35.7%) of 227 emergency physicians responded. Four (4.9%) correctly identified what was on their individual institution's urine drug screens and 17 (21%) correctly identified what was on serum screens. In other results, 74.3% erroneously relayed that all benzodiazepines can be found on urine drug screens and 46.3% incorrectly answered that acetaminophen would be found on basic quantitative serum screens. Drug screen results can be misinterpreted if the drugs the physician expects to be screened for, and what is actually screened for, are not the same. Pharmacy and laboratory personnel have a responsibility to keep the physician informed of drug screen issues. They should be proactive in advising physicians of changes in drug testing and new drug screening methods or by providing reports on the occurrence of false positive results. PMID:9682413

Durback, L F; Scharman, E J; Brown, B S



Novel screening assay for the selective detection of G-protein-coupled receptor heteromer signaling.  


Drugs targeting G-protein-coupled receptors (GPCRs) make up more than 25% of all prescribed medicines. The ability of GPCRs to form heteromers with unique signaling properties suggests an entirely new and unexplored pool of drug targets. However, current in vitro assays are ill equipped to detect heteromer-selective compounds. We have successfully adapted an approach, using fusion proteins of GPCRs and chimeric G proteins, to create an in vitro screening assay (in human embryonic kidney cells) in which only activated heteromers are detectable. Here we show that this assay can demonstrate heteromer-selective G-protein bias as well as measure transinhibition. Using this assay, we reveal that the ?-opioid receptor agonist ADL5859, which is currently in clinical trials, has a 10-fold higher potency against ?-opioid receptor homomers than ?/?-opioid receptor heteromers (pEC(50) = 6.7 ± 0.1 versus 5.8 ± 0.2). The assay enables the screening of large compound libraries to identify heteromer-selective compounds that could then be used in vivo to determine the functional role of heteromers and develop potential therapeutic agents. PMID:23097213

van Rijn, Richard M; Harvey, Jessica H; Brissett, Daniela I; DeFriel, Julia N; Whistler, Jennifer L



Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy  

PubMed Central

Abstract 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.

Kuhn, Jonas; Shaffer, Etienne; Mena, Julien; Breton, Billy; Parent, Jerome; Rappaz, Benjamin; Chambon, Marc; Emery, Yves; Magistretti, Pierre; Depeursinge, Christian; Marquet, Pierre



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


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

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



Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis.  


The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95% and 94%, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries. PMID:22310549

Fonseca, Leila de Souza; Vieira, Gisele Betzler de Oliveira; Sobral, Luciana Fonseca; Ribeiro, Elizabete Oliveira; Marsico, Anna Grazia



Urine Drug Screening of Adolescents on Request of Parents  

Microsoft Academic Search

The availability of urine screening has resulted in parents seeking this procedure when they suspect their adolescent abuses drugs. To systematically evaluate this practice, 100 consecutive adolescents were screened by use of a sensitive quantiative method which can detect low levels of drug use. A total of 43% of the adolescents tested positive for one or more drugs of abuse,




A high-throughput dual parameter assay for assessing drug-induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays.  


Mitochondrial toxicity is a major reason for safety-related compound attrition and post-market drug withdrawals, highlighting the necessity for higher-throughput screens that can identify this mechanism of toxicity during the early stages of drug discovery. Here, we present the validation of a 384-well dual parameter plate-based assay capable of measuring oxygen consumption and extracellular acidification in intact cells simultaneously. The assay showed good reproducibility and robustness and is suitable for use with both suspension cells and adherent cells. To determine if the assay provides additional value in detecting mitochondrial toxicity over existing platforms, 200 commercially available drugs were tested in the assay using HL60 suspension cells as well as in two conventional mitochondrial toxicity assays: an oxygen consumption assay that uses isolated mitochondria and a cell-based assay that uses HepG2 cells grown in glucose and galactose media. The combination of the dual parameter assay and the isolated mitochondrial oxygen consumption assay identified more compounds that caused mitochondrial impairment than any other combination of the three assays or each of the three assays on its own. Furthermore, novel information was obtained from the dual parameter assay on drugs not previously reported to cause mitochondrial impairment. PMID:23147640

Hynes, James; Nadanaciva, Sashi; Swiss, Rachel; Carey, Conn; Kirwan, Sinead; Will, Yvonne



Sensitivity and specificity of Anti-HBc screening assays--which assay is best for blood donor screening?  


Compared to HIV and hepatitis C virus, the residual infectious risk of hepatitis B virus (HBV) posed by blood products is about 10 times higher. In addition to HBsAg testing, screening for anti-HBc was recommended by the German Advisory Committee Blood in March 2005. Prevalence of anti-HBc in German blood donors was investigated at five test sites located in different geographic regions. In total, 12,000 blood donors were screened for anti-HBc by PRISM HBcore, and a statistically representative number of these were tested with Abbott Murex anti-HBc total, bioMérieux Hepanostika anti-HBc uniform, Bio-Rad Monolisa anti-HBc PLUS and Dade Behring Enzygnost anti-HBc. Anti-HBc repeat reactive samples were tested for anti-HBs, anti-HBe and HBV DNA by individual donation NAT. The mean prevalence of anti-HBc was 1.75% in donors that had not been tested for anti-HBc in the past. The percentage of anti-HBs in anti-HBc repeat reactive donors was 93.7%. Samples that were additionally reactive for anti-HBe were anti-HBc reactive in all tested assays. The sample to cut-off (S/Co) values for anti-HBc were lower (competitive assays) in samples that were also positive for anti-HBe, when compared to samples that were only anti-HBc reactive. Most commercially available anti-HBc assays provide sufficient sensitivity for routine screening purposes, and lacking specificity is no longer a serious issue for most of them. Assay differences were recognized for samples that were anti-HBc only reactive. The overall loss of 1.75% of positive testing donors can be significantly reduced to 0.45% by implementation of re-entry procedures for donors with an anti-HBs titre of over 100 IU/l and negative by sensitive ID-NAT. PMID:18673399

Hourfar, M K; Walch, L A; Geusendam, G; Dengler, T; Janetzko, K; Gubbe, K; Frank, K; Karl, A; Löhr, M; Sireis, W; Seifried, E; Schmidt, M



Molecularly imprinted polymers as antibody and receptor mimics for assays, sensors and drug discovery.  


Biological receptors play an important role in affinity-based drug assays, biosensors, and at different stages during the modern drug discovery process. The molecular imprinting technology that has recently emerged has shown great potential for producing biomimetic receptors that challenge their natural counterparts. In this paper, we will overview recent progress in the use of molecularly imprinted polymers for drug assays, assembly of biomimetic sensors, and screening of combinatorial libraries. In addition, examples of using artificially-created binding sites to control synthetic reactions will be discussed. The "screening-of-building blocks" approach is expected to accelerate generation of valuable lead compounds, without the costly synthesis of large chemical libraries. PMID:15064898

Ye, Lei; Haupt, Karsten



Evaluation of frequently used drug interaction screening programs  

Microsoft Academic Search

Objective Drug-drug interaction (DDI) screening programs are an important tool to check prescriptions of multiple drugs. The objective\\u000a of the current study was to critically appraise several DDI screening programs. Methods A DDI screening program had to fulfil minimal requirements (information on effect, severity rating, clinical management,\\u000a mechanism and literature) to be included into the final evaluation. The 100 most

Priska Vonbach; André Dubied; Stephan Krähenbühl; Jürg H. Beer



Automated microfluidic screening assay platform based on DropLab.  


This paper describes DropLab, an automated microfluidic platform for programming droplet-based reactions and screening in the nanoliter range. DropLab can meter liquids with picoliter-scale precision, mix multiple components sequentially to assemble composite droplets, and perform screening reactions and assays in linear or two-dimensional droplet array with extremely low sample and reagent consumptions. A novel droplet generation approach based on the droplet assembling strategy was developed to produce multicomponent droplets in the nanoliter to picoliter range with high controllability on the size and composition of each droplet. The DropLab system was built using a short capillary with a tapered tip, a syringe pump with picoliter precision, and an automated liquid presenting system. The tapered capillary was used for precise liquid metering and mixing, droplet assembling, and droplet array storage. Two different liquid presenting systems were developed based on the slotted-vial array design and multiwell plate design to automatically present various samples, reagents, and oil to the capillary. Using the tapered-tip capillary and the picoliter-scale precision syringe pump, the minimum unit of the droplet volume in the present system reached ~20 pL. Without the need of complex microchannel networks, various droplets with different size (20 pL-25 nL), composition, and sequence were automatically assembled, aiming to multiple screening targets by simply adjusting the types, volumes, and mixing ratios of aspirated liquids on demand. The utility of DropLab was demonstrated in enzyme inhibition assays, protein crystallization screening, and identification of trace reducible carbohydrates. PMID:21043448

Du, Wen-Bin; Sun, Meng; Gu, Shu-Qing; Zhu, Ying; Fang, Qun



Clinical evaluation and use of urine screening for drug abuse.  

PubMed Central

Urine drug screening is indicated to evaluate patients who show mental status or behavioral changes and to monitor the abstinence of drug abusers. The appropriate timing for collecting urine specimens may vary depending on the suspected drug of abuse and on laboratory factors. Laboratories use a variety of techniques to do urine screens, and these must be understood by clinicians ordering the screens to interpret results correctly. In treating drug-abusing patients, clinicians must apply structured reinforcement in conjunction with urine screen results to aid patients in achieving abstinence.

Saxon, A J; Calsyn, D A; Haver, V M; Delaney, C J



Altering drug tolerance of surface plasmon resonance assays for the detection of anti-drug antibodies.  


Anti-drug antibody (ADA) responses are a concern for both drug efficacy and safety, and high drug concentrations in patient samples may inhibit ADA assays. We evaluated strategies to improve drug tolerance of surface plasmon resonance (SPR) assays that detect ADAs against a bispecific Adnectin drug molecule that consists of an anti-VEGFR2 domain linked to an anti-IGF-1R domain (V-I-Adnectin). Samples containing ADAs against V-I-Adnectin and various drug concentrations were tested in the presence of 1M guanidine hydrochloride (Gdn), at pH values ranging from 4.5 to 7.4 and temperatures of up to 37°C. Temperature had a negligible effect in weakening the affinity of interaction of monoclonal antibodies with polyethylene glycol(PEG)-V-I-Adnectin and did not increase drug tolerance of the ADA assay. Low pH increased drug tolerance of the assay relative to pH 7.4 but caused nonspecific binding of the drug during competition experiments. The chaotropic agent Gdn lowered the affinity of interaction between an anti-V-Adnectin monoclonal antibody and the drug (from KD=0.93nM to KD=348nM). That decrease in the affinity of drug-ADA interaction correlated with an increase of assay drug tolerance. Conditions that lower drug-ADA interaction affinity could also be used to develop drug-tolerant SPR assays for other systems. PMID:23886888

Barbosa, Maria D F S; Gokemeijer, Jochem; Martin, Aaron D; Bush, Alex



Improved benzodiazepine radioreceptor assay using the MultiScreen Assay System.  


In this article, an improved benzodiazepine radioreceptor assay is described, which allows substantial reduction in assay time. The filtration in this method was performed by using the MultiScreen Assay System. The latter consists of a 96-well plate with glass fibre filters sealed at the bottom, which allows both the incubation and the filtration of the specimen in the same plate. After the filtration, the filters were punched out for quantitation of the bound labeled ligand [3H]flunitrazepam. The results obtained with the MultiScreen Assay System did not differ significantly from the data obtained with the conventional filtration manifold (48S): The Ki's of lorazepam were 2.4 +/- 0.30 and 1.9 +/- 0.15 nM, respectively. In case a radioactive label is replaced by a fluorescent label, the bound labeled-ligand usually cannot be determined in the presence of the receptor material. Here, the bound labeled-ligand has to be dissociated after the filtration step. To dissociate the ligand-receptor complex, Tris- HCl buffer, containing 10 microM flumazenil, was added to the filters and the second filtrates were collected containing the previously bound fractions in the absence of receptor material. This approach showed the same Ki for lorazepam, 2.5 +/- 0.04 nM as without dissociation, when using the radio-labeled benzodiazepine [3H]flunitrazepam. PMID:10701983

Janssen, M J; Ensing, K; de Zeeuw, R A



Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands.  


Surface plasmon resonance (SPR) biosensors recently gained an important place in drug discovery. Here we present a primary and secondary SPR biosensor screening methodology. The primary screening method is based on a direct binding assay with covalent immobilized drug target proteins. For the secondary screening method, a sequential competition assay has been developed where the captured protein is first exposed to an unknown test compound, followed directly by an exposure to a high-molecular-weight reporter ligand. Using the high-molecular-weight reporter ligand to probe the remaining free binding site on the sensor, a significant signal enhancement is obtained. Furthermore, this assay format allows the validation of the primary direct binding assay format, efficiently revealing false positive data. As a model system, acetylcholine binding protein (AChBP), which is a soluble model protein for neuronal nicotinic acetylcholine receptors, has been used. The secondary assay is lower in throughput than the primary assay; however, the signal-to-noise ratio is two times higher compared with the direct assay, and it has a z' factor of 0.96. Using both assays, we identified the compound tacrine as a ligand for AChBP. PMID:20599657

Retra, Kim; Geitmann, Matthis; Kool, Jeroen; Smit, August B; de Esch, Iwan J P; Danielson, U Helena; Irth, Hubertus



BioAssay Ontology Annotations Facilitate Cross-Analysis of Diverse High-Throughput Screening Data Sets  

Microsoft Academic Search

High-throughput screening data repositories, such as PubChem, represent valuable resources for the development of small-molecule chemical probes and can serve as entry points for drug discovery programs. Although the loose data format offered by PubChem allows for great flexibility, important annotations, such as the assay format and technologies employed, are not explicitly indexed. The authors have previously developed a BioAssay

Stephan C. Schürer; Uma Vempati; Robin Smith; Vance Lemmon



Feeder layer-free in vitro assay for screening antitrypanosomal compounds against Trypanosoma brucei brucei and T. b. evansi.  

PubMed Central

A drug-susceptible Trypanosoma brucei brucei stock, a multidrug-resistant T. b. brucei stock, and a T. b. evansi stock resistant to two commercial trypanocides were adapted to a feeder layer-free culture system. Bloodstream forms were grown continuously in a liquid medium at 37 degrees C in 4% CO2 in air. Samples of trypanosome populations in the logarithmic growth phase were incubated with various concentrations of commercial and experimental compounds. Growth inhibition was monitored after a 24-h incubation and quantified by comparing the number of generations between control and drug-treated cultures. Some of the experimental compounds [taxol, formicin B, thioridazine, Ro 15-0216, and DL-alpha-(difluoromethyl)ornithine hydrochloride monohydrate] showed activity against both drug-susceptible and drug-resistant trypanosomes. Other compounds [sinefungin, 1,3,5-triacetylbenzene tris(guanylhydrazone)trimethanesulfonate hydrate, and 9-deazainosine] which inhibited the growth of drug-susceptible trypanosomes showed little or no effect upon drug-resistant parasites. Gossypol, however, had no antitrypanosomal effect on either trypanosome stock. The results obtained in this study correlate with observations obtained from drug screening in mice. The main advantages of the described in vitro screening assay are as follows: (i) lower amounts of drugs are required, (ii) results are obtained more rapidly, (iii) animals are not necessary, and (iv) the method is less labor intensive. These advantages result in an economical and rapid assay for primary drug screening.

Kaminsky, R; Zweygarth, E



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  

Microsoft Academic Search

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

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



An evaluation of protein assays for quantitative determination of drugs  

Microsoft Academic Search

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red–Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble

Katherine M. Williams; Sarah J. Arthur; Gillian Burrell; Fionnuala Kelly; Darren W. Phillips; Thomas Marshall



Multiparametric immunotoxicity screening in mice during early drug development.  


Evaluation of potential adverse effects on the immune system should be incorporated into drug development prior to phase III clinical trials. In addition to standard toxicity results, T-dependent antibody response (TDAR) assays are widely used to evidence impaired immune function. The present study was aimed at validating a multiparametric screening approach in mice to investigate exaggerated pharmacologic or unintended immunosuppressive effects in early drug development. Male CD1 mice injected with a single IV dose of 2mg KLH displayed a robust anti-KLH IgM response that peaked on day +5. Anti-KLH IgM response, standard haematology parameters, and thymus/spleen weight and histology were examined in mice treated once daily for 4 days with cyclophosphamide (CY; 5-20mg/kg/day), cyclosporine (CS; 10-90mg/kg/day), dexamethasone (DX; 5-20mg/kg/day), prednisolone (PR; 3-30mg/kg/day) or chlorpromazine (CZ; 10-30mg/kg/day). CY and CS decreased anti-KLH IgM response at all dose levels. CY induced a marked decrease in WBC count and thymus/spleen weight with histological changes in both lymphoid organs. CS mainly decreased thymus weight (highest dose), which was associated with lymphoid depletion, without relevant effects on haematology parameters. Neither DX nor PR nor CZ induced significant changes in anti-KLH IgM response. DX and PR decreased lymphocyte counts and thymus/spleen weight, and induced histological changes in both lymphoid organs. CZ (higher doses) decreased lymphocyte count and thymus weight, and induced consistent histological changes in the thymus. This multiparametric study was able to detect 5 human drugs with variable immunosuppressive potency and thus may prove to be a useful early screening tool for predicting drug immunotoxicity. PMID:22944472

Aulí, M; Domènech, A; Andrés, A; Orta, M; Salvà, M; Descotes, J; Prats, N



Cell-based assays: fuelling drug discovery  

Microsoft Academic Search

It has been estimated that over a billion dollars in resources can be consumed to obtain clinical approval, and only a few\\u000a new chemical entities are approved by the US Food and Drug Administration (FDA) each year. Therefore it is of utmost importance\\u000a to obtain the maximum amount of information about biological activity, toxicological profile, biochemical mechanisms, and\\u000a off-target interactions

Elisa Michelini; Luca Cevenini; Laura Mezzanotte; Andrea Coppa; Aldo Roda



Drug Resistance Assays for Mycobacterium tuberculosis  

Microsoft Academic Search

The introduction of antimicrobial therapy of tuberculosis during the second half of the last century was a turning point in\\u000a the millennium-old history of this disease. However, the problem of drug resistance emerged, and with it, two levels of concern.\\u000a First, such resistance not only poses a public health threat to successful control of TB epidemics, but it also complicates

Leonid Heifets; Gerard Cangelosi


Screening of Drugs for Thermogenic Anti-Obesity Properties: Antidepressants  

Microsoft Academic Search

Twelve antidepressant drugs, currently in clinical use, were screened for thermogenic properties on the basis of their ability to stimulate the activity of the sympathetic nervous system via an inhibitory effect on noradrenaline reuptake into the sympathetic neurons. Drug screening was carried out on mice made obese by hypothalamic lesioning using monosodium glutamate. Preliminary experiments, based on changes in body

A. G. Dulloo; D. S. Miller



Evaluation of in vitro drug screening leads using experimental models of human ovarian cancer  

Microsoft Academic Search

Five compounds which were identified as potential new anticancer drugs inin vitro screening with the human tumor colony forming assay were selected for further evaluation usingin vitro andin vivo models of human ovarian cancer. Three of five compounds were found to inhibitin vitro colony formation of ovarian cancer cell lines derived from both untreated and combination chemotherapy refractory patients. One

Karen G. Louie; Thomas C. Hamilton; Robert H. Shoemaker; Robert C. Young; Robert F. Ozols



Predicting treatment-outcome in cocaine dependence from admission urine drug screen and peripheral serotonergic measures  

Microsoft Academic Search

We investigated whether urine drug screens (UDS) at admission and platelet paroxetine binding, a measure of serotonin transporter sites, were related to outcome measures for cocaine patients in treatment. Tritiated paroxetine binding sites on platelets were assayed and UDS were obtained for 105 African American cocaine-dependent outpatients. Outcome measures included number of negative urines, days in treatment, dropouts, and number

Ashwin A Patkar; Charles C Thornton; Wade H Berrettini; Edward Gottheil; Stephen P Weinstein; Kevin P Hill



Assays for membrane tyrosine kinase receptors: methods for high-throughput screening and utility for diagnostics.  


The development of novel antagonists or agonists of membrane tyrosine kinase receptors is a large focus of discovery research. This review will provide some background on membrane tyrosine kinases as well as a description of some of the better established assays used for the high-throughput screening of membrane tyrosine kinase inhibitors. Biochemical methods detailed include those using labels such as radioactivity and fluorescence (fluorescence energy transfer, fluorescence and fluorescence polarization) as well as label-free assays using luminescence. These assays are solid phase, liquid phase, as well as bead based. In addition, a discussion on which tools are available to screen for membrane tyrosine kinase receptor modulators/activators using whole-cell assays will be presented. The potential clinical need for testing receptor activation/phosphorylation as well as the possibility of using some of these tests to measure biomarkers of disease or as clinical diagnostic tools to tailor drug therapy or monitor its efficacy will also be discussed. PMID:16013974

Minor, Lisa K



A phenotypic screening assay for modulators of huntingtin-induced transcriptional dysregulation.  


Huntington's Disease is a rare neurodegenerative disease caused by an abnormal expansion of CAG repeats encoding polyglutamine in the first exon of the huntingtin gene. N-terminal fragments containing polyglutamine (polyQ) sequences aggregate and can bind to cellular proteins, resulting in several pathophysiological consequences for affected neurons such as changes in gene transcription. One transcriptional pathway that has been implicated in HD pathogenesis is the CREB binding protein (CBP)/cAMP responsive element binding (CREB) pathway. We developed a phenotypic assay to screen for compounds that can reverse the transcriptional dysregulation of the pathway caused by induced mutated huntingtin protein (µHtt). 293/T-REx cells were stably co-transfected with an inducible full-length mutated huntingtin gene containing 138 glutamine repeats and with a reporter gene under control of the cAMP responsive element (CRE). One clone, which showed reversible inhibition of µHtt-induced reporter activity upon treatment with the neuroprotective Rho kinase inhibitor Y27632, was used for the development of a high-throughput phenotypic assay suitable for a primary screening campaign, which was performed on a library of 24,000 compounds. Several hit compounds were identified and validated further in a cell viability adenosine triphosphate assay. The assay has the potential for finding new drug candidates for the treatment of HD. PMID:23562876

Lazzeroni, Giulia; Benicchi, Tiziana; Heitz, Freddy; Magnoni, Letizia; Diamanti, Daniela; Rossini, Lara; Massai, Luisa; Federico, Cesare; Fecke, Wolfgang; Caricasole, Andrea; La Rosa, Salvatore; Porcari, Valentina



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


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

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



Potential impact of drug effects, availability, pharmacokinetics, and screening on estimates of drugs implicated in cases of assault.  


Drug-facilitated sexual assault (DFSA) is a serious and troubling crime. It is important to know if and how different drugs might be used to facilitate assault in order to deter such crime. There are a number of ways in which drugs that are used for DFSA might not be detected by routine screens. The purpose of this analysis was to draw reasonable inferences regarding drugs with a high likelihood of being used for DFSA and not being detected by routine screens. National data from poison control centres, hospital emergency rooms, and law enforcement seizures were used to evaluate the relative magnitude of problems and illicit availability associated with different classes of drugs. General drug classes were examined to include additional drugs that might be used for DFSA on the basis of their amnesic effects, widespread availability, and pharmacokinetics (i.e. short half-life). The benzodiazepine-site ligands zolpidem and eszopiclone, 'club drugs' GHB and ketamine, muscle relaxants such as carisoprodol, and antihistamines such as diphenhydramine were identified as drugs that might be used for DFSA and remain undetected by routine screens. Future studies that are designed to examine the role of these drugs in DFSA cases could provide better estimates of their use for DFSA. A better understanding of what is being missed in DFSA cases might help prioritize the development of new assays, provide rationale for the availability of particular assays for routine testing, and inform practitioners and the general public of the potential DFSA risks of certain drugs. PMID:21960542

Carter, Lawrence P



Microchip assays for screening monoclonal antibody product quality.  


Microchip CE-SDS was evaluated as a high-throughput alternative to conventional CE-SDS for monitoring monoclonal antibody protein quality. A commercial instrument (LabChip) 90) was used to separate dodecyl sulfate coated proteins through a sieving polymer based on the proteins' sizes. Under reducing conditions, the microchip CE-SDS separation was similar to that of conventional CE-SDS, providing reasonable resolution of the non-glycosylated and the glycosylated heavy chains. The fluorescence detection on LabChip 90 using non-covalent fluorescent labeling method was about as sensitive as the 220 nm UV detection used in a conventional CE instrument. A simple glycan typing assay was developed for the reducing microchip CE-SDS format. Antibodies, either pure or in crude cell culture media are treated with Endoglycosidase H, which specifically cleaves the hybrid and high mannose type glycans. A heavy chain migration shift on reducing CE-SDS resulting from the loss of glycan is used to measure the level of high mannose/hybrid type glycans as a percentage of the total glycans. Microchip CE-SDS, under both non-reducing and reducing conditions, can be used in a variety of antibody product screening assays. The microchip analyses provide sufficient resolution and sensitivity for this purpose but on a time scale approximately 70 times faster (41 s versus 50 min per sample) than conventional CE separation under typical operational conditions. PMID:19130579

Chen, Xiaoyu; Tang, Kaiyan; Lee, Maximilian; Flynn, Gregory C



Use of zebrafish apoptosis assays for preclinical drug discovery.  


Introduction: Apoptosis has become an important target for drug discovery. Although the mouse remains the animal model of choice for the preclinical assessment of drug toxicity and efficacy, zebrafish are increasingly used for early drug studies. Here, we describe approaches for assessing drug effects on apoptosis in transparent zebrafish. Areas covered: In this review, the authors discuss the drug effects on developmentally regulated apoptosis using microscopy. They also discuss the effects of neuroprotectants in a chemical induced disease model using morphometric image analysis. Finally, the authors review the effects of radioprotectants in irradiated whole animals using a conventional 96-well microplate format. Expert opinion: Several challenges have limited more widespread use of zebrafish as the organism of choice for drug screening. However, zebrafish do have a number of compelling inherent advantages including: rapid organogenesis, transparency, statistically significant numbers of animals per experiment and adaptability of cell-based methods. PMID:23964640

McGrath, Patricia; Seng, Wen Lin



The changing face of screening and drug discovery.  


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

Cooper, Matthew A



Convenient cell fusion assay for rapid screening for HIV entry inhibitors  

NASA Astrophysics Data System (ADS)

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.

Jiang, Shibo; Radigan, Lin; Zhang, Li



Neural System for in silico Drug-Drug Interaction Screening  

Microsoft Academic Search

Drug usage is always associated with risk drugs interactions are considered to be one of the potential sources of undesirable action of drugs. Such a situation enforced U.S. Food and Drug Administration (FDA) as well as European Medicines Agency (EMEA) to issue a guidance for industry and researchers for in vivo and in vitro drug interactions studies. The authors proposed

Sebastian Polak; Jerzy Brandys; Aleksander Mendyk



Safety Screening of Drugs in Cancer Therapy  

Microsoft Academic Search

Development of new drugs requires a thorough investigation of efficacy and safety of pharmaceuticals. The potential risks and benefits of drugs used in chemotherapy are carefully considered such that the benefits of using a new drug outweigh the risks in terms of the side effects caused by the drug. Damage to normal cells, tissues, organs and\\/or the whole organism is

J. Nath; G. Krishna



Impact of new technologies for cellular screening along the drug value chain.  


High-information screening formats, using more physiologically relevant cellular models and readout approaches, are slowly replacing traditional, target-orientated approaches in drug discovery programs. With improved access to primary cells, as well as label-free, non-intrusive methods of compound interrogation (such as automated electrophysiology), high-thoughput screening facilities have to adapt to more complex assay scenarios. The implementation of novel cellular systems, readout technologies and data management in a drug discovery company are essential to improve the current falling productivity evident in recent years throughout the pharmaceutical industry. PMID:20206290

Möller, Clemens; Slack, Mark



High-throughput screening applied to drug synthesis process development.  


Combinatorial chemistry and high-throughput screening have greatly increased the rate of lead drug molecule discovery. The increase in the number of compounds in the early phase of the drug development pipeline has, however, created a bottleneck in the development of cost-effective, scalable chemical processes for the manufacture of these drug candidates. Thus, pharmaceutical chemical development groups and their suppliers are implementing their own forms of high-throughput screening to improve their productivity to solve this bottleneck. Developments for implementing high-throughput screening for chemical process development are reviewed. PMID:19649902

Cannarsa, M J; Uno, T; Larsen, C



Neonatal illicit drug screening practices in Iowa: the impact of utilization of a structured screening protocol  

Microsoft Academic Search

Objective:The purpose of the study was to determine the neonatal illicit drug screening practices of Iowa birthing hospitals.Study design:Cross-sectional survey design was implemented. The impact of structured screening protocols on the numbers of neonates screened and positive testing in 2004 was reviewed.Results:Of 81 birthing hospitals, 53 (65%) participated in the study. Screening and positive test rates were higher in hospitals

R Oral; T Strang



Translation of a tumor microenvironment mimicking 3D tumor growth co-culture assay platform to high-content screening.  


For drug discovery, cell-based assays are becoming increasingly complex to mimic more realistically the nature of biological processes and their diversifications in diseases. Multicellular co-cultures embedded in a three-dimensional (3D) matrix have been explored in oncology to more closely approximate the physiology of the human tumor microenvironment. High-content analysis is the ideal technology to characterize these complex biological systems, although running such complex assays at higher throughput is a major endeavor. Here, we report on adapting a 3D tumor co-culture growth assay to automated microscopy, and we compare various imaging platforms (confocal vs. nonconfocal) with correlating automated image analysis solutions to identify optimal conditions and settings for future larger scaled screening campaigns. The optimized protocol has been validated in repeated runs where established anticancer drugs have been evaluated for performance in this innovative assay. PMID:22923784

Krausz, Eberhard; de Hoogt, Ronald; Gustin, Emmanuel; Cornelissen, Frans; Grand-Perret, Thierry; Janssen, Lut; Vloemans, Nele; Wuyts, Dirk; Frans, Sandy; Axel, Amy; Peeters, Pieter Johan; Hall, Brett; Cik, Miroslav



Development of an in vitro drug sensitivity assay for Trichuris muris first-stage larvae  

PubMed Central

Background Trichuriasis represents a major public health problem in the developing world and is regarded as a neglected disease. Albendazole and mebendazole, the two drugs of choice against trichuriasis display only moderate cure rates, hence alternative drugs are needed. To identify candidate compounds, in vitro drug sensitivity testing currently relies on the adult Trichuris muris motility assay. The objective of the present study was to develop a simple and cost-effective drug sensitivity assay using Trichuris muris first-stage larvae (L1). Methods Several potential triggers that induce hatching of T. muris were studied, including gastrointestinal enzymes, acidic environment and intestinal microflora. Next, optimal culture conditions for T. muris L1 were determined assessing a wide range of culture media. T. muris L1 were incubated in the presence of mebendazole, ivermectin, nitazoxanide, levamisole or oxantel pamoate at 37°C. The viability of the parasites was evaluated microscopically after 24 hours. The usefulness of fluorescent markers (resazurin, calcein AM, ethidium homodimer-1 or fluorescein-conjugated albumin) in drug sensitivity testing was also assessed. Results The established L1 motility assay provided accurate and reproducible drug effect data in vitro. IC50 values for oxantel pamoate, levamisole and nitazoxanide were 0.05, 1.75 and 4.43 ?g/mL, respectively. Mebendazole and ivermectin failed to show any trichuricidal effect on L1. No correlation was found between data from the four fluorescent markers and the comparative motility assay. Conclusions The motility assay based on L1 was found suitable for drug sensitivity screening. It is rather simple, cost-effective, time-saving and sustains medium-throughput testing. Furthermore, it greatly reduces the need for the animal host and is therefore more ethical. None of the viability markers assessed in this study were found to be satisfactory.



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

SciTech Connect

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.

Keating, Christopher, E-mail: [Department of Biomedical Sciences, University of Sheffield, Sheffield (United Kingdom); Martinez, Vicente; Ewart, Lorna [Department of Safety Pharmacology, Safety Assessment UK, AstraZeneca, Alderley Park (United Kingdom); Gibbons, Stephen; Grundy, Luke [Department of Biomedical Sciences, University of Sheffield, Sheffield (United Kingdom); Valentin, Jean-Pierre [Department of Safety Pharmacology, Safety Assessment UK, AstraZeneca, Alderley Park (United Kingdom); Grundy, David [Department of Biomedical Sciences, University of Sheffield, Sheffield (United Kingdom)



Single-Molecule Detection Technologies in Miniaturized High Throughput Screening: Binding Assays for G Protein-Coupled Receptors Using Fluorescence Intensity Distribution Analysis and Fluorescence Anisotropy  

Microsoft Academic Search

G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based

Martin Rudiger; Ulrich Haupts; Keith J. Moore; Andrew J. Pope



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


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 discrepant results between two laboratories compared to when specimens were analyzed at the same laboratory using two different assays. PMID:15899564

Lu, Natalie T; Taylor, Bruce G



Development and optimization of a novel 384-well anti-malarial imaging assay validated for high-throughput screening.  


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

Duffy, Sandra; Avery, Vicky M



Domain-Based Biosensor Assay to Screen for Epidermal Growth Factor Receptor Modulators in Live Cells  

PubMed Central

Abstract Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein–labeled SRC homology 2 domain–based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.

Antczak, Christophe; Bermingham, Alun; Calder, Paul; Malkov, Dmitry; Song, Keming; Fetter, John



Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.  


Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries. PMID:22280060

Antczak, Christophe; Bermingham, Alun; Calder, Paul; Malkov, Dmitry; Song, Keming; Fetter, John; Djaballah, Hakim



Preemployment drug screening in a large metropolitan medical center  

Microsoft Academic Search

To assess the prevalence of illicit drug use among job applicants, a large metropolitan medical center conducted preemployment\\u000a drug screening of all applicants during January 1988. Urine samples from 172 preinformed applicants were screened using Enzyme\\u000a Multiplied Immunoassay Technique (Emit d.a.u.™) followed by confirmatory gas chromatography\\/mass spectrophotometry. 4.1%\\u000a of tests were positive for marijuana and\\/or cocaine and none was positive

Donald Angehr Smith; Raymond Hanbury



A rapid transglutaminase assay for high-throughput screening applications.  


Transglutaminases (TGs) are widely distributed enzymes that catalyze posttranslational modification of proteins by Ca(2+)-dependent cross-linking reactions. The family members of TGs participate in many significant processes of biological functions such as tissue regeneration, cell differentiation, apoptosis, and certain pathologies. A novel technique for TG activity assay was developed in this study. It was based on the rapid capturing, fluorescence quenching, and fast separation of the unreacted fluorescent molecules from the macromolecular product with magnetic dextran-coated charcoal. As few as 3 ng of guinea pig liver transglutaminase (gpTG) could be detected by the method; activities of 96 TG samples could be measured within an hour. The K(m) of gpTG determined by this method for monodansylcadaverine (dansyl-CAD) and N, N-dimethylcasein was 14 and 5 muM, respectively. A typical competitive inhibition pattern of cystamine on dansyl-CAD for gpTG activity was also demonstrated. The application of this technique is not limited to the use of dansyl-CAD as the fluorescent substrate of TG; other small fluor-labeled TG substrates may substitute dansyl-CAD. Finally, this method is rapid, highly sensitive, and inexpensive. It is suitable not only for high-throughput screening of enzymes or enzyme inhibitors but also for enzyme kinetic analysis. PMID:16928981

Wu, Yu-Wei; Tsai, Yu-Hui



Versatile Assays for High Throughput Screening for Activators or Inhibitors of Intracellular Proteases and Their Cellular Regulators  

PubMed Central

Background Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. Methods & Findings We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS) of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy. Conclusions Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

Hayashi, Hideki; Cuddy, Michael; Shu, Vincent Chih-Wen; Yip, Kenneth W.; Madiraju, Charitha; Diaz, Paul; Matsuyama, Toshifumi; Kaibara, Muneshige; Taniyama, Kohtaro; Vasile, Stefan; Sergienko, Eduard; Reed, John C.



MI-96-1: Milk Monitoring with Antimicrobial Drug Screening ...  

Center for Food Safety and Applied Nutrition (CFSAN)

... Prior to 1991, the PMO recognized only one official test method for detecting drug residues in milk, the Bacillus stearothermophilus Disc Assay ... More results from


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

PubMed Central

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 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.



Comparative drug pair screening across multiple glioblastoma cell lines reveals novel drug-drug interactions.  


Background Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults, and despite state-of-the-art treatment, survival remains poor and novel therapeutics are sorely needed. The aim of the present study was to identify new synergistic drug pairs for GBM. In addition, we aimed to explore differences in drug-drug interactions across multiple GBM-derived cell cultures and predict such differences by use of transcriptional biomarkers. Methods We performed a screen in which we quantified drug-drug interactions for 465 drug pairs in each of the 5 GBM cell lines U87MG, U343MG, U373MG, A172, and T98G. Selected interactions were further tested using isobole-based analysis and validated in 5 glioma-initiating cell cultures. Furthermore, drug interactions were predicted using microarray-based transcriptional profiling in combination with statistical modeling. Results Of the 5 × 465 drug pairs, we could define a subset of drug pairs with strong interaction in both standard cell lines and glioma-initiating cell cultures. In particular, a subset of pairs involving the pharmaceutical compounds rimcazole, sertraline, pterostilbene, and gefitinib showed a strong interaction in a majority of the cell cultures tested. Statistical modeling of microarray and interaction data using sparse canonical correlation analysis revealed several predictive biomarkers, which we propose could be of importance in regulating drug pair responses. Conclusion We identify novel candidate drug pairs for GBM and suggest possibilities to prospectively use transcriptional biomarkers to predict drug interactions in individual cases. PMID:24101737

Schmidt, Linnéa; Kling, Teresia; Monsefi, Naser; Olsson, Maja; Hansson, Caroline; Baskaran, Sathishkumar; Lundgren, Bo; Martens, Ulf; Häggblad, Maria; Westermark, Bengt; Forsberg Nilsson, Karin; Uhrbom, Lene; Karlsson-Lindahl, Linda; Gerlee, Philip; Nelander, Sven



Inverse Drug Screens: A Rapid and Inexpensive Method for Implicating Molecular Targets  

PubMed Central

Summary Identification of gene products that function in some specific process of interest is a common goal in developmental biology. Although use of drug compounds to probe biological systems has a very long history in teratology and toxicology, systematic hierarchical drug screening has not been capitalized upon by the developmental biology community. This “chemical genetics” approach can greatly benefit the study of embryonic and regenerative systems, and we have formalized a strategy for using known pharmacological compounds to implicate specific molecular candidates in any chosen biological phenomenon. Taking advantage of a hierarchical structure that can be imposed on drug reagents in a number of fields such as ion transport, neurotransmitter function, metabolism, and cytoskeleton, any assay can be carried out as a binary search algorithm. This inverse drug screen methodology is much more efficient than exhaustive testing of large numbers of drugs, and reveals the identity of a manageable number of specific molecular candidates that can then be validated and targeted using more expensive and specific molecular reagents. Here, we describe the process of this loss-of-function screen and illustrate its use in uncovering novel bioelectrical and serotonergic mechanisms in embryonic patterning. This technique is an inexpensive and rapid complement to existing molecular screening strategies. Moreover, it is applicable to maternal proteins, and model species in which traditional genetic screens are not feasible, significantly extending the opportunities to identify key endogenous players in biological processes.

Adams, Dany S.; Levin, Michael



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

PubMed Central

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.

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



Thermodynamic Studies for Drug Design and Screening  

PubMed Central

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.

Garbett, Nichola C.; Chaires, Jonathan B.



Rapid Identification of Antifungal Compounds against Exserohilum rostratum Using High Throughput Drug Repurposing Screens.  


A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens. PMID:23990907

Sun, Wei; Park, Yoon-Dong; Sugui, Janyce A; Fothergill, Annette; Southall, Noel; Shinn, Paul; McKew, John C; Kwon-Chung, Kyung J; Zheng, Wei; Williamson, Peter R



Rapid Identification of Antifungal Compounds against Exserohilum rostratum Using High Throughput Drug Repurposing Screens  

PubMed Central

A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.

Sugui, Janyce A.; Fothergill, Annette; Southall, Noel; Shinn, Paul; McKew, John C.; Kwon-Chung, Kyung J.; Zheng, Wei; Williamson, Peter R.



Plasma Drug Activity Assay for Treatment Optimization in Tuberculosis Patients ? †  

PubMed Central

Low antituberculosis (TB) drug levels are common, but their clinical significance remains unclear, and methods of measurement are resource intensive. Subjects initiating treatment for sputum smear-positive pulmonary TB were enrolled from Kibong'oto National TB Hospital, Tanzania, and levels of isoniazid, rifampin, ethambutol, and pyrazinamide were measured at the time of typical peak plasma concentration (C2 h). To evaluate the significance of the effect of observed drug levels on Mycobacterium tuberculosis growth, a plasma TB drug activity (TDA) assay was developed using the Bactec MGIT system. Time to detection of plasma-cocultured M. tuberculosis versus time to detection of control growth was defined as a TDA ratio. TDA assays were later performed using the subject's own M. tuberculosis isolate and C2 h plasma from the Tanzanian cohort and compared to drug levels and clinical outcomes. Sixteen subjects with a mean age of 37.8 years ± 10.7 were enrolled. Fourteen (88%) had C2 h rifampin levels and 11 (69%) had isoniazid levels below 90% of the lower limit of the expected range. Plasma spiked with various concentrations of antituberculosis medications found TDA assay results to be unaffected by ethambutol or pyrazinamide. Yet with a range of isoniazid and rifampin concentrations, TDA exhibited a statistically significant correlation with drug level and drug MIC, and a TDA of ?1.0 indicated the presence of multidrug-resistant TB. In Tanzania, low (?2.0) TDA was significantly associated with both lower isoniazid and rifampin C2 h levels, and very low (?1.5) TDA corresponded to a trend toward lack of cure. Study of TDA compared to additional clinical outcomes and as a therapeutic management tool is warranted.

Heysell, Scott K.; Mtabho, Charles; Mpagama, Stellah; Mwaigwisya, Solomon; Pholwat, Suporn; Ndusilo, Norah; Gratz, Jean; Aarnoutse, Rob E.; Kibiki, Gibson S.; Houpt, Eric R.



Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells.  


Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease-specific iPSCs have been generated, there has been limited progress in iPSC-based drug screening/discovery for liver diseases, and the low gene-targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency, for which there is currently no drug or gene therapy available, we established a platform to discover new drug candidates and correct disease-causing mutation with a high efficiency. A high-throughput format screening assay, based on our hepatic differentiation protocol, was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients, we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large-scale drug screening, five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition, using the recently developed transcription activator-like effector nuclease technology, we achieved high gene-targeting efficiency in AAT-deficiency patient iPSCs with 25%-33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications. Conclusions: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs, both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases. PMID:23325555

Choi, Su Mi; Kim, Yonghak; Shim, Joong Sup; Park, Joon Tae; Wang, Rui-Hong; Leach, Steven D; Liu, Jun O; Deng, Chuxia; Ye, Zhaohui; Jang, Yoon-Young



Kinetic assay for high-throughput screening of in vitro transthyretin amyloid fibrillogenesis inhibitors.  


Stabilization of tetrameric transthyretin (TTR) by binding of small ligands is a current strategy aimed at inhibiting amyloid fibrillogenesis in transthyretin-associated pathologies, such as senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). A kinetic assay is developed for rapid evaluation of compounds as potential in vitro inhibitors in a high-throughput screening format. It is based on monitoring the time-dependent increase of absorbance due to turbidity occurring by acid-induced protein aggregation. The method uses the highly amyloidogenic Y78F mutant of human transthyretin (heterogously expressed in Escherichia coli cells). Initial rates of protein aggregation at different inhibitor concentrations follow a monoexponential dose-response curve from which inhibition parameters are calculated. For the assay development, thyroid hormones and nonsteroidal antiinflamatory drugs were chosen among other reference compounds. Some of them are already known to be in vitro inhibitors of TTR amyloidogenesis. Analysis time is optimized to last 1.5 h, and the method is implemented in microtiter plates for screening of libraries of potential fibrillogenesis inhibitors. PMID:15762752

Dolado, Ignacio; Nieto, Joan; Saraiva, Maria João M; Arsequell, Gemma; Valencia, Gregori; Planas, Antoni


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

PubMed Central

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.

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



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...


High-throughput screening assays for the identification of chemical probes  

Microsoft Academic Search

High-throughput screening (HTS) assays enable the testing of large numbers of chemical substances for activity in diverse areas of biology. The biological responses measured in HTS assays span isolated biochemical systems containing purified receptors or enzymes to signal transduction pathways and complex networks functioning in cellular environments. This Review addresses factors that need to be considered when implementing assays for

Ronald L Johnson; Anton Simeonov; Menghang Xia; Wei Zheng; Christopher P Austin; Douglas S Auld; James Inglese



Improved microbial screening assay for the detection of quinolone residues in poultry and eggs  

Microsoft Academic Search

An improved microbiological screening assay is reported for the detection of quinolone residues in poultry muscle and eggs. The method was validated using fortified tissue samples and is the first microbial assay to effectively detect enrofloxacin, difloxacin, danofloxacin, as well as flumequine and oxolinic acid, at or below their EU maximum residue limits (MRL). The accuracy of the assay was

M. G. Pikkemaat; P. P. J. Mulder; J. W. A. Elferink; A. De Cocq; M. W. F. Nielen; H. J. Van Egmond



Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF) with silver colloids in 96-well plates: Application to ultra fast and sensitive immunoassays, High Throughput Screening and drug discovery  

Microsoft Academic Search

Fluorescence detection is the basis of most assays used in drug discovery and High Throughput Screening (HTS) today. In all of these assays, assay rapidity and sensitivity is a primary concern, the sensitivity determined by both the quantum yield of the fluorophores and efficiency of the detection system, while rapidity is determined by the physical and biophysical parameters of temperature,

Kadir Aslan; Patrick Holley; Chris D. Geddes



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

PubMed Central

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.

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



Rapid Diagnosis of Extensively Drug-Resistant Tuberculosis by Use of a Reverse Line Blot Hybridization Assay?†  

PubMed Central

Drug resistance in tuberculosis (TB) is a matter of grave concern for TB control programs, as there is currently no cure for some extensively drug-resistant (XDR) strains. There is concern that this resistance could transmit, stressing the need for additional control measures, rapid diagnostic methods, and newer drugs for treatment. We developed an in-house assay that can rapidly detect resistance to drugs involved in the definition of XDR-TB directly from smear-positive specimens. Two hundred fifteen phenotypically XDR-TB isolates and 50 pansusceptible isolates were analyzed using a reverse line blot hybridization (RLBH) assay. The assay was also successfully applied to 73 smear-positive clinical specimens. The RLBH assay exhibited good sensitivity for the detection of resistance to isoniazid (99%), rifampin (99%), fluoroquinolones (95.3%), and second-line aminoglycosides (94.8%). The results from application of this assay on direct smear-positive clinical specimens revealed 93% concordance with the phenotypic drug susceptibility test (DST) results for the above-mentioned drugs. The time to accurate DST results was significantly reduced from weeks to 3 days. This molecular assay is a highly accurate tool for screening for XDR-TB, which achieves a substantial reduction in diagnostic delays.

Ajbani, Kanchan; Shetty, Anjali; Mehta, Ajita; Rodrigues, Camilla



A real-time screening assay for GIRK1/4 channel blockers.  


The cardiac acetylcholine-activated K(+) channel (I(K,Ach)) represents a novel target for drug therapy in the treatment of atrial fibrillation (AF). This channel is a member of the G-protein-coupled inward rectifier K(+) (GIRK) channel superfamily and is composed of the GIRK1/4 (Kir3.1 and Kir3.4) subunits. The goal of this study was to develop a cell-based screening assay for identifying new blockers of the GIRK1/4 channel. The mouse atrial HL-1 cell line, expressing the GIRK1/4 channel, was plated in 96-well plate format, loaded with the fluorescent membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) and measured using a fluorescent imaging plate reader (FLIPR). Application of the muscarinic agonist carbachol to the cells caused a rapid, time-dependent decrease in the fluorescent signal, indicative of K(+) efflux through the GIRK1/4 channel (carbachol vs. control solution, Z' factor = 0.5-0.6). The GIRK1/4 channel fluorescent signal was blocked by BaCl(2) and enhanced by increasing the driving force for K(+) across the cell membrane. To test the utility of the assay for screening GIRK1/4 channel blockers, cells were treated with a small compound library of Na(+) and K(+) channel modulators. Analogues of amiloride and propafenone were identified as channel blockers at concentrations less than 1 µM. Thus, the GIRK1/4 channel assay may be used in the development of new and selective agents for treating AF. PMID:20938046

Walsh, Kenneth B



Development of High Throughput Screening Assays Using Fluorescence Polarization: Nuclear Receptor-Ligand-Binding and Kinase\\/Phosphatase Assays  

Microsoft Academic Search

Fluorescence polarization (FP) has been used to develop high throughput screening (HTS) assays for nuclear receptor-ligand displacement and kinase inhibition. FP is a solution-based, homogeneous technique requiring no immobilization or separation of reaction components. The FP-based estrogen receptor (ER) assay is based on the competition of fluorescein-labeled estradiol and estrogen-like compounds for binding to ER. These studies determined the Kd

Gregory J. Parker; Tong Lin Law; Francis J. Lenoch; Randall E. Bolger



Predicting phospholipidosis: a fluorescence noncell based in vitro assay for the determination of drug-phospholipid complex formation in early drug discovery.  


This paper describes for the first time, a high-throughput fluorescence noncell based assay to screen for the drug-phospholipid interaction, which correlates to phospholipidosis. Anionic amphiphilic phospholipids can form complexes in aqueous solution, and its critical micelle concentration (CMC) can be determined using the fluorescence probe N,N-dimethyl-6-propionyl-2-naphthylamine (Prodan). Upon interaction with drug candidates, this CMC may shift to a lower value due to the association between lipids and drug candidates, the stronger the interaction, the greater the shift. Metabolism of a drug can change the degree of phospholipidosis depending on the rate of metabolism and the nature of the metabolite(s). Our data from 45 drugs and metabolites of 10 drugs using this fluorescence approach demonstrate a good correlation with phospholipidosis as reported with human studies, in vivo testing, and cellular assays. This assay therefore offers a fast, reliable, and cost-effective screening tool for early prediction of the phospholipidosis-inducing potential of drug candidates. PMID:21790130

Zhou, Liping; Geraci, Gina; Hess, Sloan; Yang, Linhong; Wang, Jianling; Argikar, Upendra



Cellular assays for drug discovery in genetic disorders of intracellular trafficking.  


Intracellular membrane trafficking is essential for organelle biogenesis, structure, and function; the exchange of material between organelles; and communication between the cell and its external environment. Genetic disorders affecting intracellular trafficking can lead to a variety of human diseases, but specific therapies for these diseases are notably lacking. In this article, we focus on how current knowledge about genetic disorders that affect intracellular trafficking can be used to develop strategies for cell-based assays in order to identify drugs using high-content screening approaches. PMID:23662666

De Matteis, Maria Antonietta; Vicinanza, Mariella; Venditti, Rossella; Wilson, Cathal



A programmable microfluidic cell array for combinatorial drug screening.  


We describe the development of a fully automatic and programmable microfluidic cell culture array that integrates on-chip generation of drug concentrations and pair-wise combinations with parallel culture of cells for drug candidate screening applications. The device has 64 individually addressable cell culture chambers in which cells can be cultured and exposed either sequentially or simultaneously to 64 pair-wise concentration combinations of two drugs. For sequential exposure, a simple microfluidic diffusive mixer is used to generate different concentrations of drugs from two inputs. For generation of 64 pair-wise combinations from two drug inputs, a novel time dependent variable concentration scheme is used in conjunction with the simple diffusive mixer to generate the desired combinations without the need for complex multi-layer structures or continuous medium perfusion. The generation of drug combinations and exposure to specific cell culture chambers are controlled using a LabVIEW interface capable of automatically running a multi-day drug screening experiment. Our cell array does not require continuous perfusion for keeping cells exposed to concentration gradients, minimizing the amount of drug used per experiment, and cells cultured in the chamber are not exposed to significant shear stress continuously. The utility of this platform is demonstrated for inducing loss of viability of PC3 prostate cancer cells using combinations of either doxorubicin or mitoxantrone with TRAIL (TNF-alpha Related Apoptosis Inducing Ligand) either in a sequential or simultaneous format. Our results demonstrate that the device can capture the synergy between different sensitizer drugs and TRAIL and demonstrate the potential of the microfluidic cell array for screening and optimizing combinatorial drug treatments for cancer therapy. PMID:22456798

Kim, Jeongyun; Taylor, David; Agrawal, Nitin; Wang, Han; Kim, Hyunsoo; Han, Arum; Rege, Kaushal; Jayaraman, Arul



Genotoxicity screening via the ?H2AX by flow assay.  


The measurement of serine139-phosphorylated histone H2AX (?H2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for ?H2AX detection (hereafter termed the ?H2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in ?H2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold ?H2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the ?H2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). ?H2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that ?H2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on ?H2AX were atypical and limited to a small sub-population of S-phase nuclei. Nevertheless, the ?H2AX by flow assay represents a novel genotoxicity assay with the potential to flag both pro- and proximate genotoxins. PMID:21824484

Smart, D J; Ahmedi, K P; Harvey, J S; Lynch, A M



A Comparison of Assay Performance Measures in Screening Assays: Signal Window, Z' Factor, and Assay Variability Ratio  

Microsoft Academic Search

In this article, the authors compare the assay performance measures, signalwindow, Z' factor, and assay variability ratio. They examine their mathematical formulae for similarities and differences, describe their statistical sampling properties using the results of a computer simulation, and illustrate their usewith example data. Based on these results, the authors recommend the Z' factor as a preferredmeasure of assay performance

Philip W. Iversen; Brian J. Eastwood; G. Sitta Sittampalam; Karen L. Cox



Reliability of the Drug Use Screening Inventory among Adolescent Alcoholics.  

ERIC Educational Resources Information Center

|Examines psychometric reliability of Drug Use Screening Inventory (DUSI) utilizing adolescents with DSM-III-R diagnosis of Psychoactive Substance Use Disorder. Concludes that split-half, internal, and test-retest reliability is superior. Suggests that DUSI may be useful for identifying and quantifying substance use and related problems. Includes…

Tarter, Ralph E.; And Others



Screens and assays for agents useful in controlling parasitic nematodes  

US Patent & Trademark Office Database

The invention provides a method of identifying anti-nematode compounds and further provides transgenic nematodes that may be used to practice the method. In particular, the invention provides a screen for compounds that inhibit a nematode secretion pathway e.g, compounds that inhibit the secretion of proteins by nematodes. The transgenic nematodes express reporters for nematode secreted proteins. In preferred embodiments of the invention the screen is performed using C. elegans, i.e., certain embodiments of the invention utilize C. elegans and C. elegans secretory pathways as a model system for parasitic nematodes and parasitic nematode secretion pathways. The invention also provides pharmaceutical compositions that may be used in the treatment and prevention of nematode infection in humans and animals and anti-nematode agents that may be used to protect plants from plant-parasitic nematodes. In addition, the invention provides a genetic screen for identifying additional targets for anti-nematode compounds.




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...



Technology Transfer Automated Retrieval System (TEKTRAN)

A simple, rapid fluorescence assay was developed for screening both enrofloxacin (ENRO) and tetracyclines in chicken muscle at the U.S. tolerance levels (300 ng/g and 2000 ng/g, respectively). Screening for both classes of antibiotics is accomplished using one extraction, thus simplifying and exped...


Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries.  


The EPXH2 gene encodes soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of arachidonic acid epoxides that play important roles in blood pressure, cell growth, inflammation, and pain. While recent findings suggested complementary biological roles for Nterm-phos, research is limited by the lack of potent bioavailable inhibitors of this phosphatase activity. Also, a potent bioavailable inhibitor of this activity could be important in the development of therapy for cardiovascular diseases. We report herein the development of an HTS enzyme-based assay for Nterm-phos (Z'>0.9) using AttoPhos as the substrate. This assay was used to screen a wide variety of chemical entities, including a library of known drugs that have reached through clinical evaluation (Pharmakon 1600), as well as a library of pesticides and environmental toxins. We discovered that ebselen inhibits sEH phosphatase activity. Ebselen binds to the N-terminal domain of sEH (K(I)=550 nM) and chemically reacts with the enzyme to quickly and irreversibly inhibit Nterm-phos, and subsequently Cterm-EH, and thus represents a new class of sEH inhibitor. PMID:23219563

Morisseau, Christophe; Sahdeo, Sunil; Cortopassi, Gino; Hammock, Bruce D



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

Code of Federal Regulations, 2010 CFR for predicting drug resistance/susceptibility based on...genomic mutations that confer resistance to specific antiretroviral drugs, as an...Document: In Vitro HIV Drug Resistance Genotype Assay.â See §...



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

Code of Federal Regulations, 2010 CFR for predicting drug resistance/susceptibility based on...genomic mutations that confer resistance to specific antiretroviral drugs, as an...Document: In Vitro HIV Drug Resistance Genotype Assay.â See §...



21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.  

Code of Federal Regulations, 2010 CFR

...Identification. A neuroleptic drugs radioceptor assay test system is a device intended to measure in serum or plasma the dopamine receptor blocking activity of neuroleptic drugs and their active metabolites. A neuroleptic drug has...



21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.  

Code of Federal Regulations, 2010 CFR

...Identification. A neuroleptic drugs radioceptor assay test system is a device intended to measure in serum or plasma the dopamine receptor blocking activity of neuroleptic drugs and their active metabolites. A neuroleptic drug has...



Neuraminidase activity provides a practical read-out for a high throughput influenza antiviral screening assay  

Microsoft Academic Search

BACKGROUND: The emergence of influenza strains that are resistant to commonly used antivirals has highlighted the need to develop new compounds that target viral gene products or host mechanisms that are essential for effective virus replication. Existing assays to identify potential antiviral compounds often use high throughput screening assays that target specific viral replication steps. To broaden the search for

Maryna C Eichelberger; Arash Hassantoufighi; Meng Wu; Min Li



Microplate screening assay for the detection of arsenite-oxidizing and arsenate-reducing bacteria  

Microsoft Academic Search

An efficient, inexpensive microplate colorimetric assay for screening of bacteria which can be used in bioremediation of arsenic was developed. The assay is based on the colorimetric analysis of the precipitates formed upon reaction of silver nitrate with arsenic. The method proved reliable and sensitive for the detection of As[III] oxidizers and As[V] reducers and can be used over a

Diliana D. Simeonova; Didier Lièvremont; Florence Lagarde; Daniel A. E. Muller; Veneta I. Groudeva; Marie-Claire Lett



Highly sensitive method for assay of drug-induced apoptosis using fluorescence correlation spectroscopy.  


Apoptosis plays a crucial role in many biological processes and pathogenesis of various malignancies and diseases of the immune system. In this paper, we described a novel method for sensitive detection of drug-induced apoptosis by using fluorescence correlation spectroscopy (FCS). The principle of this method is based on the assay of DNA fragmentation in the process of the drug-induced apoptosis. FCS is a single molecule method, and it can be used for sensitive and selective assay of DNA fragmentation without separation. We first developed a highly sensitive method for characterization of DNA fragments using a home-built FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin. Furthermore, FCS method established was used to directly detect the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data. The characteristic diffusion time of DNA fragments was used as an important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of lidamycin concentration, which implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gel electrophoresis. Compared to current methods, the method described here is sensitive and simple, and more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nL level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs. PMID:22876965

Ruan, Lingao; Xu, Zhancheng; Lan, Tao; Wang, Jinjie; Liu, Heng; Li, Chaodong; Dong, Chaoqing; Ren, Jicun



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

Microsoft Academic Search

Objective  To screen commonly used prescription drugs for possible carcinogenic effects.\\u000a \\u000a \\u000a \\u000a Methods  In a large health care program we identified 105 commonly used drugs, not previously screened. Recipients were followed for\\u000a up to 12½ years for incident cancer. Nested case–control analyses of 55 cancer sites and all combined included up to ten matched\\u000a controls per case, with lag of at least 2 years between

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



A high content screening assay for identifying lysosomotropic compounds  

Microsoft Academic Search

Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable.Hence, we developed a

Sashi Nadanaciva; Shuyan Lu; David F. Gebhard; Bart A. Jessen; William D. Pennie; Yvonne Will



Mining small-molecule screens to repurpose drugs.  


Repurposing and repositioning drugs--discovering new uses for existing and experimental medicines-is an attractive strategy for rescuing stalled pharmaceutical projects, finding treatments for neglected diseases, and reducing the time, cost and risk of drug development. As this strategy emerged, academic researchers began performing high-throughput screens (HTS) of small molecules--the type of experiments once exclusively conducted in industry--and making the data from these screens available to all. Several methods can mine this data to inform repurposing and repositioning efforts. Despite these methods' limitations, it is hopeful that they will accelerate the discovery of new uses for known drugs, but this hope has not yet been realized. PMID:21715466

Swamidass, S Joshua



Quantitative analysis of preservatives in drug preparations by microbiological assay*  

PubMed Central

The stability of a preservative selected for incorporation into a drug preparation can only be determined by suitable tests applied at regular intervals, preferably over long periods. Chemical tests are available for a number of preservatives and some of them appear to be adequate but difficulties have occurred from time to time and studies were undertaken to develop microbiological tests. Chemical methods are usually more precise than biological assays but do not always measure the antimicrobial activity of the preservative as accurately. It is always possible that a preservative may break down, losing some of its effectiveness even though the chemical determinant upon which the test is based remains intact. Two assay methods were studied. One, a plate-diffusion procedure, was found suitable for assaying formol and preservatives containing mercury. Preservatives such as phenol, benzethonium chloride and others do not diffuse in an agar plate and a tube—plate bacterial count procedure was developed for this group. Procedures for both tests are described and some examples given. ImagesFIG. 2FIG. 4

Greenberg, L.; Naubert, J.



Method comparison of EMIT 700 and EMIT II with RIA for drug screening.  


The EMIT 700 and EMIT II assays for marijuana, cocaine, opiates, barbiturates, and phencyclidine, as performed on a Hitachi 717 Analyzer, were compared with Roche Abuscreen RIA tests for high-volume drug abuse screening. The EMIT II kits offer some advantages over the EMIT 700 kits for testing large work loads. The EMIT II marijuana test in particular exhibits a calibration curve that promises improved ability to separate negative from positive samples due to the increased absorbance rate separation between the negative and cutoff calibrators. Both EMIT formulations are preferable in terms of speed and ease of analysis in comparison with our laboratory's current RIA procedures. Over 50,000 urine samples were screened by EMIT 700 and RIA and by EMIT II and RIA. The performance of both EMIT assays was approximately equivalent to RIA in terms of their ability to detect urine samples that confirmed positive for cocaine and opiates. Both EMIT assays detected approximately 90% of the urine samples screened positive by RIA and confirmed positive for marijuana. Both EMIT assays performed better than RIA in detecting confirmed-positive barbiturate samples. PMID:8207931

Armbruster, D A; Schwarzhoff, R H; Pierce, B L; Hubster, E C


Peptides as drugs: From screening to application.  


Peptides are ideally suited to mimic natural ligands and thereby function in an antagonistic or agonistic way. Furthermore they are able to physiologically disrupt functional complexes due to their small size and specific binding properties. Proteins form homo- or heteromeric (macro)molecular complexes and intricate networks by interacting with small molecules, peptides, nucleic acids or other proteins. On average, five interaction partners have been estimated for any given cellular protein, illustrating the complexity of the formed 'interactomes' and the impact of their investigation. Many protein-protein interactions are mediated by hot-spots, which comprise only a small part of the large binding interface but account for 80% of the binding energy. Thus, these hot spots provide an 'Achilles heel' for pharmaceutical interventions aiming at the disruption of functional protein-protein complexes. Methods to select peptides for defined target structures include display technologies on phages, ribosomes or yeast, and the yeast-two-hybrid system. Once selected, these peptides can be optimized for their binding affinity using peptide arrays. Stabilization of biologically unstable peptides is achieved by the introduction of non-natural amino acids to form so-called peptidomimetics that are resistant to cellular proteases. Moreover, lipocalins and peptide aptamers represent scaffolded binding structures with unique binding characteristics and enhanced stability. In case of extracellular targets, like cell surface receptors or pathogens in patients` plasma, peptide inhibitors have direct access. Addressing intracellular targets with peptides is more difficult since short hydrophilic peptides generally do not cross plasma membranes on their own. However, intracellular uptake of peptides can be achieved by coupling to carrier systems like liposomes or nanoparticles or upon fusion to a protein transduction domain. Alternatively, peptides may be expressed within cells after transduction with viral vectors in a gene therapy setting. This review will summarize the broad potential of peptides as drugs, with special emphasis on peptides which inhibit protein-protein interactions. PMID:22429133

Dietrich, Ursula; Dürr, Ralf; Koch, Joachim



Evaluation of a Novel Metric for Quality Control in an RNA Interference High Throughput Screening Assay  

Microsoft Academic Search

The application of genome scale RNA interference (RNAi) relies on the development of high quality RNAi high throughput screening (HTS) assays. An important quality control (QC) characteristic in an HTS assay is how well the positive controls, samples, and negative controls can be separated from each other in the assay. Signal-to-noise ratio, signal-to-background ratio, Z-factor and Z'-factor have been adopted

Xiaohua Douglas Zhang; Amy S. Espeseth; Namjin Chung; Marc Ferrer



Development of a novel ?-secretase binding assay using the AlphaScreen platform.  


Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. ?-secretase-1 (BACE1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form A? is a validated target for AD. Herein, the authors develop and validate a novel binding assay for BACE1 using the AlphaScreen platform that is amenable for high-throughput screening (HTS). Small-molecule BACE1 inhibitors of the hydroxyethylamine, hydantoin, and sulfamide classes were functionalized by biotin PEG linkers of varying lengths forming probes that were bound to streptavidin donor beads. BACE1 was coupled to nickel-chelate acceptor beads. Upon mixing, probes designed from all three classes registered high signal-to-background values in the AlphaScreen binding assay, where the interaction between probe and BACE1 was completely blocked by free parent compound. A probe from the hydantoin class was chosen for further optimization, where the final assay conditions of 50 nM BACE and 250 nM probe were used and Z(') values >0.75 were commonly observed. IC50 values determined by the AlphaScreen assay format exhibited ~10-fold greater sensitivity when compared with a fluorescence polarization-based activity assay. The assay was miniaturized to a 1536-well format for HTS, in which 525 000 compounds were screened. PMID:23543430

Ren, Zhao; Tam, Danny; Xu, Ying-Zi; Wone, David; Yuan, Shendong; Sham, Hing L; Cheung, Harry; Regnstrom, Karin; Chen, Xiaohua; Rudolph, Donald; Jobling, Michael F; Artis, Dean R; Bova, Michael P



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

PubMed Central

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.

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



An AlphaScreen®-based assay for high-throughput screening for specific inhibitors of nuclear import.  


Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)-1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen(®)) technology, which is high throughput, requires low amounts of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin ?/?, successfully identifying several inhibitors of the IN/importin ?/? interaction. Importantly, they demonstrate that one of the identified compounds, mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin ?/? inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach. PMID:21297106

Wagstaff, Kylie M; Rawlinson, Stephen M; Hearps, Anna C; Jans, David A



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

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.

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



High-Content Drug Screening with Engineered Musculoskeletal Tissues  

PubMed Central

Tissue engineering for in vitro drug-screening applications based on tissue function is an active area of translational research. Compared to targeted high-throughput drug-screening methods that rapidly analyze hundreds of thousands of compounds affecting a single biochemical reaction or gene expression, high-content screening (HCS) with engineered tissues is more complex and based on the cumulative positive and negative effects of a compound on the multiple pathways altering tissue function. It may therefore serve as better predictor of in vivo activity and serve as a bridge between high-throughput drug screening and in vivo animal studies. In the case of the musculoskeletal system, tissue function includes determining improvements in the mechanical properties of bone, tendon, cartilage, and, for skeletal muscle, contractile properties such as rate of contraction/relaxation, force generation, fatigability, and recovery from fatigue. HCS of compound banks with engineered tissues requires miniature musculoskeletal organs as well as automated functional testing. The resulting technologies should be rapid, cost effective, and reduce the number of small animals required for follow-on in vivo studies. Identification of compounds that improve the repair/regeneration of damaged tissues in vivo would have extensive clinical applications for treating musculoskeletal disorders.



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

SciTech Connect

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.

Tani, Hidenori [Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Akimitsu, Nobuyoshi [Radioisotope Center, University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Fujita, Osamu; Matsuda, Yasuyoshi [Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Miyata, Ryo [Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Tsuneda, Satoshi [Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan); Igarashi, Masayuki [Microbial Chemistry Research Center, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan); Sekiguchi, Yuji [Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Noda, Naohiro [Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan); Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan)], E-mail:



Quantitative screening for anticestode drugs based on changes in baseline enzyme secretion by Taenia crassiceps.  


Neurocysticercosis (NCC), an infection of the brain with the larval stage of the Taenia solium tapeworm, is responsible for an estimated one-third of adult-onset epilepsy cases in regions of the world where it is endemic. Currently, anthelmintic drugs used for treatment of NCC are only partially effective, and there is, therefore, a pressing need for new therapeutic agents. Discovery of new anthelmintics with activity against T. solium has been limited by the lack of suitable sensitive assays that allow high-throughput screening. Using an in vitro culture system with Taenia crassiceps metacestodes, we demonstrate that changes in secretion of parasite-associated alkaline phosphatase (AP) and phosphoglucose isomerase (PGI) can be used to detect and quantify anthelmintic effects of praziquantel (PZQ), a drug with activity against T. solium. We applied two enzyme release assays to screen for anti-T. crassiceps activity in nonconventional antiparasitic drugs and demonstrate that nitazoxanide and artesunate induced release of both AP and PGI in differing time- and dose-related patterns. Furthermore, imatinib, a tyrosine kinase inhibitor previously reported to have parasiticidal activity against Schistosoma mansoni, also induced release of both AP and PGI in a dose-dependent manner, similar in pattern to that observed with the other anthelmintics. We also evaluated release of ATP into cyst supernatants as an indicator of drug effects but did not see any differences between treated and untreated cysts. These data provide the basis for rapid and quantitative screening assays for testing for anthelmintic activity in candidate anticestode agents. PMID:23229489

Mahanty, Siddhartha; Madrid, Elise M; Nash, Theodore E



Electrochemical Biochip for Drug Screening At Cellular Level  

NASA Astrophysics Data System (ADS)

Drug screening at cellular level has becomes an attractive field of research. Different researchers have tried to record cellular response to drugs by electrical or optical approach using both invasive and non-invasive methods. Silicon-based microelectrode integrated microchips are useful tools for in situ temporal recording of neurotransmitter releasing from neural cells. A microfabricated electrochemical biochip is presented in this paper. Using dopaminergic cells grown on the chip, the dopamine excytosis can be electrochemical amperomatric detected non-invasively from drug incubated dopaminegic cells by the microelectrode integrated on chip. This silicon-based electrochemical chip has been designed with an electrode array located on the cell culture chamber bottom. Each electrode is individually electrical controlled. MN9D and PC12 dopaminergic cell lines have been demonstrated on this chip for drug effects study. This silicon-based electrochemical microchip provides a non-invasive, in situ, temporal detection of dopamine exocytosis from dopaminegic cells, and holds the potential for applications in studying the mechanisms of dopamine exocytosis and drug screening. It is also extendable for other cell culture and drug effects study.

Chen, Yu; Cui, Hui-fang; Ye, Jian-shan; Chong, Ser-choong; Lim, Tit-meng; Sheu, Fwu-shan; Hui, Wing-cheong



Activity of Clinically Relevant Antimalarial Drugs on Plasmodium falciparum Mature Gametocytes in an ATP Bioluminescence "Transmission Blocking" Assay  

PubMed Central

Background Current anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes. Methods and Findings Difficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV–V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs. Conclusions The work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti-malarial drugs to target gametocyte-specific metabolic pathways.

Lozano, Sonia; Miguel, Celia; Franco, Virginia; Leroy, Didier; Herreros, Esperanza



The automated micronucleus assay for early assessment of genotoxicity in drug discovery.  


Recent publications on the automated in vitro micronucleus assay show predictive values higher than 85% for the classification of in vitro aneugens, clastogens and non-genotoxic compounds. In the present work, the CHO-k1 micronucleus assay in combination with cellular imaging was further evaluated. Firstly, the effect of a range of S9 concentrations on micronucleus formation and cytotoxicity was investigated. Subsequently, the reproducibility and predictivity of the micronucleus assay on CHO-k1 cells was investigated with a set of four compounds. Then, a larger set of compounds (n=44) was tested on CHO-k1 cells and inter-laboratory correlation was calculated. Finally, cellular imaging was compared with flow cytometry for in vivo assessment of micronucleus formation. The concentration of S9 had a significant impact on micronucleus formation and cytotoxicity. In addition, calculations of relative cell count (RCC) and cytokinesis-block proliferation index (CBPI) showed to be complementary to cytotoxicity assessment. The CHO-k1 micronucleus assay correctly classified the four reference compounds, with a dose-response relationship and low variability. Based on a larger set of compounds, the assay proved to be reliable with a sensitivity of 94% (n=31) and a specificity of 85% (n=13). A correlation coefficient of 97% was obtained when the lowest observable adverse effect levels (LOAELs) from our study were compared with those published by Diaz et al. (2007) [10]. In conclusion, the in vitro CHO-k1 micronucleus assay combined with cellular imaging is a predictive assay appropriate for genotoxicity screening at early stages of drug development. In addition, for in vivo assessment of micronucleus formation, we preferred to use flow cytometry rather than cell imaging. PMID:23159395

Tilmant, K; Gerets, H H J; De Ron, P; Cossu-Leguille, C; Vasseur, P; Dhalluin, S; Atienzar, F A



Drosophila modifier screens to identify novel neuropsychiatric drugs including aminergic agents for the possible treatment of Parkinson's disease and depression.  


Small molecules that increase the presynaptic function of aminergic cells may provide neuroprotection in Parkinson's disease (PD) as well as treatments for attention deficit hyperactivity disorder (ADHD) and depression. Model genetic organisms such as Drosophila melanogaster may enhance the detection of new drugs via modifier or 'enhancer/suppressor' screens, but this technique has not been applied to processes relevant to psychiatry. To identify new aminergic drugs in vivo, we used a mutation in the Drosophila vesicular monoamine transporter (dVMAT) as a sensitized genetic background and performed a suppressor screen. We fed dVMAT mutant larvae ?1000 known drugs and quantitated rescue (suppression) of an amine-dependent locomotor deficit in the larva. To determine which drugs might specifically potentiate neurotransmitter release, we performed an additional secondary screen for drugs that require presynaptic amine storage to rescue larval locomotion. Using additional larval locomotion and adult fertility assays, we validated that at least one compound previously used clinically as an antineoplastic agent potentiates the presynaptic function of aminergic circuits. We suggest that structurally similar agents might be used to development treatments for PD, depression and ADHD, and that modifier screens in Drosophila provide a new strategy to screen for neuropsychiatric drugs. More generally, our findings demonstrate the power of physiologically based screens for identifying bioactive agents for select neurotransmitter systems.Molecular Psychiatry advance online publication, 11 December 2012; doi:10.1038/mp.2012.170. PMID:23229049

Lawal, H O; Terrell, A; Lam, H A; Djapri, C; Jang, J; Hadi, R; Roberts, L; Shahi, V; Chou, M-T; Biedermann, T; Huang, B; Lawless, G M; Maidment, N T; Krantz, D E



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

PubMed Central

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.

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



Improving high-content-screening assay performance by using division-arrested cells.  


As cell-based assays are used more commonly in robotic high-throughput compound screening, cells themselves have become critical reagents. Thus, it has become essential to produce cell reagents with high consistency and quality. We experimented with cells division-arrested with low-level mitomycin C treatment and demonstrate that they perform with better consistency than non-division-arrested counterparts in high-content screening imaging assays. We propose that for cell-based screening, it is possible to uncouple the cell production process from the screening process. Cells can be produced en masse, treated to become irreversibly division-arrested, and cryopreserved. These "ready-to-use" reagents can be thawed, plated, and used in screening with improved consistency and convenience. PMID:16305308

Vasudevan, Chandrasekaran; Fursov, Natalie; Maunder, Patrick; Cong, Mei; Federici, Mark; Haskins, Jeffrey R; Livelli, Thomas; Zhong, Zhong



Screening of Dengue virus antiviral activity of marine seaweeds by an in situ enzyme-linked immunosorbent assay.  


Dengue is a significant public health problem worldwide. Despite the important social and clinical impact, there is no vaccine or specific antiviral therapy for prevention and treatment of dengue virus (DENV) infection. Considering the above, drug discovery research for dengue is of utmost importance; in addition natural marine products provide diverse and novel chemical structures with potent biological activities that must be evaluated. In this study we propose a target-free approach for dengue drug discovery based on a novel, rapid, and economic in situ enzyme-linked immunosorbent assay and the screening of a panel of marine seaweed extracts. The in situ ELISA was standardized and validated for Huh7.5 cell line infected with all four serotypes of DENV, among them clinical isolates and a laboratory strain. Statistical analysis showed an average S/B of 7.2 and Z-factor of 0.62, demonstrating assay consistency and reliability. A panel of fifteen seaweed extracts was then screened at the maximum non-toxic dose previously determined by the MTT and Neutral Red cytotoxic assays. Eight seaweed extracts were able to reduce DENV infection of at least one serotype tested. Four extracts (Phaeophyta: Canistrocarpus cervicornis, Padina gymnospora; Rhodophyta: Palisada perforate; Chlorophyta: Caulerpa racemosa) were chosen for further evaluation, and time of addition studies point that they might act at an early stage of the viral infection cycle, such as binding or internalization. PMID:23227238

Koishi, Andrea Cristine; Zanello, Paula Rodrigues; Bianco, Éverson Miguel; Bordignon, Juliano; Nunes Duarte dos Santos, Claudia



Development of an AlphaScreen-based HIV-1 integrase dimerization assay for discovery of novel allosteric inhibitors.  


In recent years, HIV-1 integrase (IN) has become an established target in the field of antiretroviral drug discovery. However, its sole clinically approved inhibitor, the integrase strand transfer inhibitor (INSTI) raltegravir, has a surprisingly low genetic barrier for resistance. Furthermore, the only two other integrase inhibitors currently in advanced clinical trials, elvitegravir and dolutegravir, share its mechanism of action and certain resistance pathways. To maintain a range of treatment options, drug discovery efforts are now turning toward allosteric IN inhibitors, which should be devoid of cross-resistance with INSTIs. As IN requires a precise and dynamic equilibrium between several oligomeric species for its activities, the modulation of this equilibrium presents an interesting allosteric target. We report on the development, characterization, and validation of an AlphaScreen-based assay for high-throughput screening for modulators of HIV-1 IN dimerization. Compounds identified as hits in this assay proved to act as allosteric IN inhibitors. Additionally, the assay offers a flexible platform to study IN dimerization. PMID:22337657

Demeulemeester, Jonas; Tintori, Cristina; Botta, Maurizio; Debyser, Zeger; Christ, Frauke



Screening for drug-resistant Candida yeasts with chromogenic agar.  


We examined the utility of agar dilution to screen yeasts for reduced susceptibility to several newer antifungal drugs including echinocandins and azoles. We compared agar dilution susceptibility screening with the Clinical and Laboratory Standards Institute (CLSI) method for Candida isolates. We added echinocandins and azoles to CHROMagar Candida medium prior to its solidification. Assessment of resistance was based on growth characteristics, wherein decreased colony size in the presence of antifungal drugs was used as an indicator of susceptibility. Clinical Candida isolates of C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. guilliermondii, C. lusitaniae, C. rugosa and C. dubliniensis were screened for drug susceptibility. Overall, antifungal susceptibility of the yeasts to anidulafungin, caspofungin, micafungin, posaconazole and voriconazole, determined using CHROMagar agar dilution, were shown to be 96, 80, 94, 90 and 97% as accurate, respectively, as those determined by the CLSI method, i.e., within one tube dilution of CLSI MICs. Categorical errors by percentage had a broader range. Major errors noted with anidulafungin, caspofungin and micafungin were 3, 6 and 0%, respectively, while very major errors were 15, 55 and 38%, respectively. Major errors with posaconazole and voriconazole were 12 and 0%, respectively, while very major errors were 0 and 22%, respectively, compared to CLSI standards. Most of the assessment errors were found with C. glabrata and C. parapsilosis. Agar dilution screening for drug susceptibility with the current panel of antifungal drugs is rapid, accurate and effective. However, the determination of resistance or non-susceptibility in yeasts may be more problematic, and may be species dependent. PMID:20109095

Kirkpatrick, William R; Zimmerman, Joseph D; Haikal, Fadi P; Broker, Michael J; Brockway, Erin; Fothergill, Annette W; McCarthy, Dora I; Patterson, Thomas F; Redding, Spencer W



Identification of Rift Valley Fever Virus Nucleocapsid Protein-RNA Binding Inhibitors Using a High-Throughput Screening Assay  

PubMed Central

Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential anti-viral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug screening assay and tested 26,424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of FDA approved drugs, drug-like molecules and natural products extracts we identified several lead compounds that are promising candidates for medicinal chemistry.

Ellenbecker, Mary; Lanchy, Jean-Marc; Lodmell, J. Stephen



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

ERIC Educational Resources Information Center

|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)|

Cannell, M. Barry; Favazza, Armando R.



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

ERIC Educational Resources Information Center

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)

Cannell, M. Barry; Favazza, Armando R.



Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish  

PubMed Central

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ?0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current “high-content” whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.

Mathias, Jonathan R.; Coothankandaswamy, Veena; Xie, Xiayang; Distel, Martin; Koster, Reinhard W.; Parsons, Michael J.; Bhalla, Kapil N.; Saxena, Meera T.; Mumm, Jeff S.



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

SciTech Connect

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.

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



The development of a high-content screening binding assay for the smoothened receptor.  


In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-µM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling. PMID:22644265

Bee, Weilin Tiger; Xie, Wensheng; Truong, Maggie; Will, Matthew; Turunen, Brandon; Zuercher, William J; McMillan, Lynette; Li, Hu; Hornberger, Keith R; Davenport, Elizabeth A; Ames, Robert S; Kallal, Lorena A



poolHiTS: A Shifted Transversal Design based pooling strategy for high-throughput drug screening  

PubMed Central

Background A key goal of drug discovery is to increase the throughput of small molecule screens without sacrificing screening accuracy. High-throughput screening (HTS) in drug discovery involves testing a large number of compounds in a biological assay to identify active compounds. Normally, molecules from a large compound library are tested individually to identify the activity of each molecule. Usually a small number of compounds are found to be active, however the presence of false positive and negative testing errors suggests that this one-drug one-assay screening strategy can be significantly improved. Pooling designs are testing schemes that test mixtures of compounds in each assay, thereby generating a screen of the whole compound library in fewer tests. By repeatedly testing compounds in different combinations, pooling designs also allow for error-correction. These pooled designs, for specific experiment parameters, can be simply and efficiently created using the Shifted Transversal Design (STD) pooling algorithm. However, drug screening contains a number of key constraints that require specific modifications if this pooling approach is to be useful for practical screen designs. Results In this paper, we introduce a pooling strategy called poolHiTS (Pooled High-Throughput Screening) which is based on the STD algorithm. In poolHiTS, we implement a limit on the number of compounds that can be mixed in a single assay. In addition, we show that the STD-based pooling strategy is limited in the error-correction that it can achieve. Due to the mixing constraint, we show that it is more efficient to split a large library into smaller blocks of compounds, which are then tested using an optimized strategy repeated for each block. We package the optimal block selection algorithm into poolHiTS. The MATLAB codes for the poolHiTS algorithm and the corresponding decoding strategy are also provided. Conclusion We have produced a practical version of STD algorithm for pooled drug screens. This pooling strategy provides both assay compression and error-correction capabilities that can both accelerate and reduce the overall cost of HTS in drug discovery.

Kainkaryam, Raghunandan M; Woolf, Peter J



Automation and miniaturization of the bioluminescent UGT-Glo assay for screening of UDP-glucuronosyltransferase inhibition by various compounds.  


The uridine 5'-diphospho-glucuronosyltransferase (UGT) family of enzymes is involved in the metabolism of various compounds. These enzymes transfer a hydrophilic glucuronic acid moiety to their substrates, rendering them more water soluble and amenable to excretion. The UGTs act on various endogenous substrates, such as bilirubin, 17?-estradiol, and testosterone, and drugs and other xenobiotics. The function of these enzymes is essential for the clearance of drugs and toxicants, and alteration of UGT activity is a potential cause of adverse drug-drug interactions in vivo. This has stimulated an increased interest in the study of UGT function and inhibition, and the desire to profile new drug entities against UGT enzymes, similar to CYP450 profiling. However, certain factors have hindered the development of a robust method for UGT profiling. Current methods for assessing UGT enzyme activity are laborious and involve protein precipitation and/or chromatographic separation steps, which are not amenable to rapid screening applications for UGT inhibitors or substrates. The approach presented here is a bioluminescent assay for measuring UGT enzyme activity and inhibition in vitro. Using flexible, robust instrumentation in a 384-well microplate format, this study highlights the quick and easy assay implementation for estimation of inhibition kinetics with a variety of known and suspected UGT substrates and inhibitors. PMID:21609684

Larson, Brad; Kelts, Jessica L; Banks, Peter; Cali, James J



Colorimetric assays for quantitative analysis and screening of epoxide hydrolase activity.  


Focusing on directed evolution to tailor enzymes as usable biocatalysts for fine chemistry, we have studied in detail several colorimetric assays for quantitative analysis of epoxide hydrolase (EH) activity. In particular, two assays have been optimized to characterize variants issued from the directed evolution of the EH from Aspergillus niger. Assays described in this paper are sufficiently reliable for quantitative screening of EH activity in microtiter plates and are low cost alternatives to GC or MS analysis. Moreover, they are usable for various epoxides and not restricted to a type of substrate, such as those amenable to assay by UV absorbancy. They can be used to assay EH activity on any epoxide and to directly assay enantioselectivity when both (R) and (S) substrates are available. The advantages and drawbacks of these two methods to assay EH activity of a large number of natural samples are summarized. PMID:16328991

Cedrone, F; Bhatnagar, T; Baratti, Jacques C



Development of a novel ectonucleotidase assay suitable for high-throughput screening.  


5'-Ectonucleotidase (NT5E) catalyzes the conversion of adenosine monophosphate to adenosine and free phosphate. The role of this ectonucleotidase and its production of adenosine are linked with immune function, angiogenesis, and cancer. NT5E activity is typically assayed either by chromatographic quantification of substrates and products using high-performance liquid chromatography (HPLC) or by quantification of free phosphate using malachite green. These methods are not suitable for robust screening assays of NT5E activity. HPLC is not readily suitable for the rapid and efficient assay of multiple samples and malachite green is highly sensitive to the phosphate-containing buffers common in various media and sample buffers. Here the development and validation of a novel high-throughput ectonucleotidase screening assay are described, which makes use of a luciferase-based assay reagent, the Promega CellTiter-Glo kit, to measure the catabolism of AMP by NT5E. This multiwell plate-based assay facilitates the screening of potential ectonucleotidase antagonists and is unaffected by the presence of contaminating phosphate molecules present in screening samples. PMID:22522649

Sachsenmeier, Kris F; Hay, Carl; Brand, Erin; Clarke, Lori; Rosenthal, Kim; Guillard, Sandrine; Rust, Steven; Minter, Ralph; Hollingsworth, Robert



Systematic analysis of large screening sets in drug discovery.  


Each year large pharmaceutical companies produce massive amounts of primary screening data for lead discovery. To make better use of the vast amount of information in pharmaceutical databases, companies have begun to scrutinize the lead generation stage to ensure that more and better qualified lead series enter the downstream optimization and development stages. This article describes computational techniques for end to end analysis of large drug discovery screening sets. The analysis proceeds in three stages: In stage 1 the initial screening set is filtered to remove compounds that are unsuitable as lead compounds. In stage 2 local structural neighborhoods around active compound classes are identified, including similar but inactive compounds. In stage 3 the structure-activity relationships within local structural neighborhoods are analyzed. These processes are illustrated by analyzing two large, publicly available databases. PMID:16472218

Blower, Paul E; Cross, Kevin P; Fligner, Michael A; Myatt, Glenn J; Verducci, Joseph S; Yang, Chihae



Fast Standardized Therapeutic-Efficacy Assay for Drug Discovery against Tuberculosis?  

PubMed Central

Murine models of Mycobacterium tuberculosis infection are essential tools in drug discovery. Here we describe a fast standardized 9-day acute assay intended to measure the efficacy of drugs against M. tuberculosis growing in the lungs of immunocompetent mice. This assay is highly reproducible, allows good throughput, and was validated for drug lead optimization using isoniazid, rifampin, ethambutol, pyrazinamide, linezolid, and moxifloxacin.

Rullas, Joaquin; Garcia, Juan Ignacio; Beltran, Manuela; Cardona, Pere-Joan; Caceres, Neus; Garcia-Bustos, Jose Francisco; Angulo-Barturen, Inigo



Reliability of the Drug Use Screening Inventory Among Adolescent Alcoholics  

Microsoft Academic Search

Psychometric reliability of the Drug Use Screening Inventory (DUSI) was examined in 191 adolescents who qualified for a DSM-III-R diagnosis of Psychoactive Substance Use Disorder. Internal, split-half and test-retest reliability was found to be superior. Caucasian subjects exhibited more severe substance use problems than African-American subjects but did not differ in any other domain. Females reported more severe disturbances than

Ralph E. Tarter; Ada C. Mezzich; Levent Kirisci; Nancy Kaczynksi



Test concentration setting for fish in vivo endocrine screening assays.  


Fish in vivo screening methods to detect endocrine active substances, specifically interacting with the hypothalamic-pituitary-gonadal axis, have been developed by both the Organization for Economic Co-operation and Development (OECD) and United States Environmental Protection Agency (US-EPA). In application of these methods, i.e. regulatory testing, this paper provides a proposal on the setting of test concentrations using all available acute and chronic data and also discusses the importance of avoiding the confounding effects of systemic toxicity on endocrine endpoints. This guidance is aimed at reducing the number of false positives and subsequently the number of inappropriate definitive vertebrate studies potentially triggered by effects consequent to systemic, rather than endocrine, toxicity. At the same time it provides a pragmatic approach that maximizes the probability of detecting an effect, if it exists, thus limiting the potential for false negative outcomes. PMID:23481302

Wheeler, James R; Panter, Grace H; Weltje, Lennart; Thorpe, Karen L



Luciferin IPA-based higher throughput human hepatocyte screening assays for CYP3A4 inhibition and induction.  


The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIPA). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1-aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (maximum inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metabolism. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (maximum inhibition of <50%) or no apparent inhibitory effects in the HTS assay. In the 96-well plate induction assay, the CYP3A4 inducers rifampin, phenobarbital, carbamazepine, phenytoin, troglitazone, rosiglitazone, and pioglitazone yielded dose-dependent induction of LIPA metabolism, whereas the CYP1A2 inducers omeprazole and 3-methylcholanthrene did not display any induction in the CYP3A4 activity. The high sensitivity and specificity of the assays, the relative ease of execution, and reduced cost, time, and test material requirements suggest that the HTS assays may be applied routinely for screening a large number of chemicals in the drug discovery phase for CYP3A4 inhibitory and inducing potential. PMID:21832258

Doshi, Utkarsh; Li, Albert P



A New In Vivo Screening Paradigm to Accelerate Antimalarial Drug Discovery  

PubMed Central

The emergence of resistance to available antimalarials requires the urgent development of new medicines. The recent disclosure of several thousand compounds active in vitro against the erythrocyte stage of Plasmodium falciparum has been a major breakthrough, though converting these hits into new medicines challenges current strategies. A new in vivo screening concept was evaluated as a strategy to increase the speed and efficiency of drug discovery projects in malaria. The new in vivo screening concept was developed based on human disease parameters, i.e. parasitemia in the peripheral blood of patients on hospital admission and parasite reduction ratio (PRR), which were allometrically down-scaled into P. berghei-infected mice. Mice with an initial parasitemia (P0) of 1.5% were treated orally for two consecutive days and parasitemia measured 24 h after the second dose. The assay was optimized for detection of compounds able to stop parasite replication (PRR?=?1) or induce parasite clearance (PRR >1) with statistical power >99% using only two mice per experimental group. In the P. berghei in vivo screening assay, the PRR of a set of eleven antimalarials with different mechanisms of action correlated with human-equivalent data. Subsequently, 590 compounds from the Tres Cantos Antimalarial Set with activity in vitro against P. falciparum were tested at 50 mg/kg (orally) in an assay format that allowed the evaluation of hundreds of compounds per month. The rate of compounds with detectable efficacy was 11.2% and about one third of active compounds showed in vivo efficacy comparable with the most potent antimalarials used clinically. High-throughput, high-content in vivo screening could rapidly select new compounds, dramatically speeding up the discovery of new antimalarial medicines. A global multilateral collaborative project aimed at screening the significant chemical diversity within the antimalarial in vitro hits described in the literature is a feasible task.

Jimenez-Diaz, Maria Belen; Viera, Sara; Ibanez, Javier; Mulet, Teresa; Magan-Marchal, Noemi; Garuti, Helen; Gomez, Vanessa; Cortes-Gil, Lorena; Martinez, Antonio; Ferrer, Santiago; Fraile, Maria Teresa; Calderon, Felix; Fernandez, Esther; Shultz, Leonard D.; Leroy, Didier; Wilson, David M.; Garcia-Bustos, Jose Francisco; Gamo, Francisco Javier; Angulo-Barturen, Inigo



Drug abuse in the workplace: employee screening techniques  

SciTech Connect

Recent studies show that as many as three to five percent of the employees of a medium- to large-sized plant may be dependent on drugs as a way of life. The detrimental effects of drug abuse in the workplace can be measured in lost productivity, poor quality control and other areas at an annual cost to the American economy of $30 billion. However, a price tag cannot be attached to the lives affected by this unrelenting problem. The purpose of this paper is to provide an overview of the employee screening and hiring techniques available to industry to detect and eliminate potentially dangerous or fatal situations involving drug abuse in the workplace. The techniques are universal and can be effectively applied by the nuclear industry as well as other businesses to ensure that its work force is a reputable and reliable one.

Buzzeo, R.W.



New scaffolds of natural origin as Integrase-LEDGF/p75 interaction inhibitors: Virtual screening and activity assays.  


The disruption of crucial interactions between HIV-1 Integrase and cellular cofactor LEDGF/p75 represents an emerging approach for the design and development of new antiretroviral agents. In this study we report the successful application of a structure-based virtual screening strategy for the discovery of natural hit structures able to inhibit Integrase-LEDGF/p75 interaction. The application of sequential filters (drug-likeness, 3D-pharmacophore mapping, docking, molecular dynamics simulations) yielded a hit list of compounds, out of which 9 were tested in the in vitro AlphaScreen assays and 8 exhibited a detectable inhibition of the interaction between the two proteins. The best inhibitors belong to different chemical classes and could be represent a good starting point for further optimization and structure-activity relationship studies. PMID:23994868

De Luca, Laura; Morreale, Francesca; Christ, Frauke; Debyser, Zeger; Ferro, Stefania; Gitto, Rosaria



Fragment screening at adenosine-A(3) receptors in living cells using a fluorescence-based binding assay.  


G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is determined in the correct cellular environment and preferably using an unmodified receptor. We developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to determine binding affinity constants of competing ligands at human adenosine-A(1) and -A(3) receptors. This method was validated as a tool to screen a library of low molecular weight fragments, and identified a hit with submicromolar binding affinity (K(D)). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A(3) receptor. This technology represents a significant advance that will allow the determination of ligand and fragment affinities at receptors in their native membrane environment. PMID:22999879

Stoddart, Leigh A; Vernall, Andrea J; Denman, Jessica L; Briddon, Stephen J; Kellam, Barrie; Hill, Stephen J



A Comparison of Self-Reported Drug Use With a Urine Drug Screen in a Working Population  

Microsoft Academic Search

This study summarizes information on 162 workers who completed a urine screen and self-report concerning drug use. It is the first to compare self-report of drug use in the workplace with a urine screen in which individual participant (nonaggregate) data were used. The findings indicate that agreement between the 2 methods of drug detection, although statistically significant, is at best

Rodabe Bharucha-Reid; Daisy McCann; M. Anthony Schork; Betsy Foxman; Alan Bass; Winifred Fraser; Sandra Cook; Rachel Kaufman



A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening  

PubMed Central

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.

Gilbert, Daniel F.; Erdmann, Gerrit; Zhang, Xian; Fritzsche, Anja; Demir, Kubilay; Jaedicke, Andreas; Muehlenberg, Katja; Wanker, Erich E.; Boutros, Michael



Biomimetic membrane platform containing hERG potassium channel and its application to drug screening.  


The hERG (human ether-à-go-go-related gene) potassium channel has been extensively studied by both academia and industry because of its relation to inherited or drug-induced long QT syndrome (LQTS). Unpredicted hERG and drug interaction affecting channel activity is of main concern for drug discovery. Although there are several methods to test hERG and drug interaction, it is still necessary to develop some efficient and economic ways to probe hERG and drug interactions. To contribute this aim, we have developed a biomimetic lipid membrane platform into which the hERG channel can be folded. Expression and integration of the hERG channel was achieved using a cell-free (CF) expression system. The folding of hERG in the biomimetic membrane system was investigated using Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) and Imaging Surface Plasmon Resonance (iSPR). In addition, the hERG channel folded into our biomimetic membrane platform was used for probing the channel and drug interactions through fluorescence polarization (FP) assay. Our results suggest that the biomimetic system employed is capable of detecting the interaction between hERG and different channel blockers at varied concentrations. We believe that our current approach could be applied to other membrane proteins for drug screening or other protein-related interactions. PMID:23423263

Arslan Yildiz, Ahu; Kang, CongBao; Sinner, Eva-Kathrin



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

PubMed Central

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.

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



Multiplex inhibitor screening and kinetic constant determinations for yeast hexokinase using mass spectrometry based assays  

Microsoft Academic Search

An electrospray ionization mass spectrometry based assay was developed for kinetic measurements and inhibitor screening of\\u000a yeast hexokinase. There is considerable discrepancy in the literature as to the accuracy of kinetic data obtained for hexokinase.\\u000a In the assay described herein, the product, glucose 6-phosphate was directly monitored by ion trap mass spectrometry and quantified\\u000a using an internal standard, 2 deoxy-glucose

Hong Gao; Julie A. Leary



A solid-phase assay to screen monoclonal antibodies against DNA-binding protein  

Microsoft Academic Search

A method is described for selecting monoclonal antibodies (mAb) against DNA-binding protein. The protocol involves a non-radioactive solid-phase DNA binding assay using a 96-well plate. Because the solid-phase assay is highly specific and sensitive, partially purified antigen is sufficient for the immuniz- ation, and mAb screening can be performed with crude cell extract as the antigen. MAbs obtained by this

N. Ishii; H. Nakayama; J. Katayama; M. Arisawa; Y. Aoki



An Image-Based Drug Susceptibility Assay Targeting the Placental Sequestration of Plasmodium falciparum-Infected Erythrocytes  

PubMed Central

Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria.

Genovesio, Auguste; Ayong, Lawrence; Freitas-Junior, Lucio H.



Drug design and testing: profiling of antiproliferative agents for cancer therapy using a cell-based methyl-[3H]-thymidine incorporation assay.  


Drug design is an iterative process requiring cycles of compound synthesis and testing, with each successive synthesis phase yielding molecules predicted to have improved characteristics over the previous set of compounds. In the field of cancer drug discovery, a key early-stage element of the drug design and testing process usually involves the screening of compounds in cell-based in vitro assays. One of the most frequent parameters assessed in cancer drug discovery is the impact of a given molecule on the proliferation of a cancer cell. The methyl-[3H]-thymidine incorporation assay is a widely used, gold standard, method for measuring inhibition of cell proliferation and has been used successfully to screen and optimize potential new cancer drugs. The assay is based on measuring incorporation of methyl-[3H]-thymidine (the radiolabeled form of the DNA precursor thymidine) into the DNA of dividing cancer cells. The screen is used to generate concentration effect relationships for test compounds and for the derivation of IC50 values. IC50 value is defined as the concentration of a test compound required to achieve half maximal inhibition of methyl-[3H]-thymidine incorporation, a parameter that is indicative of antiproliferative potency. IC50 values derived from cell-based assays help drive the medicinal chemistry efforts toward improved drug design, and it is, therefore, critical that the screen provides consistent, robust data over the lifetime of the project - a requirement that necessitates good-quality cell culture practices. The methyl-[3H]-thymidine incorporation assay has been adapted to high-throughput format to facilitate screening of large numbers of compounds. The detailed description of this method, exemplified using the COLO-205 colorectal cancer cell line in a 96-well format, should give the reader a thorough account of how to conduct proliferation assays, as well as some notes and tips on how to ensure success and avoid potential pitfalls. PMID:21516428

Griffiths, Matthew; Sundaram, Hardy



Using molecular similarity to highlight the challenges of routine immunoassay-based drug of abuse/toxicology screening in emergency medicine  

PubMed Central

Background Laboratory tests for routine drug of abuse and toxicology (DOA/Tox) screening, often used in emergency medicine, generally utilize antibody-based tests (immunoassays) to detect classes of drugs such as amphetamines, barbiturates, benzodiazepines, opiates, and tricyclic antidepressants, or individual drugs such as cocaine, methadone, and phencyclidine. A key factor in assay sensitivity and specificity is the drugs or drug metabolites that were used as antigenic targets to generate the assay antibodies. All DOA/Tox screening immunoassays can be limited by false positives caused by cross-reactivity from structurally related compounds. For immunoassays targeted at a particular class of drugs, there can also be false negatives if there is failure to detect some drugs or their metabolites within that class. Methods Molecular similarity analysis, a computational method commonly used in drug discovery, was used to calculate structural similarity of a wide range of clinically relevant compounds (prescription and over-the-counter medications, illicit drugs, and clinically significant metabolites) to the target ('antigenic') molecules of DOA/Tox screening tests. These results were compared with cross-reactivity data in the package inserts of immunoassays marketed for clinical testing. The causes for false positives for phencyclidine and tricyclic antidepressant screening immunoassays were investigated at the authors' medical center using gas chromatography/mass spectrometry as a confirmatory method. Results The results illustrate three major challenges for routine DOA/Tox screening immunoassays used in emergency medicine. First, for some classes of drugs, the structural diversity of common drugs within each class has been increasing, thereby making it difficult for a single assay to detect all compounds without compromising specificity. Second, for some screening assays, common 'out-of-class' drugs may be structurally similar to the target compound so that they account for a high frequency of false positives. Illustrating this point, at the authors' medical center, the majority of positive screening results for phencyclidine and tricyclic antidepressants assays were explained by out-of-class drugs. Third, different manufacturers have adopted varying approaches to marketed immunoassays, leading to substantial inter-assay variability. Conclusion The expanding structural diversity of drugs presents a difficult challenge for routine DOA/Tox screening that limit the clinical utility of these tests in the emergency medicine setting.

Krasowski, Matthew D; Pizon, Anthony F; Siam, Mohamed G; Giannoutsos, Spiros; Iyer, Manisha; Ekins, Sean



Development of a high-content screening assay panel to accelerate mechanism of action studies for oncology research.  


Efficient elucidation of the biological mechanism of action of novel compounds remains a major bottleneck in the drug discovery process. To address this need in the area of oncology, we report the development of a multiparametric high-content screening assay panel at the level of single cells to dramatically accelerate understanding the mechanism of action of cell growth-inhibiting compounds on a large scale. Our approach is based on measuring 10 established end points associated with mitochondrial apoptosis, cell cycle disruption, DNA damage, and cellular morphological changes in the same experiment, across three multiparametric assays. The data from all of the measurements taken together are expected to help increase our current understanding of target protein functions, constrain the list of possible targets for compounds identified using phenotypic screens, and identify off-target effects. We have also developed novel data visualization and phenotypic classification approaches for detailed interpretation of individual compound effects and navigation of large collections of multiparametric cellular responses. We expect this general approach to be valuable for drug discovery across multiple therapeutic areas. PMID:22706350

Towne, Danli L; Nicholl, Emily E; Comess, Kenneth M; Galasinski, Scott C; Hajduk, Philip J; Abraham, Vivek C




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...


Facilitating drug discovery: an automated high-content inflammation assay in zebrafish.  


Zebrafish larvae are particularly amenable to whole animal small molecule screens due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a <1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos or tailfin amputation. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED2 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes. In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts. In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development. PMID:22825322

Wittmann, Christine; Reischl, Markus; Shah, Asmi H; Mikut, Ralf; Liebel, Urban; Grabher, Clemens



A Mycobacterium marinum zone of inhibition assay as a method for screening potential antimycobacterial compounds from marine extracts.  


A novel screening method for antimycobacterial agents using Mycobacterium marinum was developed. M. marinum was selected as a model organism because it has a close phylogenetic relationship to M. tuberculosis, a relatively rapid doubling time, similar drug susceptibilities to M. tuberculosis, and less stringent safety requirements. More than 1000 crude marine and plant extracts were screened against M. marinum in a Zone of Inhibition (ZOI) assay, and twenty-one target extracts were identified. The crude organic extract of the marine sponge, Haliclona sp.10, was chosen for further investigation as it yielded a ZOI of 20 mm at a concentration of 80 microg/disk. Following bioassay-guided fractionation, (-)-papuamine was isolated, and yielded a 15 mm ZOI at a concentration of 25 microg/disk. In standard assays using M. marinum, (-)-papuamine exhibited both an MIC and an MBC95 of 6.25 microg/mL. This is the first report of antimycobacterial activity for (-)-papuamine. In addition, when (-)-papuamine and other natural product extracts were tested for activity against both M. marinum and M. tuberculosis, activity was comparable between the two species. These data indicate that (-)-papuamine is a promising lead for the development of new antimycobacterial agents and that M. marinum is a useful surrogate for the screening of antimycobacterial compounds. PMID:17534789

Barker, Lucia P; Lien, Benjamin A; Brun, Olivier S; Schaak, Damen D; McDonough, Kathleen A; Chang, Leng Chee



Development of a colorimetric and a fluorescence phosphatase-inhibitor assay suitable for drug discovery approaches.  


Protein phosphatases (PP) are interesting drug targets. However, their ubiquitous presence and involvement in different, partially opposing signal pathways suggest that specificity may be achieved rather by targeting their interaction with subunits determining substrate specificity than the enzyme itself. An interesting subunit is phosphatase inhibitor-1 (I-1), which, in its protein kinase A-phosphorylated form (I-1(P)), inhibits the catalytic subunit of type 1 phosphatase (PP1c). In the current study, we established a colorimetric and a fluorescence-based assay system for the identification of compounds interfering with the inhibitory effect of I-1(P) on PP1c. The fluorescence assay exhibited 500-fold higher sensitivity toward PP1c. A nine-residue peptide containing the PP1c-binding motif (RVxF) of I-1 stimulated PP1c activity in the presence of I-1(P) (EC50 27 µM and 2.3 µM in the colorimetric and fluorescence assay, respectively). This suggests that the peptide interfered with the inhibitory effect of I-1(P) on PP1c and represents a proof-of-principle. The calculated Z' factor for PP1c (0.84) and the PP1c-I-1(P) complex (0.73) confirmed the suitability of the fluorescence assay for high-throughput screenings (HTS). By testing several thousand small molecules, we suggest the advantages of kinetic measurements over single-point measurements using the fluorescence-based assay in an HTS format. PMID:23606651

Sotoud, Hannieh; Gribbon, Philip; Ellinger, Bernhard; Reinshagen, Jeanette; Boknik, Peter; Kattner, Lars; El-Armouche, Ali; Eschenhagen, Thomas



A powerful yeast-based screening assay for the identification of inhibitors of indoleamine 2,3-dioxygenase.  


Activation of the enzyme indoleamine 2,3-dioxygenase 1 (IDO1) underlies the course of several human pathological conditions and, to date, no efficacious therapeutic IDO inhibitors are available. We proposed to develop a robust screening system based on the use of yeast cells to identify new lead compounds for the pharmacological inhibition of IDO-the BLOCKADE platform. Yeast combines the advantages of a relevant surrogate model for eukaryotic cell processes with the amenity to miniaturization and automation. We brought added value to the system by increasing the stringency of our assay, as the BLOCKADE strain was not deleted for any efflux pump, thus creating additional challenges for test compounds to be identified as hits. Screening of a library of 50 080 small molecules led to the identification of 101 potential IDO inhibitors, a low hit rate of 0.2%, reflecting the stringent assay conditions imposed. Most important, secondary pharmacology assays in mammalian cells confirmed activity for 76% of the hits, whereas hepatotoxicity testing indicated that 87% of them displayed a safe profile. The high predictivity rates obtained using the BLOCKADE platform clearly validate our system as a powerful tool for drug discovery. PMID:22791376

Cerejo, Marta; Andrade, Gonçalo; Roca, Christophe; Sousa, José; Rodrigues, Cátia; Pinheiro, Ricardo; Chatterjee, Sukalyan; Vieira, Helena; Calado, Patrícia



Optical detection of pesticides and drugs based on chemiluminescence–fluorescence assays  

Microsoft Academic Search

The paper illustrates two different concepts for monitoring pesticides and drugs. In the first approach double stranded deoxyribonucleic acid (dsDNA) was used as the sensing material, which intercalated drugs with high affinity. The intercalation of drugs such as Chlorpromazine (CP), Clomipramine (CM) and Imipramine (IP) were investigated. The assay was based on competition between the drug molecule and a fluorescent

Bengt Danielsson; Ioana Surugiu; Anatoli Dzgoev; Michael Mecklenburg; Kumaran Ramanathan



The holistic integration of virtual screening in drug discovery.  


During the past decade, virtual screening (VS) has come of age. In this review, we document the evolution and maturation of VS from a rather exotic, stand-alone method toward a versatile hit and lead identification technology. VS campaigns have become fully integrated into drug discovery campaigns, evenly matched and complementary to high-throughput screening (HTS) methods. Here, we propose a novel classification of VS applications to help to monitor the advances in VS and to support future improvement of computational hit and lead identification methods. Several relevant VS studies from recent publications, in both academic and industrial settings, were selected to demonstrate the progress in this area. Furthermore, we identify challenges that lie ahead for the development of integrated VS campaigns. PMID:23340112

Tanrikulu, Yusuf; Krüger, Björn; Proschak, Ewgenij



Human Papillomavirus Testing with the Hybrid Capture 2 Assay and PCR as Screening Tools  

Microsoft Academic Search

The recognition of high-risk human papillomaviruses (HPVs) as etiological agents of cervical cancer has in- creased the demands to use testing for HPV for the detection of abnormal cervical smears and for cervical cancer screening. The present study compared the performance of the Hybrid Capture 2 (HC2) assay with that of PCR for the detection of significant cervical lesions in

S.-M. Kulmala; S. Syrjanen; I. Shabalova; N. Petrovichev; V. Kozachenko; J. Podistov; O. Ivanchenko; S. Zakharenko; R. Nerovjna; L. Kljukina; M. Branovskaja; V. Grunberga; A. Juschenko; P. Tosi; R. Santopietro; K. Syrjanen



A mechanism-based whole-cell screening assay to identify inhibitors of protein export in Escherichia coli by the Sec pathway.  


More than 20% of bacterial proteins are noncytoplasmic, and most of these pass through the SecYEG channel en route to the periplasm, cell membrane, or surrounding environment. The Sec pathway, encompassing SecYEG and several associated proteins (SecA, SecB, YidC, SecDFYajC), is of interest as a potential drug target because it is distinct from targets of current drugs, is essential for bacterial growth, and exhibits dissimilarities in eukaryotes and bacteria that increase the likelihood of selectively inhibiting the microbial pathway. As a step toward validating the pathway as a drug target, we have adapted a mechanism-based whole-cell assay in a manner suitable for high-throughput screening (HTS). The assay uses an engineered strain of Escherichia coli that accumulates beta-galactosidase (?-gal) in its cytoplasm if translocation through SecYEG is blocked. The assay should facilitate rapid identification of compounds that specifically block the Sec pathway because widely, toxic compounds and nonspecific protein synthesis inhibitors prevent ?-gal production and thus do not register as hits. Testing of current antibiotics confirmed that they do not generally act through the Sec pathway. A mini-screen of 800 compounds indicated the assay's readiness for larger screening projects. PMID:22233648

Crowther, Gregory J; Quadri, S Arshiya; Shannon-Alferes, Benjamin J; Van Voorhis, Wesley C; Rosen, Henry



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

PubMed Central

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.

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



TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors.  


UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13. PMID:22034497

Madiraju, Charitha; Welsh, Kate; Cuddy, Michael P; Godoi, Paulo H; Pass, Ian; Ngo, Tram; Vasile, Stefan; Sergienko, Eduard A; Diaz, Paul; Matsuzawa, Shu-Ichi; Reed, John C



High-Throughput RT-PCR for small-molecule screening assays  

PubMed Central

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis.

Bittker, Joshua A.



Screening assay of residual antibiotics in livestock samples by LC-MS/MS.  


A LC-MS/MS screening assay of multi-class antibiotics was developed for 19 residual antibiotics in livestock samples. Sample preparation employed the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach using 0.5% formic acid in acetonitrile-methanol (8 : 2), with salting-out using magnesium sulfate, trisodium citrate and sodium chloride. Recovery values from 5 different livestock samples ranged from 45.5 to 121.6%, and the RSDs were under 18% at two concentration levels. The limit of quantification values of 19 analytes were under 10 µg/kg in all livestock samples, and the procedure can detect almost all analytes under the MRL. Screening capability was confirmed by employing spiked samples. This new screening assay for residual antibiotics in livestock samples is expected to be useful for routine laboratory tests. PMID:22688024

Nakajima, Takayuki; Sasamoto, Takeo; Hayashi, Hiroshi; Kanda, Maki; Takeba, Kazue; Kanai, Setsuko; Kusano, Tomoko; Matsushima, Yoko; Takano, Ichiro



Identification of inhibitors for a virally encoded protein kinase by 2 different screening systems: in vitro kinase assay and in-cell activity assay.  


The human cytomegalovirus (HCMV) protein kinase pUL97 represents an important determinant for viral replication and thus is a promising target for the treatment of HCMV. The authors screened a compound library of nearly 5000 entities based on known kinase inhibitors in 2 distinct ways. A radioactive in vitro kinase assay was performed with recombinant pUL97, purified from baculovirus-infected insect cells, on myelin basic protein-coated FlashPlates. About 20% of all compounds tested inhibited pUL97 kinase activity by more than 50% at a concentration of 10 microM. These hits belonged to various structural classes. To elucidate their potential to inhibit pUL97 in a cellular context, all compounds of the library were also tested in a cell-based activity assay. For this reason, a HEK293 cell line was established that ectopically expressed pUL97. When these cells were incubated with ganciclovir (GCV), pUL97 phosphorylated GCV to its monophosphate, which subsequently became phosphorylated to cytotoxic metabolites by cellular enzymes. Thereby, pUL97 converted cells into a GCV-sensitive phenotype. Inhibition of the pUL97 kinase activity resulted in protection of the cells against the cytotoxic effects of GCV. In total, 199 compounds of the library were cellular active at nontoxic concentrations, and 93 of them inhibited pUL97 in the in vitro kinase assay. Among these, promising inhibitors of HCMV replication were identified. The 2-fold screening system described here should facilitate the development of pUL97 inhibitors into potent drug candidates. PMID:15695342

Mett, Helmut; Hölscher, Kerstin; Degen, Heidrun; Esdar, Christina; De Neumann, Birgit Felden; Flicke, Birgit; Freudenreich, Tatjana; Holzer, Gaby; Schinzel, Sieglinde; Stamminger, Thomas; Stein-Gerlach, Matthias; Marschall, Manfred; Herget, Thomas



A Screening Assay to Identify Agents That Enhance T-Cell Recognition of Human Melanomas  

PubMed Central

Abstract Although a series of melanoma differentiation antigens for immunotherapeutic targeting has been described, heterogeneous expression of antigens such as Melan-A/MART-1 and gp100 results from a loss of antigenic expression in many late stage tumors. Antigen loss can represent a means for tumor escape from immune recognition, and a barrier to immunotherapy. However, since antigen-negative tumor phenotypes frequently result from reversible gene regulatory events, antigen enhancement represents a potential therapeutic opportunity. Accordingly, we have developed a cell-based assay to screen for compounds with the ability to enhance T-cell recognition of melanoma cells. This assay is dependent on augmentation of MelanA/MART-1 antigen presentation by a melanoma cell line (MU89). T-cell recognition is detected as interleukin-2 production by a Jurkat T cell transduced to express a T-cell receptor specific for an HLA-A2 restricted epitope of the Melan-A/MART-1 protein. This cellular assay was used to perform a pilot screen by using 480 compounds of known biological activity. From the initial proof-of-principle primary screen, eight compounds were identified as positive hits. A panel of secondary screens, including orthogonal assays, was used to validate the primary hits and eliminate false positives, and also to measure the comparative efficacy of the identified compounds. This cell-based assay, thus, yields consistent results applicable to the screening of larger libraries of compounds that can potentially reveal novel molecules which allow better recognition of treated tumors by T cells.

Dunn, Ian S.; Rose, Lenora B.; Newton, Estelle E.; Kurnick, James T.



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

PubMed Central

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.

McKim, James M



Development and validation of a high-throughput screening assay for human long-chain fatty acid transport proteins 4 and 5.  


Dietary long-chain fatty acid (LCFA) uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). Six subtypes of this transporter are differentially expressed throughout the human and rodent body. To facilitate drugs discovery against FATP subtypes, the authors used mammalian cell lines stably expressing the recombinant human FATP4 and 5 and developed a high-throughput screening (HTS) assay using a 96-well fluorometric imaging plate reader (FLIPR). LCFA uptake signal-to-background ratios were between 3- and 5-fold. Two 4-aryl-dihydropyrimidinones, j3 and j5, produced inhibition of FATP4 with a half-maximal inhibitory concentration (IC(50)) of 0.21 and 0.63 microM, respectively, and displayed approximately 100-fold selectivity over FATP5. The US Drug Collection library was screened against the FATP5. A hit rate of around 0.4% was observed with a Z' factor of 0.6 +/- 0.2. Two confirmed hits are bile acids, chenodiol and ursodiol with an IC(50) of 2.4 and 0.22 microM, respectively. To increase throughput, a single time point measurement in a 384-well format was developed using the Analyst HT, and the results are comparable with the 96-well format. In conclusion, the FATP4 and 5 cell-based fluorescence assays are suitable for a primary drug screen, whereas differentiated cell lines are useful for a secondary drug screen. PMID:20448275

Zhou, Wei; Madrid, Peter; Fluitt, Amy; Stahl, Andreas; Xie, Xinmin Simon



Chemical and biological stability of anticancer drugs used in a human tumor clonogenic assay  

Microsoft Academic Search

Human tumor clonogenic assays (HTCA) are being used to evaluate the chemosensitivity of human cancers to both standard and experimental anticancer drugs as well as to predict clinical tumor response and resistance to these agents. To enable us to design and accurately interpret drug assay data we quantitated the chemical and biological stability characteristics of various cell cycle-specific and cell

Ruth Ludwig; David S. Alberts



Pharmacologic studies of anticancer drugs with the human tumor stem cell assay  

Microsoft Academic Search

To optimize the human tumor stem cell assay (HTSCA) for clinical and research purposes we have carried out in vitro pharmacology studies. Useful observations were made in four areas. (1) Drug assay design: The predictive accuracy of the HTSCA depends on the in vitro testing of drug concentrations of less than 10% of those which are pharmacologically achievable with standard

David S. Alberts; Sydney E. Salmon; H.-S. George Chen; Thomas E. Moon; Laurie Young; Earl A. Surwit



Progress toward virtual screening for drug side effects.  


The development and application of a computational protocol for conducting virtual screens of drug side interactions is described. A conventional drug-docking algorithm (AutoDock) is used to conduct two separate studies. First, a series of docking simulations is performed by using guanosine diphosphate and adenosine diphosphate as prototype drugs with the goal of successfully differentiating known receptors from a large number of bait receptors. Using the electrostatic energy of the purine ring as a basis for discrimination allows the correct identification of receptors in blind studies with 100% specificity and 94% sensitivity. In a second study, similar methodology is used to investigate the binding of clinically relevant inhibitors (Gleevec, purvalanol A, and hymenialdisine) to a variety of protein kinase targets. Overall, excellent agreement between experimental and predicted preferences for kinase targets is obtained. An important conclusion from the latter study is that homology-modeled structures of putative receptors may reasonably be used as targets for docking when directly solved crystal structures are not available. The prospects for routine application of the methodology as a means of identifying potential side interactions of candidate drugs are discussed. PMID:12211034

Rockey, William M; Elcock, Adrian H



Attitudes of Matriculating First-Year Pharmacy Students Toward a Mandatory, Random Drug-Screening Program  

PubMed Central

Objective. To determine the attitudes of incoming pharmacy students toward a mandatory, random urine drug-screening program. Methods. This was an anonymous, voluntary survey of students at the McWhorter School of Pharmacy (MSOP) using an instrument composed of 40 items. The instrument was administered during orientation week prior to the session during which the policies and procedures of MSOP's drug-screening program were to be discussed. Results. The survey instrument was completed by all 129 (100%) students in the class. Two-thirds of the students were aware of MSOP's drug-screening program prior to applying, but only a few felt uneasy about applying to the school because of the program. The greatest concerns expressed by the students included what would happen if a student unintentionally missed a drug screen or was busy with other matters when called for screening, how much time a drug-screening would take, and the possibility of false-positive drug screen results. The vast majority of students agreed with statements regarding the potential benefits of drug testing. Students who consumed alcohol in a typical week and those with current or past use of an illegal substance held less favorable attitudes toward MSOP’s mandatory drug-screening program compared with students who did not share those characteristics. Conclusion. Although there were definite concerns expressed regarding pragmatic issues surrounding drug screening, the first-year pharmacy students held generally favorable opinions about the school's mandatory drug-screening program.

Oliver, Maggee; Hogue, Michael D.; Alverson, Susan P.; Woolley, Thomas W.



High-throughput screening for Kv1.3 channel blockers using an improved FLIPR-based membrane-potential assay.  


Voltage-gated K(+) channels are potential drug targets for an increasing number of disease indications. Searching for compounds that modulate K(+) channel activities by high-throughput screening (HTS) is becoming a standard approach in the drug discovery effort. Here the authors report an improved fluorometric imaging plate reader (FLIPR) membrane potential assay for Kv1.3 K(+) channel HTS. They have found that the Chinese hamster ovary (CHO) cells have endogenous membrane electrogenic transporters that contribute to maintaining membrane potential. Blocking the recombinant K(+) channels in the overexpressing CHO cell line hardly changed the membrane potential. Inhibition of the endogenous transporters is essential to achieve the required assay robustness. The authors identified the optimal assay conditions and designed a simple assay format. After an HTS campaign using this assay, various chemical series of Kv1.3 channel blockers have been identified and confirmed by the automated electrophysiological IonWorks assay. The correlation in dose response between FLIPR and IonWorks was established by biophysical modeling and experimental data. After characterization using patch-clamp recording, both use-dependent and use-independent compounds were identified. Some compounds possess nanomolar potency, indicating that the FLIPR assay is effective for successfully identifying K(+) channel blockers as novel drug candidates. PMID:20044579

Liu, Kun; Samuel, Manoj; Tillett, Jeff; Hennan, James K; Mekonnen, Belew; Soloveva, Veronica; Harrison, Richard K; Paslay, Jeff W; Larocque, James



Drug screening to identify suppressors of GFAP expression  

PubMed Central

Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein of astrocytes in the vertebrate central nervous system. Increased levels of GFAP are the hallmark feature of gliosis, a non-specific response of astrocytes to a wide variety of injuries and disorders of the CNS, and also occur in Alexander disease where the initial insult is a mutation within the coding region of GFAP itself. In both settings, excess GFAP may cause or exacerbate astrocyte dysfunction. With the goal of finding drugs that reduce the expression of GFAP, we have devised screens to detect changes in GFAP promoter activity or protein levels in primary cultures of mouse astrocytes in a 96-well format. We have applied these screens to libraries enriched in compounds that are already approved for human use by the FDA. We report that several compounds are active at micromolar levels in suppressing the expression of GFAP. Treatment of mice for 3 weeks with one of these drugs, clomipramine, causes nearly 50% reduction in the levels of GFAP protein in brain.

Cho, Woosung; Brenner, Michael; Peters, Noel; Messing, Albee



Development of a Cell-Based, High-Throughput Screening Assay for Cholesterol Efflux Using a Fluorescent Mimic of Cholesterol  

PubMed Central

Abstract Reverse cholesterol transport is the process by which extrahepatic cells, including macrophage-derived foam cells in arterial atherosclerotic plaque, transport excessive cholesterol back to the liver for bile acid synthesis and excretion, thus lowering the peripheral lipid burden. Cholesterol efflux from peripheral cells is the first step in this process, and finding drugs and interventions that promote this event is an important endeavor. Radioisotope-labeled cholesterol traditionally has been employed in measuring efflux efficiency, but this reagent has limitations for high-throughput screening. We developed an alternative method to measure cholesterol efflux in macrophage-derived foam cells using a novel fluorescent cholesterol mimic comprising the Pennsylvania Green fluorophore, attached by a linker containing a glutamic acid residue, to a derivative of N-alkyl-3?-cholesterylamine. Compared with the traditional radioisotope-based assay, this fluorescence-based assay gave similar results in the presence of known modulators of cholesterol efflux, such as cyclic AMP, and different cholesterol acceptors. When the fluorescent probe was employed in a high-throughput screening format, a variety of chemicals and bioactive compounds with known and unknown effects on cholesterol efflux could be tested simultaneously by plate-reader in a short period of time. Treatment of THP-1-derived macrophages with inhibitors of the membrane transporter ATP-binding cassette A1, such as glyburide or a specific antibody, significantly reduced the export of this fluorescent compound, indicating that ATP-binding cassette A1 represents the primary mediator of its cellular efflux. This fluorescent mimic of cholesterol provides a safe, sensitive, and reproducible alternative to radioactive assays in efflux experiments and has great potential as a valuable tool when incorporated into a drug discovery program.

Zhang, Jun; Cai, Sutang; Peterson, Blake R.; Kris-Etherton, Penny M.



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

PubMed Central

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

Kellam, P; Larder, B A



Development of novel, 384-well high-throughput assay panels for human drug transporters: drug interaction and safety assessment in support of discovery research.  


Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities. PMID:24062352

Tang, Huaping; Shen, Ding Ren; Han, Yong-Hae; Kong, Yan; Balimane, Praveen; Marino, Anthony; Gao, Mian; Wu, Sophie; Xie, Dianlin; Soars, Matthew G; O'Connell, Jonathan C; Rodrigues, A David; Zhang, Litao; Cvijic, Mary Ellen



An antioxidant screening assay based on oxidant-induced growth arrest in Saccharomyces cerevisiae.  


This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants. PMID:21375688

Wu, Ming J; O'Doherty, Patrick J; Fernandez, Harvey R; Lyons, Victoria; Rogers, Peter J; Dawes, Ian W; Higgins, Vincent J



Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay.  


Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world. PMID:23482387

Bell, Sara C; Alford, Ross A; Garland, Stephen; Padilla, Gabriel; Thomas, Annette D



Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents  

PubMed Central

Prion diseases such as Creutzfeldt–Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP–FRET-enabled high throughput assay (PrP–FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood–brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform.

Karapetyan, Yervand Eduard; Sferrazza, Gian Franco; Zhou, Minghai; Ottenberg, Gregory; Spicer, Timothy; Chase, Peter; Fallahi, Mohammad; Hodder, Peter; Weissmann, Charles; Lasmezas, Corinne Ida



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


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

Mashalidis, Ellene H; Sled?, Pawe?; Lang, Steffen; Abell, Chris



Development and Validation of a High-Throughput Screening Assay for Human Long-Chain Fatty Acid Transport Proteins 4 and 5  

Microsoft Academic Search

Dietary long-chain fatty acid (LCFA) uptake across cell membranes is mediated principally by fatty acid transport proteins (FATPs). Six subtypes of this transporter are differentially expressed throughout the human and rodent body. To facilitate drugs discovery against FATP subtypes, the authors used mammalian cell lines stably expressing the recombinant human FATP4 and 5 and developed a high-throughput screening (HTS) assay

Wei Zhou; Peter Madrid; Amy Fluitt; Andreas Stahl; Xinmin Xie



Effects of Genetic Mutations and Chemical Exposures on Caenorhabditis elegans Feeding: Evaluation of a Novel, High-Throughput Screening Assay  

PubMed Central

Background Government agencies have defined a need to reduce, refine or replace current mammalian-based bioassays with testing methods that use alternative species. Invertebrate species, such as Caenorhabditis elegans, provide an attractive option because of their short life cycles, inexpensive maintenance, and high degree of evolutionary conservation with higher eukaryotes. The C. elegans pharynx is a favorable model for studying neuromuscular function, and the effects of chemicals on neuromuscular activity, i.e., feeding. Current feeding methodologies, however, are labor intensive and only semi-quantitative. Methodology/Principal Findings Here a high-throughput assay is described that uses flow cytometry to measure C. elegans feeding by determining the size and intestinal fluorescence of hundreds of nematodes after exposure to fluorescent-labeled microspheres. This assay was validated by quantifying fluorescence in feeding-defective C. elegans (eat mutants), and by exposing wild-type nematodes to the neuroactive compounds, serotonin and arecoline. The eat mutations previously determined to cause slow pumping rates exhibited the lowest feeding levels with our assay. Concentration-dependent increases in feeding levels after serotonin exposures were dependent on food availability, while feeding levels decreased in arecoline-exposed nematodes regardless of the presence of food. The effects of the environmental contaminants, cadmium chloride and chlorpyrifos, on wild-type C. elegans feeding were then used to demonstrate an application of the feeding assay. Cadmium exposures above 200 µM led to a sharp drop in feeding levels. Feeding of chlorpyrifos-exposed nematodes decreased in a concentration-dependent fashion with an EC50 of 2 µM. Conclusions/Significance The C. elegans fluorescence microsphere feeding assay is a rapid, reliable method for the assessment of neurotoxic effects of pharmaceutical drugs, industrial chemicals or environmental agents. This assay may also be applicable to large scale genetic or RNAi screens used to identify genes that are necessary for the development or function of the pharynx or other neuromuscular systems.

Boyd, Windy A.; McBride, Sandra J.; Freedman, Jonathan H.



Exploration of novel in vitro assays to study drugs against Trichuris spp.  


Though trichuriasis is a significant public health problem, few effective drugs are available underscoring the need for new drug therapies. For the evaluation of trichuricidal activity of test compounds in vitro an accurate, reliable, sensitive, fast and cheap drug sensitivity assay is essential. The aim of the present investigation was to evaluate the performance of different in vitro drug sensitivity assays in comparison to the standard motility assay. Trichuris muris L4 larvae or adult worms were isolated from the intestinal tract from infected female C57BL/10 mice and incubated in the presence of ivermectin, levamisole and nitazoxanide (200, 100 and 50 ?g/ml) for 72 h. The health status of the worms was either evaluated microscopically using a motility scale from 0 to 3 (motility assay), by examination of absorbance or emission in response to metabolic activity (MTT (Thiazolyl Blue Tetrazolium Bromide) and Alamar Blue assay), through analysis of absorbance of an enzyme-substrate reaction (acid phosphatase activity assay), by measuring the noise amplitudes (isothermal microcalorimetry and xCELLigence System) or the heat flow (isothermal microcalorimetry) of T. muris. The Alamar Blue assay, xCELLigence and microcalorimetry compared favorably to the standard motility assay. These three assays precisely determined the trichuricidal activity of the three test drugs. The acid phosphatase and the MTT assays showed a poorer performance than the motility assay. In conclusion, the colorimetric Alamar Blue in vitro assay is a good alternative to the motility assay to study drug effects against T. muris L4 and adults, since it is easy to perform, precise and of low cost. PMID:21889548

Silbereisen, Angelika; Tritten, Lucienne; Keiser, Jennifer



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


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

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



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


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

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



Time-schedule dependency of the inhibiting activity of various anticancer drugs in the clonogenic assay  

Microsoft Academic Search

To analyze the discrepancy between the in vitro response in the clonogenic assay and the clinical response, the time-schedule dependencies of various anticancer drugs were determined by comparing the inhibiting effect against colony formation by PC-7 cells treated with the drugs for 1 h with that of those treated for 24h. According to their schedule dependency the drugs can be

Yuka Matsushima; Fumihiko Kanzawa; Akio Hoshi; Eiji Shimizu; Hiroaki Nomori; Yasutsuna Sasaki; Nagahiro Saijo



Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium.  

PubMed Central

In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.

Collins, L; Franzblau, S G



[Problem of screening of antineoplastic drugs in tissue and cell cultures].  


Problems on the anticancer drugs' screening in vitro are reviewed on the basis of generalized data from literature. The probability to screen in vitro the active substances is not less than the chance to screen the drugs active against the human tumours in experiments on animals. The use of special test-systems permits screening specific substances including those inducing the tumour cell differentiation. PMID:4006851

Dobrynin, Ia V



Affordable luciferase reporter assay for cell-based high-throughput screening.  


The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z' factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS. PMID:23112084

Siebring-van Olst, Ellen; Vermeulen, Christie; de Menezes, Renee X; Howell, Michael; Smit, Egbert F; van Beusechem, Victor W



Dictyostelium discoideum developmental cycle (DDDC) assay: a tool for Hg toxicity assessment and soil health screening.  


The social amoeba Dictyostelium discoideum has been proposed for assessing stress responses to pollutants in soil and it has already been successfully employed in the aquatic environment. Presently, we developed the DDDC assay (D. discoideum developmental cycle assay) for both soil toxicity assessment and soil health screening. The DDDC assay is primarily aimed at determining the capacity of D. discoideum to undergo its developmental programme forming a fruiting body, measured in terms of fruiting body formation inhibition and fruiting body size factor, which may be considered an indication of its ecological fitness (potential for spore dispersal). A second objective of the solid phase DDDC assay is to identify potential mechanisms of toxic action on the developmental cycle, for which three checkpoints are examined: (a) aggregation arrest, (b) migration arrest, and (c) culmination arrest. Presently, conditions for the DDDC assay such as soil texture, soil water content, soil pH, food availability and incubation time were investigated and optimized. In addition, both solid and liquid phase variants of the DDDC assay were applied to assess the toxicity of Hg, at regulatory concentrations. The developmental cycle and ecological fitness were affected from the exposure to 0.3 mg Hg/kg dry-wt soil onwards. The DDDC assay has been shown to be a high sensitivity test. PMID:23454908

Rodríguez-Ruiz, Amaia; Marigómez, Ionan; Boatti, Lara; Viarengo, Aldo



Drug screening in a zebrafish model of Duchenne muscular dystrophy  

PubMed Central

Two known zebrafish dystrophin mutants, sapje and sapje-like (sapc/100), represent excellent small-animal models of human muscular dystrophy. Using these dystrophin-null zebrafish, we have screened the Prestwick chemical library for small molecules that modulate the muscle phenotype in these fish. With a quick and easy birefringence assay, we have identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish without restoration of dystrophin expression. Three of seven candidate chemicals restored normal birefringence and increased survival of dystrophin-null fish. One chemical, aminophylline, which is known to be a nonselective phosphodiesterase (PDE) inhibitor, had the greatest ability to restore normal muscle structure and up-regulate the cAMP-dependent PKA pathway in treated dystrophin-deficient fish. Moreover, other PDE inhibitors also reduced the percentage of affected sapje fish. The identification of compounds, especially PDE inhibitors, that moderate the muscle phenotype in these dystrophin-null zebrafish validates the screening protocol described here and may lead to candidate molecules to be used as therapeutic interventions in human muscular dystrophy.

Kawahara, Genri; Karpf, Jeremy A.; Myers, Jennifer A.; Alexander, Matthew S.; Guyon, Jeffrey R.; Kunkel, Louis M.



Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis  

Microsoft Academic Search

Screening for new antimicrobial agents is routinely conducted only against actively replicating bacteria. However, it is now widely accepted that a physiological state of nonreplicating persistence (NRP) is responsible for antimicrobial tolerance in many bacterial infections. In tuberculosis, the key to shortening the 6-month regimen lies in targeting this NRP subpopulation. Therefore, a high-throughput, luminescence-based low- oxygen-recovery assay (LORA) was

Sang Hyun Cho; Saradee Warit; Baojie Wan; Chang Hwa Hwang; Guido F. Pauli; Scott G. Franzblau



A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening  

Microsoft Academic Search

A novel fluorescent assay to continuously monitor fatty acid amide hydrolase (FAAH) activity that is simple, sensitive, and amenable to high-throughput screening (HTS) of compound libraries is described in this article. Stable Chinese hamster ovary (CHO) cell lines expressing either human FAAH or an inactive mutant, FAAH-S241A, were established. Arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a novel fluorogenic substrate for

Manjunath K. Ramarao; Elizabeth A. Murphy; Marina W. H. Shen; Yuren Wang; Kristen N. Bushell; Nelson Huang; Ning Pan; Cara Williams; James D. Clark



A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase.  


Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent V(max)=2.31 pmol min(-1)U(-1). Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery. PMID:20561598

Mina, John G; Mosely, Jackie A; Ali, Hayder Z; Shams-Eldin, Hosam; Schwarz, Ralph T; Steel, Patrick G; Denny, Paul W



Therapeutic Drug Monitoring: A High-Throughput Assay Using ...  

Center for Biologics Evaluation and Research (CBER)

... Assay Using Liquid Chromatography-Tandem Mass Spectrometry for Simultaneous in vivo Phenotyping of 5 Major Cytochrome p450 Enzymes in ... More results from


Validation of a high-content screening assay using whole-well imaging of transformed phenotypes.  


Automated microscopy was introduced two decades ago and has become an integral part of the discovery process as a high-content screening platform with noticeable challenges in executing cell-based assays. It would be of interest to use it to screen for reversers of a transformed cell phenotype. In this report, we present data obtained from an optimized assay that identifies compounds that reverse a transformed phenotype induced in NIH-3T3 cells by expressing a novel oncogene, KP, resulting from fusion between platelet derived growth factor receptor alpha (PDGFR?) and kinase insert domain receptor (KDR), that was identified in human glioblastoma. Initial image acquisitions using multiple tiles per well were found to be insufficient as to accurately image and quantify the clusters; whole-well imaging, performed on the IN Cell Analyzer 2000, while still two-dimensional imaging, was found to accurately image and quantify clusters, due largely to the inherent variability of their size and well location. The resulting assay exhibited a Z' value of 0.79 and a signal-to-noise ratio of 15, and it was validated against known effectors and shown to identify only PDGFR? inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFR? inhibitors as reversers of the KP induced transformed phenotype. In conclusion, our optimized and validated assay using whole-well imaging is robust and sensitive in identifying compounds that reverse the transformed phenotype induced by KP with a broader applicability to other cell-based assays that are challenging in HTS against chemical and RNAi libraries. PMID:21182456

Ramirez, Christina N; Ozawa, Tatsuya; Takagi, Toshimitsu; Antczak, Christophe; Shum, David; Graves, Robert; Holland, Eric C; Djaballah, Hakim



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


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

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



Assays for high-throughput screening of E2 AND E3 ubiquitin ligases.  


We developed a series of assays for biochemical activities involving ubiquitin. These assays use electrochemiluminescence detection to measure the ubiquitylation of target proteins. To enable electrochemiluminescence detection, the target proteins were prepared as bacterially expressed fusion proteins and captured on the surface of specially designed microtiter plates having integrated electrodes. Ubiquitylation was quantitated directly, through the use of ubiquitin labeled with an electrochemiluminescent label, or indirectly, through the use of labeled antiubiquitin antibodies. Assays were carried out in both 96-well and 384-well plates. The success of the assay with this variety of formats allowed the selection of optimal work flows for specific applications on the basis of ease of use and overall reagent consumption and availability. We used our ubiquitylation assays to measure the activities of E2 ubiquitin-conjugating enzymes and E3 class ubiquitin ligases. Signal/background ratios for many of our assays were greater than 50, significantly facilitating their conversion to high-throughput practice in a convenient manner. The speed, sensitivity, and convenience of the assay formats makes them well suited for comprehensive interrogations of libraries of compounds or genes in applications like drug and substrate discovery for ubiquitin ligases. PMID:16338389

Kenten, John H; Davydov, Ilia V; Safiran, Yassamin J; Stewart, David H; Oberoi, Pankaj; Biebuyck, Hans A



Dot-immunogold filtration assay as a screening test for syphilis.  


A dot-immunogold filtration assay (DIGFA) for the rapid detection of reaginic antibody in the serum of syphilitic patients was developed. The assay was simple, rapid, and reproducible. The test completion time was 2 min, and the assay required no equipment. The positive dot was very obvious, and the results could easily be determined with the naked eye. A total of 350 serum samples were examined by DIGFA, the rapid plasma reagin test, and the fluorescent treponemal antibody-absorption test. The levels of agreement between DIGFA and the rapid reagin test and between DIGFA and the fluorescent treponemal antibody-absorption test were 100 and 98%, respectively. The results of clinical application indicated that DIGFA could be used as a routine screening test for syphilis. PMID:8818901

Huang, Q; Lan, X; Tong, T; Wu, X; Chen, M; Feng, X; Liu, R; Tang, Y; Zhu, Z



High-throughput screening with a miniaturized radioligand competition assay identifies new modulators of human ?2-adrenoceptors.  


Human ?(2)-adrenoceptors (?(2)-ARs) are rhodopsin-like G-protein coupled receptors, and potential drug targets. The three different human ?(2)-AR subtypes ?(2A), ?(2B) and ?(2C) are widely distributed in tissues, but so far only a few subtype-selective ligands have been identified. In this project, we set off to conduct a large chemical screen for activity on the human ?(2B)-AR and studied the selectivity of the active compounds towards the human ?(2A)- and ?(2C)-AR subtypes. We employed a radioligand competition binding assay that was optimized and miniaturized into a robotic environment. Membrane fractions containing recombinant human receptor subtypes were prepared from stably transfected Chinese hamster ovary (CHO) cell lines. Initially identified hits were followed up and characterized, and chemoinformatics tools were applied to gain better understanding of the relevance of the results. After a primary screen against ?(2B)-AR, 176 compounds of the 17,952 included in the library were declared as active at 10 ?M, of which 89 compounds were further selected for potency and affinity determinations using the three human ?(2)-AR subtypes. One of the identified positive hits was 2?,2??-Bisepigallocatechin digallate, which was found to have high affinity at all three human ?(2)-AR subtypes. This represents the first non-protonable molecule identified as able to interact with these receptors. Additionally, results obtained with a functional assay (agonist-induced stimulation of [(35)S]GTP?S binding) supported the identification of another positive hit, lysergol, as a partial agonist of the human ?(2)-AR subtypes. The dataset of confirmed active chemical species represents a readily available, high quality source for follow-up studies. Altogether, these results provide novel research approaches for drug discovery of modulators of the ?(2)-AR subtypes. PMID:22982401

Fallarero, Adyary; Pohjanoksa, Katariina; Wissel, Gloria; Parkkisenniemi-Kinnunen, Ulla-Mari; Xhaard, Henri; Scheinin, Mika; Vuorela, Pia



Modification of screening immunoassays to detect sub-threshold concentrations of cocaine, cannabinoids, and opiates in urine: use for detecting maternal and neonatal drug exposures.  


Testing for drugs of abuse in urine is commonplace in emergency departments and neonatal units. However, the clinical sensitivity of immunochemical screening methods is limited by the threshold concentrations used to distinguish between positive and negative specimens. Immunochemical screening methods for cocaine metabolite (benzoylecgonine), cannabinoids, and opiates in urine were recalibrated to detect drugs at lower threshold concentrations. The precision and linearity of the signals at the modified thresholds were verified by diluting drug-positive urine specimens to concentrations below the conventional cutoff concentration and measuring the rate signals in triplicate. To assess the clinical performance of the modified methods, specimens that tested negative using the unmodified assays were re-screened at the lower threshold, and specimens that re-screened positive were submitted for gas chromatographic/mass spectrometric (GC/MS) confirmation. Reproducibility of sub-threshold measurements was comparable to the unmodified assays, and rate separations between successive dilutions were sufficient to give semi-quantitative results. Using the lower thresholds, drugs were detected in 4-5% of the subjects that had screened negative at the conventional threshold concentration. GC/MS analysis confirmed the presence of cannabinoids and cocaine metabolite in 74% and 84%, respectively, of urine specimens that re-screened positive. Morphine, codeine, hydromorphone, or hydrocodone was detected by GC/MS analysis in 31% of opiate-positive re-screens. PMID:10678589

Hattab, E M; Goldberger, B A; Johannsen, L M; Kindland, P W; Ticino, F; Chronister, C W; Bertholf, R L



Automated High-Content Live Animal Drug Screening Using C. elegans Expressing the Aggregation Prone Serpin ?1-antitrypsin Z  

PubMed Central

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 ?1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling ?1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.

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.



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

SciTech Connect

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.

Krepinsky, A. (Bio-Mutatech Inc., Toronto, Quebec (Canada)); Bryant, D.W.; Davison, L.; McCalla, D.R. (McMaster Univ. Health Sciences Centre, Hamilton, Ontario (Canada)); Young, B. (Toronto General Hospital, Quebec (Canada)); Heddle, J. (York Univ., Toronto, Quebec (Canada)); Douglas, G. (Health and Welfare Canada, Ottawa, Ontario (Canada)); Michalko, K. (Drugs Directorate, Ottawa, Ontario (Canada))



Establishment of an In Vitro Assay for Assessing the Effects of Drugs on the Liver Stages of Plasmodium vivax Malaria  

PubMed Central

Plasmodium vivax (Pv) is the second most important human malaria parasite. Recent data indicate that the impact of Pv malaria on the health and economies of the developing world has been dramatically underestimated. Pv has a unique feature in its life cycle. Uninucleate sporozoites (spz), after invasion of human hepatocytes, either proceed to develop into tens of thousands of merozoites within the infected hepatocytes or remain as dormant forms called hypnozoites, which cause relapses of malaria months to several years after the primary infection. Elimination of malaria caused by Pv will be facilitated by developing a safe, highly effective drug that eliminates Pv liver stages, including hypnozoites. Identification and development of such a drug would be facilitated by the development of a medium to high throughput assay for screening drugs against Pv liver stages. We undertook the present pilot study to (1) assess the feasibility of producing large quantities of purified, vialed, cryopreserved Pv sporozoites and (2) establish a system for culturing the liver stages of Pv in order to assess the effects of drugs on the liver stages of Pv. We used primaquine (PQ) to establish this assay model, because PQ is the only licensed drug known to clear all Pv hepatocyte stages, including hypnozoites, and the effect of PQ on Pv hepatocyte stage development in vitro has not previously been reported. We report that we have established the capacity to reproducibly infect hepatoma cells with purified, cyropreserved Pv spz from the same lot, quantitate the primary outcome variable of infected hepatoma cells and demonstrate the inhibitory activity of primaquine on the infected hepatoma cells. We have also identified small parasite forms that may be hypnozoites. These data provide the foundation for finalizing a medium throughput, high content assay to identify new drugs for the elimination of all Pv liver stages.

Chattopadhyay, Rana; Velmurugan, Soundarapandian; Chakiath, Chinnamma; Andrews Donkor, Lucy; Milhous, Wilbur; Barnwell, John W.; Collins, William E.; Hoffman, Stephen L.



21 CFR 862.3645 - Neuroleptic drugs radioreceptor assay test system.  

Code of Federal Regulations, 2013 CFR

...DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Toxicology Test Systems § 862.3645 Neuroleptic drugs radioreceptor assay test system. (a)...



Activity-based assay for human mono-ADP-ribosyltransferases ARTD7/PARP15 and ARTD10/PARP10 aimed at screening and profiling inhibitors.  


Poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin like ADP-ribosyl transferases (ARTDs) are enzymes that catalyze the covalent modification of proteins by attachment of ADP-ribose units to the target amino acid residues or to the growing chain of ADP-ribose. A subclass of the ARTD superfamily consists of mono-ADP-ribosyl transferases that are thought to modify themselves and other substrate proteins by covalently adding only a single ADP-ribose moiety to the target. Many of the ARTD enzymes are either established or potential drug targets and a functional activity assay for them will be a valuable tool to identify selective inhibitors for each enzyme. Existing assays are not directly applicable for screening of inhibitors due to the different nature of the reaction and different target molecules. We modified and applied a fluorescence-based assay previously described for PARP1/ARTD1 and tankyrase/ARTD5 for screening of PARP10/ARTD10 and PARP15/ARTD7 inhibitors. The assay measures the amount of NAD(+) present after chemically converting it to a fluorescent analog. We demonstrate that by using an excess of a recombinant acceptor protein the performance of the activity-based assay is excellent for screening of compound libraries. The assay is homogenous and cost effective, making it possible to test relatively large compound libraries. This method can be used to screen inhibitors of mono-ARTDs and profile inhibitors of the enzyme class. The assay was optimized for ARTD10 and ARTD7, but it can be directly applied to other mono-ARTDs of the ARTD superfamily. Profiling of known ARTD inhibitors against ARTD10 and ARTD7 in a validatory screening identified the best inhibitors with submicromolar potencies. Only few of the tested ARTD inhibitors were potent, implicating that there is a need to screen new compound scaffolds. This is needed to create small molecules that could serve as biological probes and potential starting points for drug discovery projects against mono-ARTDs. PMID:23485441

Venkannagari, Harikanth; Fallarero, Adyary; Feijs, Karla L H; Lüscher, Bernhard; Lehtiö, Lari



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

Microsoft Academic Search

Background  High-throughput screening (HTS) is one of the main strategies to identify novel entry points for the development of small\\u000a molecule chemical probes and drugs and is now commonly accessible to public sector research. Large amounts of data generated\\u000a in HTS campaigns are submitted to public repositories such as PubChem, which is growing at an exponential rate. The diversity\\u000a and quantity

Ubbo Visser; Saminda Abeyruwan; Uma Vempati; Robin P Smith; Vance Lemmon; Stephan C Schürer



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

SciTech Connect

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.

Taxvig, Camilla, E-mail:; Olesen, Pelle Thonning; Nellemann, Christine



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


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

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



Apparent Activity in High-Throughput Screening: Origins of Compound-Dependent Assay Interference  

PubMed Central

Summary of recent advances Expansive compound collections made up of structurally heterogeneous chemicals, the activities of which are largely undefined, present challenging problems for high-throughput screening (HTS). Foremost is differentiating whether the activity for a given compound in an assay is directed against the targeted biology, or is the result of surreptitious compound activity involving the assay detection system. Such compound interference can be especially difficult to identify if it is reproducible and concentration-dependent – characteristics generally attributed to compounds with genuine activity. While reactive chemical groups on compounds were once thought to be the primary source of compound interference in assays used in HTS, recent work suggests that other factors, such as compound aggregation, may play a more significant role in many assay formats. Considerable progress has been made to profile representative compound libraries in an effort to identify chemical classes susceptible to producing compound interference, such as compounds commonly found to inhibit the reporter enzyme firefly luciferase. Such work has also led to the development of practices that have the potential to significantly reduce compound interference, for example, through the addition of non-ionic detergent to assay buffer to reduce aggregation-based inhibition.

Thorne, Natasha; Auld, Douglas S.; Inglese, James



Screening for drugs of abuse in hair with ion spray LC-MS-MS  

Microsoft Academic Search

Analyzing hair for many substances can be tedious and expensive, and a rapid screening method should prove helpful. Generally, screening has been performed using immunological tests, mainly in workplace drug testing, where the number of samples has been high. The aim of this study was to develop an LC-MS-MS method for the simultaneous analysis of several drugs of abuse in

Robert Kronstrand; Ingrid Nystrom; Joakim Strandberg; Henrik Druid


Shortcomings of Urine-Preferred Drug Screening on Post-Mortem Specimens  

Microsoft Academic Search

In counties with limited budgets, in order to save money on toxicology work, the request often comes from local medical examiners that screening for drugs on decedents be performed initially on urine and, if positive, to send blood for confirmation; negative urine results are not further evaluated. A study of known urine and blood drug screens was performed to evaluate

Henry J. Carson; Mary H. Dudley; Steven W. Fleming; Donald J. Linder



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

ERIC Educational Resources Information Center

|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…

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



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

SciTech Connect

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.

Varesco, L.; Gismondi, V.; Bafico, A. [Istituto Nazionale Ricerca sul Cancro, Genova (Italy)] [and others



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

PubMed Central

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 ?4?1-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 ?1-integrin and that derived from the first ? 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.



The Trade-Off between Accuracy and Accessibility of Syphilis Screening Assays  

PubMed Central

The availability of rapid and sensitive methods to diagnose syphilis facilitates screening of pregnant women, which is one of the most cost-effective health interventions available. We have evaluated two screening methods in Tanzania: an enzyme immunoassay (EIA), and a point-of-care test (POCT). We evaluated the performance of each test against the Treponema pallidum particle agglutination assay (TPPA) as the reference method, and the accessibility of testing in a rural district of Tanzania. The POCT was performed in the clinic on whole blood, while the other assays were performed on plasma in the laboratory. Samples were also tested by the rapid plasma Reagin (RPR) test. With TPPA as reference assay, the sensitivity and specificity of EIA were 95.3% and 97.8%, and of the POCT were 59.6% and 99.4% respectively. The sensitivity of the POCT and EIA for active syphilis cases (TPPA positive and RPR titer ?1/8) were 82% and 100% respectively. Only 15% of antenatal clinic attenders in this district visited a health facility with a laboratory capable of performing the EIA. Although it is less sensitive than EIA, its greater accessibility, and the fact that treatment can be given on the same day, means that the use of POCT would result in a higher proportion of women with syphilis receiving treatment than with the EIA in this district of Tanzania.

Smit, Pieter W.; Mabey, David; Changalucha, John; Mngara, Julius; Clark, Benjamin; Andreasen, Aura; Todd, Jim; Urassa, Mark; Zaba, Basia; Peeling, Rosanna W.



The Trade-Off between Accuracy and Accessibility of Syphilis Screening Assays.  


The availability of rapid and sensitive methods to diagnose syphilis facilitates screening of pregnant women, which is one of the most cost-effective health interventions available. We have evaluated two screening methods in Tanzania: an enzyme immunoassay (EIA), and a point-of-care test (POCT). We evaluated the performance of each test against the Treponema pallidum particle agglutination assay (TPPA) as the reference method, and the accessibility of testing in a rural district of Tanzania. The POCT was performed in the clinic on whole blood, while the other assays were performed on plasma in the laboratory. Samples were also tested by the rapid plasma Reagin (RPR) test. With TPPA as reference assay, the sensitivity and specificity of EIA were 95.3% and 97.8%, and of the POCT were 59.6% and 99.4% respectively. The sensitivity of the POCT and EIA for active syphilis cases (TPPA positive and RPR titer ?1/8) were 82% and 100% respectively. Only 15% of antenatal clinic attenders in this district visited a health facility with a laboratory capable of performing the EIA. Although it is less sensitive than EIA, its greater accessibility, and the fact that treatment can be given on the same day, means that the use of POCT would result in a higher proportion of women with syphilis receiving treatment than with the EIA in this district of Tanzania. PMID:24066175

Smit, Pieter W; Mabey, David; Changalucha, John; Mngara, Julius; Clark, Benjamin; Andreasen, Aura; Todd, Jim; Urassa, Mark; Zaba, Basia; Peeling, Rosanna W



A high-throughput screening fluorescence polarization assay for fatty acid adenylating enzymes in Mycobacterium tuberculosis  

PubMed Central

Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), encodes for an astonishing 34 fatty acid adenylating enzymes (FadDs), which play key roles in lipid metabolism. FadDs involved in lipid biosynthesis are functionally nonredundant and serve to link fatty acid and polyketide synthesis to produce some of the most architecturally complex natural lipids including the essential mycolic acids as well as the virulence-conferring phthiocerol dimycocerosates, phenolic glycolipids, and mycobactins. Here we describe the systematic development and optimization of a fluorescence polarization assay to identify small molecule inhibitors as potential antitubercular agents. We fluorescently labeled a bisubstrate inhibitor to generate a fluorescent probe/tracer, which bound with a KD of 245 nM to FadD28. Next, we evaluated assay performance by competitive binding experiments with a series of known ligands and assessed the impact of control parameters including incubation time, stability of the signal, temperature, and DMSO concentration. As a final level of validation the LOPAC1280™ library was screened in a 384-well plate format and the assay performed with a Z-factor of 0.75 demonstrating its readiness for high-throughput screening.

Grimes, Kimberly D.; Aldrich, Courtney C.



Colorimetric, high-throughput assay for screening Angiotensin I-converting enzyme inhibitors.  


Angiotensin I-converting enzyme (ACE) plays a pivotal role in blood pressure regulation. A colorimetric assay to measure hippuric acid (HA) was transformed into a rapid ACE assay wherein the released HA from the substrate hippuryl-histidyl-leucine (HHL) is mixed with pyridine and benzene sulfonyl chloride. The resulting yellow color with a lambda(max) at 410 nm is directly proportional to the released HA. The limit of detection and limit of quantitation were 1.46 x 10(-7) and 4.43 x 10(-7) M HA. ACE activities of different tissues using this method were comparable to the standard high-performance liquid chromatography (HPLC) method. Kinetic studies showed a K(m) of 30.8 +/- 0.1 x 10(-6) M for HHL and V(max) of 1.3 +/- 0.01 x 10(-6) mol/min for porcine lung ACE. This assay coupled with captopril and lisinopril showed IC(50) values of 1.1 +/- 0.05 x 10(-9) and 2.5 +/- 0.03 x 10(-9) M, respectively. A 96-well microplate format of this method was used to screen the ACE inhibitory potential of peptides fractionated from an enzymatic hydrolysate of arachin. The precision, accuracy, reproducibility, and excellent correlation demonstrated between the colorimetric and the often-used HPLC method renders this extraction-free method a powerful tool for high-throughput screening of ACE inhibitors. PMID:19839596

Jimsheena, V K; Gowda, Lalitha R



Two high throughput screening assays for Aberrant RNA-protein interactions in Myotonic Dystrophy Type-1  

PubMed Central

Myotonic dystrophy type-1 (DM1), the most prevalent form of adult muscular dystrophy, is caused by expansion of a CTG repeat in the 3? untranslated region of the DM protein kinase (DMPK) gene. The pathogenic effects of the CTG expansion arise from the deleterious effects of the mutant transcript. RNA with expanded CUG tracts alters the activities of several RNA binding proteins, including muscleblind-like 1 (MBNL1). MBNL1 becomes sequestered in nuclear foci in complex with the expanded CUG repeat RNA. The resulting loss of MBNL1 activity causes mis-regulated alternative splicing of multiple genes, leading to symptoms of DM1. The binding interaction between MBNL1 and mutant RNA could be a key step in the pathogenesis of DM1 and serves as a potential target for therapeutic intervention. We have developed two high throughput screen (HTS) suitable assays using both homogenous time-resolved fluorescence energy transfer (HTRF) and AlphaScreen technologies to detect the binding of a C-terminally His-tagged MBNL1 and a biotinylated (CUG)12 RNA. These assays are homogenous and successfully miniaturized to 1536-well plate format. Both assays were validated and show robust signal-to-basal ratios and Z’ factors.

Chen, Catherine Z.; Sobczak, Krzysztof; Hoskins, Jason; Southall, Noel; Marugan, Juan J.; Zheng, Wei; Thornton, Charles A.; Austin, Christopher P.



A high-throughput screening fluorescence polarization assay for fatty acid adenylating enzymes in Mycobacterium tuberculosis.  


Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), encodes for an astonishing 34 fatty acid adenylating enzymes (FadDs), which play key roles in lipid metabolism. FadDs involved in lipid biosynthesis are functionally nonredundant and serve to link fatty acid and polyketide synthesis to produce some of the most architecturally complex natural lipids including the essential mycolic acids as well as the virulence-conferring phthiocerol dimycocerosates, phenolic glycolipids, and mycobactins. Here we describe the systematic development and optimization of a fluorescence polarization assay to identify small molecule inhibitors as potential antitubercular agents. We fluorescently labeled a bisubstrate inhibitor to generate a fluorescent probe/tracer, which bound with a K(D) of 245 nM to FadD28. Next, we evaluated assay performance by competitive binding experiments with a series of known ligands and assessed the impact of control parameters including incubation time, stability of the signal, temperature, and DMSO concentration. As a final level of validation the LOPAC1280 library was screened in a 384-well plate format and the assay performed with a Z-factor of 0.75, demonstrating its readiness for high-throughput screening. PMID:21771578

Grimes, Kimberly D; Aldrich, Courtney C



Development and Testing of an In vitro Assay for Screening of Potential Therapeutic Agents against Na Channel Neurotoxins.  

National Technical Information Service (NTIS)

The objective was to develop a rapid, reliable and simple screening assay of broad scope and sensitivity for applications in the identification of compounds with potential therapeutic value in the treatment of intoxication induced by a variety of sodium c...

G. B. Brown



Protocols for a Rapid Clean-up/Extraction Procedure and an Improved P45ORGS Dioxin Screening Assay for Sediments.  

National Technical Information Service (NTIS)

The purpose of this technical note is to describe protocols for performance of an improved biomarker-based screening assay for dioxin toxic equivalents (TCDD TEQs) in sediments and other environmental samples using a recombinant human hepatoma cell line. ...



Evaluation of the BBMEC model for screening the CNS permeability of drugs  

Microsoft Academic Search

Combinatorial synthesis and high-throughput pharmacology screening have greatly increased compound throughput in modern drug-discovery programs. For CNS drugs, it is also important to determine permeability to the blood–brain barrier. Yet, given the increased pace of discovery, it difficult to conduct this screen in a timely fashion. In this presentation, we describe several improvements to an existing CNS permeability screen, the

Kenneth W. Otis; Michael L. Avery; Suzanne M. Broward-Partin; Dannette K. Hansen; Herbert W. Behlow; Dennis O. Scott; Thomas N. Thompson



Universal Screening for Alcohol and Drug Use and Racial Disparities in Child Protective Services Reporting  

Microsoft Academic Search

This study examines racial disparities in Child Protective Services (CPS) reporting at delivery in a county with universal\\u000a screening for alcohol\\/drug use in prenatal care. It also explores two mechanisms through which universal screening could reduce\\u000a reporting disparities: Equitable Surveillance and Effective Treatment. Equitable Surveillance is premised on the assumptions that identification of drug use through screening in prenatal care

Sarah C. M. Roberts; Amani Nuru-Jeter


Two plate-based colorimetric assays for screening ?-amino acid ester hydrolase with high synthesis/hydrolysis ratio.  


?-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of ?-lactam antibiotics with ?-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of ?-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes' S/H ratio can be increased by protein engineering, such approaches require a suitable screening assay. Such a screening assay has not yet been described for AEHs. In this paper, we report a 96-well plate format screening procedure for AEHs based on two spectrophotometric assays. To reduce the hydrolysis reaction while maintaining synthesis activity, and to evaluate the effectiveness of the screening strategy, we introduced random mutations in part of the aeh gene from Xanthomonas rubrillineans by error-prone PCR. By a parallel plate-based screening strategy, three mutants with improved S/H ratio, R87L, T132N and N219I, were obtained. PMID:22664195

Wang, Lu; Ye, Li-Juan; Pan, Yue; Cao, Yi



Characterization of eptifibatide during drug formulation stability assays  

Microsoft Academic Search

The objective of the study was to determine the identity of a new impurity detected in HPLC chromatograms of research samples of eptifibatide manufactured by a new process and formulated into drug product. The identification of the unknown impurity was required in order to understand the mechanism of its formation. The analysis was performed by using tandem mass spectrometers coupled

Ronghua Wang; Debby Feder; Frank Hsieh



High Throughput and High Content Screening Capabilities of the University of Cincinnati Drug Discovery Center  

PubMed Central

The Drug Discovery Center collaborates with a wide range of academic and industrial research centers to facilitate the identification of active small molecules with high potential for use as biological probes or as starting points for drug discovery programs. The DDC operates state-of-the-art high throughput and high content screening instrumentation and a diverse 350,000 compound library. The center's personnel provide collaborators with advice in assay design, analytical technology selection, and library design via cheminformatics and/or structure-based approaches. Typical programs are exemplified by an HTS program targeting the identification of novel atypical PKC inhibitors for potential use in cancer and an HCS program targeting the identification of stimulators of the differentiation of oligodendrocyte progenitor cells to oligodendrocytes for potential use in multiple sclerosis. Activities of atypical PKCs, PKCiota and PKCzeta, were measured using both fluorescent detection of ADP production and MALDI-TOF detection of substrate phosphorylation. 30,000 compounds were screened for their effect on PKC activity using these orthogonal detection methods to identify potential inhibitors. Oligodendrocyte differentiation was measured in a High Content Screen by pairing a selective marker of oligodendrocytes with Alexa Fluor 488 secondary antibody for the detection of mature ODs. DAPI stain was used for nucleus detection. Confocal microscope images were acquired and analyzed using an algorithm for neurite outgrowth adapted for the characterization of oligodendrocyte processes. Three measurements: Mean Maximum Process Length per Cell, Mean Process Signal Intensity per Cell, and Percentage Differentiated Cells per Well, were quantified by the image analysis script and afforded a statistically significant separation of promoter controls and inhibitor controls from non-treated (neutral) controls with Mean Maximum Process Length as measurement.

Kirby, Jason; Tang, Hong; Najm, Fadi J.; Tesar, Paul J; Greis, Ken; Seibel, William; Papoian, Ruben; Rathore, Rakesh



An Automated Time-of-Drug-Addition Assay to Routinely Determine the Mode of Action of HIV-1 Inhibitors.  


Abstract Cell-based high-throughput screening campaigns are widely used to identify novel antiviral compounds, for example, against human immunodeficiency virus type 1 (HIV-1). Typically, these assays enable identification of compounds that potentially target any viral or cellular factor involved in the viral replication cycle. Unraveling the mechanism of action of these active compounds is an important step to facilitate further drug development. Time-of-addition (TOA) assays are an elegant tool to achieve this goal by comparing the TOA profile of novel compounds with those of well-studied reference compounds. Downscaling to a 384-well format and automation significantly increase the capacity of the TOA assay, enabling compound handling around the clock. Mechanical liquid dispensing with optimized time points for compound addition ensures robustness (Z?'>0.8) and maximal resolution in profiling novel antiviral compounds. The presented methodology has been optimized for routine use and allows for fully automated high-throughput screening to support the process in search for novel inhibitors of HIV-1. PMID:24144343

Van Loock, Marnix; Van den Eynde, Christel; Hansen, John; Geluykens, Peggy; Ivens, Tania; Sauviller, Sarah; Bunkens, Lieve; Van Acker, Koen; Nijs, Erik; Dams, Géry



A simple heterogeneous one-step assay for screening estrogenic compounds.  


Estrogen receptor (ER) modulators are a serious health issue but estrogenic compounds, especially antagonists of ER function, are widely screened for in search of novel therapeutics against hormonal diseases such as the breast cancer. Here we report a novel and a simple bioassay for estrogenic and anti-estrogenic compounds based on ligand-dependent recruitment of ER co-activator steroid receptor co-activator 1 (SRC-1) to purified Renilla luciferase-tagged ER?. In this assay, in vivo-biotinylated (E. coli) SRC-1, purified Renilla luciferase-ER?, and the analyte sample are mixed and incubated for 2 h in a streptavidin-coated microtiter wells, and after one washing step, luminescence is measured with a simple instrument. The assay does not require chemical labeling of the components and shows good sensitivity (25 pM E(2)) and wide dynamic range of more than four orders of magnitude. PMID:22986538

Huovinen, Tuomas; Rytkönen, Kalle; Lamminmäki, Urpo; Pellinen, Teijo



Use of hybridot assay to screen for BK and JC polyomaviruses in non-immunosuppressed patients.  

PubMed Central

Urine samples from 50 patients attending a genitourinary outpatient clinic and from 13 renal allograft recipients were investigated for evidence of infection with human BK and JC polyomaviruses using cytology and a new DNA hybridot assay. Forty four per cent of samples from the renal allograft recipients were positive by cytology and 75% by DNA hybridisation, indicating that hybridot assay is more sensitive than cytological screening. BK and JC viral DNA was found in 20% of the patients attending the genitourinary clinic, showing infection with BK virus and JC virus in a group of patients with clinical conditions not normally associated with immunological deficiency-a finding that has not been reported before. Images Figure

Cobb, J J; Wickenden, C; Snell, M E; Hulme, B; Malcolm, A D; Coleman, D V



Data Display and Analysis Strategies for The NCI Disease-Oriented in Vitro Antitumor Drug Screen  

Microsoft Academic Search

\\u000a For the past five years, NCI staff and collaborators have been pursuing the development and implementation of an unprecedented\\u000a new investigational antitumor drug discovery screen. These efforts have not only been controversial, but have also been undertaken\\u000a during an era of diminishing resources for antitumor drug screening and drug development at NCI. Moreover, numerous unanticipated\\u000a obstacles, not only in the

M. R. Boyd; K. D. Paull; L. R. Rubinstein


A high-throughput pH indicator assay for screening glycosyltransferase saturation mutagenesis libraries.  


Protein engineering using directed evolution or saturation mutagenesis at hot spots is often used to improve enzyme properties such as their substrate selectivity or stability. This requires access to robust high-throughput assays to facilitate the analysis of enzyme libraries. However, relatively few studies on directed evolution or saturation mutagenesis of glycosyltransferases have been reported in part due to a lack of suitable screening methods. In the present study we report a general screening assay for glycosyltransferases that has been developed using the blood group alpha-(1-->3)-galactosyltransferase (GTB) as a model. GTB utilizes UDP-Gal as a donor substrate and alpha-L-Fucp-(1-->2)-beta-D-Galp-O-R (H antigen) as an acceptor substrate and synthesizes the blood group B antigen alpha-D-Galp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-R. A closely related alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) uses UDP-GalNAc as a donor with the same H acceptor, yielding the A antigen alpha-D-Galp-NAc-(1-->3)-[alpha-L-Fuc(1-->2)]-beta-D-Gal-O-R. GTA and GTB are highly homologous enzymes differing in only 4 of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. The screening assay is based on the color change of the pH indicator bromothymol blue when a proton is released during the transfer of Gal/GalNAc from UDP-Gal/UDP-GalNAc to the acceptor substrate. Saturation mutagenesis of GTB enzyme at M214, a hot spot adjacent to the (211)DVD(213) metal binding motif, was performed and the resulting library was screened for increases in UDP-GalNAc transfer activity. Two novel mutants, M214G and M214S, identified by pH indicator screening, were purified and kinetically characterized. M214S and M214G both exhibited two-fold higher k(cat) and specific activity than wild-type GTB for UDP-GalNAc. The results confirm the importance of residue M214 for donor enzyme specificity. PMID:18405657

Persson, Mattias; Palcic, Monica M



Drug screening of hair by liquid chromatography-tandem mass spectrometry.  


Hair has become an important matrix for drug analysis, complementary to blood and urine as a matrix. A prolonged detection window makes hair analysis suitable for the detection of exposure to illegal and medicinal drugs for periods up to 12 months. In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for drug screening in hair was developed and validated. To 20 mg of hair, 0.45 mL of acetonitrile/25 mM formic acid (5:95 v/v) and 50 microL of deuterated internal standards were added, and the sample was incubated in a water bath at 37 degrees C for 18 h. LC separation was achieved with a Zorbax SB-Phenyl column (2.1 x 100 mm, 3.5-microm particle). Mass detection was performed by positive ion mode electrospray LC-MS-MS and included the following drugs/metabolites: nicotine, cotinine, morphine, 6-monoacetylmorphine, codeine, amphetamine, methamphetamine, 3,4-methylenedioxymeth-amphetamine, cocaine, benzoylecgonine, 7-aminonitrazepam, 7-aminoclonazepam, 7-aminoflunitrazepam, oxazepam, diazepam, alprazolam, zopiclone, zolpidem, carisoprodol, meprobamate, buprenorphine, and methadone. Within- and between-assay relative standard deviations varied from 2.0% to 12% and 2.7% to 15%, respectively. The accuracies were in the range of -24% to 16%, and recoveries ranged from 25% to 100%. The LC-MS-MS method proved to be simple and robust for the determination of drugs in hair. It has been used for authentic samples in our laboratory in the past year. PMID:18544222

Hegstad, S; Khiabani, H Z; Kristoffersen, L; Kunøe, N; Lobmaier, P P; Christophersen, A S



A Strategy for High-Throughput Assay Development Using Leads Derived from Nuclear Magnetic Resonance-Based Screening  

Microsoft Academic Search

A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and

Philip J. Hajduk; Stephen F. Betz; Jamey Mack; Xiaoan Ruan; Danli L. Towne; Claude G. Lerner; Bruce A. Beutel; Stephen W. Fesik



Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors.  


In Gram-negative bacteria, production of bis-(3',5')-cyclic diguanylic acid (c-di-GMP) by diguanylate cyclases (DGCs) is the main trigger for production of extracellular polysaccharides and for biofilm formation. Mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation, thus making DGCs interesting targets for new antimicrobial agents with anti-biofilm activity. In this report, we describe a strategy for the screening for DGC inhibitors consisting of a combination of three microbiological assays. The primary assay utilizes an Escherichia coli strain overexpressing the adrA gene, encoding the DGC protein AdrA, and relies on detection of AdrA-dependent cellulose production as red colony phenotype on solid medium supplemented with the dye Congo red (CR). Presence of DGC inhibitors blocking AdrA activity would result in a white phenotype on CR medium. The CR assay can be performed in 96-well microtiter plates, making it suitable for high-throughput screenings. To confirm specific inhibition of c-di-GMP biosynthesis, chemical compounds positive in the CR assay are tested for their ability to inhibit biofilm formation and in a reporter gene assay which monitors expression of curli-encoding genes as a function of DGC activity. Screening of a chemical library using the described approach allowed us to identify sulfathiazole, an antimetabolite drug, as an inhibitor of c-di-GMP biosynthesis. Sulfathiazole probably affects c-di-GMP biosynthesis in an indirect fashion rather than by binding to DGCs; however, sulfathiazole represents the first example of drug able to affect biofilm formation by interfering with c-di-GMP metabolism. PMID:19707751

Antoniani, Davide; Bocci, Paola; Maciag, Anna; Raffaelli, Nadia; Landini, Paolo



A Single-Question Screening Test for Drug Use in Primary Care  

PubMed Central

BACKGROUND Drug use (illicit drug use and nonmedical use of prescription drugs) is common but under-recognized in primary care settings. We validated a single-question screening test for drug use and drug use disorders in primary care. METHODS Adult patients recruited from primary care waiting rooms were asked the single screening question, “How many times in the past year have you used an illegal drug or used a prescription medication for non-medical reasons?” A response of ?1 was considered positive. They were also asked the 10-item Drug Abuse Screening Test (DAST). The reference standard was the presence or absence of current (past year) drug use or a drug use disorder (abuse or dependence) as determined by a standardized diagnostic interview. Drug use was also determined by oral fluid testing for common drugs of abuse. RESULTS Of 394 eligible primary care patients, 286 (73%) completed the interview. The single screening question was 100% sensitive (95% CI 90.6% to 100%) and 73.5% specific (95% CI 67.7% to 78.6%) for the detection of a drug use disorder. It was less sensitive for the detection of self-reported current drug use (92.9%, 95% CI 86.1% to 96.5%) and drug use detected by oral fluid testing or self-report (81.8%, 95% CI 72.5% to 88.5%). Test characteristics were similar to that of the DAST, and were affected very little by subject demographic characteristics. CONCLUSIONS The single screening question accurately identified drug use in this sample of primary care patients, supporting the utility of this brief screen in primary care.

Smith, Peter C.; Schmidt, Susan M.; Allensworth-Davies, Donald; Saitz, Richard



Efficiency of Donor Screening for Human Parvovirus B19 by the Receptor–Mediated Hemagglutination Assay Method  

Microsoft Academic Search

Background and Objectives: Mass screening for parvovirus B19 (B19) by receptor–mediated hemagglutination assay (RHA) may be inadequate to eliminate the virus from plasma pools. We characterized B19 carriers detected in blood donor screening by RHA to explain why some carriers were not detected by RHA. Materials and Methods: Donor plasma was screened for B19 by RHA, B19 DNA by nested

H. Sakata; H. Ihara; S. Sato; T. Kato; H. Ikeda; S. Sekiguchi



Development and Testing of an In-vitro Assay for Screening of Potential Therapeutic Agents Active against Na Channel Neurotoxins.  

National Technical Information Service (NTIS)

A rapid screening assay based upon the ability of (3H)BTX-B binding to rat brain synaptoneurosomes to report sensitively on ligand-receptor interactions at any of at least five distinct sodium channel binding domains in a single assay has been refined and...

G. B. Brown



Simulating Henipavirus Multicycle Replication in a Screening Assay Leads to Identification of a Promising Candidate for Therapy  

Microsoft Academic Search

humans, with fatality rates of up to 75%. We designed a new high-throughput screening (HTS) assay for inhibitors of infection based on envelope glycoprotein pseudotypes. The assay simulates multicycle replication and thus identifies inhibitors that target several stages of the viral life cycle, but it still can be carried out under biosafety level 2 (BSL-2) conditions. These features permit a

Matteo Porotto; Gianmarco Orefice; Christine C. Yokoyama; Bruce A. Mungall; Ronald Realubit; Michael L. Sganga; Mohamad Aljofan; Michael Whitt; Fraser Glickman; Anne Moscona



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

PubMed Central

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.

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.



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

NASA Astrophysics Data System (ADS)

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.

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.



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


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

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



Characterizing the Diversity and Biological Relevance of the MLPCN Assay Manifold and Screening Set  

PubMed Central

The NIH Molecular Libraries Initiative (MLI), launched in 2004 with initial goals of identifying chemical probes for characterizing gene function and druggability, has produced PubChem, a chemical genomics knowledgebase for fostering translation of basic research into new therapeutic strategies. This paper assesses progress toward these goals by evaluating MLI target novelty and propensity for undergoing biochemically or therapeutically relevant modulations and the degree of chemical diversity and biogenic bias inherent in the MLI screening set. Our analyses suggest that while MLI target selection has not yet been fully optimized for biochemical diversity, it covers biologically interesting pathway space that complements established drug targets. We find the MLI screening set to be chemically diverse and to have greater biogenic bias than comparable collections of commercially available compounds. Biogenic enhancements such as incorporation of more metabolite-like chemotypes are suggested.

Zhang, Jintao; Lushington, Gerald H.; Huan, Jun




PubMed Central

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.

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



Screening for Drug Abuse Among Medical and Nonmedical Users of Prescription Drugs in a Probability Sample of College Students  

PubMed Central

Objectives To determine the prevalence of medical and nonmedical use of 4 classes of prescription drugs (opioid, stimulant, sleeping, and sedative or anxiety) and to assess probable drug abuse among 4 mutually exclusive groups of medical and nonmedical use of prescription drugs. Design In 2005, a Web survey was self-administered by a probability sample of 3639 college students (68% response rate). Setting A large, midwestern 4-year university. Participants The sample had a mean age of 19.9 years, and respondents were 53.6% female, 67.4% white, 12.1% Asian, 6.0% African American, 4.2% Hispanic, and 10.2% other racial categories. Main Outcome Measures Medical and nonmedical use of prescription drugs was measured. Probable drug abuse was assessed using a modified version of the Drug Abuse Screening Test, Short Form. Results A total of 40.1% of respondents reported no lifetime use of at least 1 of 4 classes of prescription drugs, 39.7% reported medical use only, 15.8% reported both medical and nonmedical use, and 4.4% reported nonmedical use only. The odds of a positive screening result for drug abuse were greater among medical and nonmedical users (adjusted odds ratio, 5.5; 95% confidence interval, 3.4–7.3) and nonmedical users only (adjusted odds ratio, 6.5; 95% confidence interval, 4.0–10.6) compared with nonusers. The odds of a positive screening result for drug abuse did not differ between medical users only and nonusers. Conclusions Nonmedical users of prescription drugs are at heightened risk for drug abuse, whereas medical users without a history of nonmedical use are generally not at increased risk. Drug abuse screening should be routine for college students, especially among individuals with any history of nonmedical use of prescription drugs.

McCabe, Sean Esteban



Drugs of abuse screening in urine as part of a metabolite-based LC-MSn screening concept.  


Today, immunoassays and several chromatographic methods are in use for drug screening in clinical and forensic toxicology and in doping control. For further proof of the authors' new metabolite-based liquid chromatography-mass spectrometry (LC-MS(n)) screening concept, the detectability of drugs of abuse and their metabolites using this screening approach was studied. As previously reported, the corresponding reference library was built up with MS(2) and MS(3) wideband spectra using a LXQ linear ion trap with electrospray ionization in the positive mode and full scan information-dependent acquisition. In addition to the parent drug spectra recorded in methanolic solution, metabolite spectra were identified after protein precipitation of urine from rats after administration of the corresponding drugs and added to the library. This consists now of data of over 900 parent compounds, including 87 drugs of abuse, and of over 2,300 metabolites and artifacts, among them 436 of drugs of abuse. Recovery, process efficiency, matrix effects, and limits of detection for selected drugs of abuse were determined using spiked human urine, and the resulting data have been acceptable. Using two automatic data evaluation tools (ToxID and SmileMS), the intake of 54 of the studied drugs of abuse could be confirmed in urine samples of drug users after protein precipitation and LC separation. The following drugs classes were covered: stimulants, designer drugs, hallucinogens, (synthetic) cannabinoids, opioids, and selected benzodiazepines. The presented LC-MS(n) method complements the well-established gas chromatography-mass spectroscopy procedure in the authors' laboratory. PMID:21533799

Wissenbach, Dirk K; Meyer, Markus R; Remane, Daniela; Philipp, Anika A; Weber, Armin A; Maurer, Hans H



A novel screening method for competitive FRET-aptamers applied to E. coli assay development.  


A novel high-throughput screening method is described in which a family of DNA aptamers selected against E. coli outer membrane proteins (OMPs) is subjected to PCR in the presence of fluorophore-dUTP conjugates using Deep Vent® exo- polymerase. The fluorophore-doped aptamers and their complementary strands are then heated to render them single-stranded and screened in filter well microtiter plates for fluorescence resonance energy transfer (FRET) assay potential. Using this system, a superior competitive FRET-aptamer designated EcO 4R was identified and the location of its putative binding pocket was determined by individually testing FRET potential in each of the secondary loop structures. By labeling the binding pocket with Alexa Fluor (AF) 647 and binding the aptamer to heavily Black Hole Quencher-3 (BHQ-3)-labeled E. coli bacteria, detection of as few as 30 live unlabeled E. coli per ml was achieved in a competitive displacement FRET assay format. The far red fluorescence emission enables detection in largely blue-green autofluorescent matrices. In addition, the competitive transfer of AF 647-EcO-4R aptamer to unlabeled E. coli cells after a 15 min equilibration period was verified by fluorescence microscopy. The present study also demonstrated that high aptamer affinity is not well correlated with competitive FRET potential. PMID:20443050

Bruno, John G; Carrillo, Maria P; Phillips, Taylor; Andrews, Carrie J



GFP assay as a sensitive eukaryotic screening model to detect toxic and genotoxic activity of azaarenes.  


Azaarenes are nitrogen-containing polyaromatic heterocyclic compounds (NPAHs). The majority of the azaarenes found in the environment originate from anthropogenic sources. Concentrations of NPAHs found in the environment are reported to be one to two orders of magnitude lower than polycyclic aromatic hydrocarbons (PAHs) concentrations, yet their biological effects can be of similar magnitude. Very few studies on the genotoxicity of azaarenes are available in the literature. In the present study, a preliminary profile of both the toxic and genotoxic potential of 5 PAHs and their 20 aza-analogues were investigated. To assess the toxic and genotoxic activity, a green fluorescent protein (GFP) assay based on the yeast Saccharomyces cerevisiae was selected. To compare the sensitivity of this eukaryotic short-term assay with bacterial screening tests, the Toxi-Chromotest for toxicity and SOS-Chromotest for genotoxicity assessment were also performed. This comparison indicates that in most cases, the yeast GFP assay is apparently of comparable specificity to the bacterial toxicity or genotoxicity tests with respect to the correlation of positive/negative responses, but much more sensitive with respect to the effective concentration values. In the cases of phenazine, phenanthridine, 1,10-phenanthroline, or 4,7-phenanthroline, one to two orders of magnitude lower IC20 and minimum genotoxic concentration values in the yeast GFP assay were observed. In this study, the authors present evidence that genotoxicity assessment using the yeast GFP assay can provide a simple system to monitor the activity of these environmental pollutants that could possess mutagenic potential at low concentrations. PMID:16841313

Bartos, T; Letzsch, S; Skarek, M; Flegrová, Z; Cupr, P; Holoubek, I



Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death.  


Parkinson's disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by ?-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z' factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD. PMID:20705047

Yoon, Il Sang; Au, Qingyan; Barber, Jack R; Ng, Shi Chung; Zhang, Bin



An interactive visualization-based approach for high throughput screening information management in drug discovery.  


While high throughput screening (HTS) techniques are capable of generating large amounts of biologically significant data, assimilating and mining this information can be extremely complex and potentially crucial information patterns can easily be lost in the mounds of data. The predominantly life-science oriented scientific training of the researchers in this area furthermore, precludes their using complex querying or data-mining algorithms. Keeping in account these challenges, our goal in this paper is to provide a highly intuitive environment for storing and interacting with large amounts of HTS assay data. The principal modes of user-data interactions supported in the proposed paradigm are interaction and visualization rich. Moreover, they span the heterogeneous data modalities common to drug discovery, including but not limited to chemical structures, high-throughput assay formats, graphical information, and alpha-numeric data types. Case studies and experiments demonstrate the efficacy of the proposed approach in terms of its ease of use as well as its capability to discern complex information patterns in the data. PMID:17947168

Pui Shan Chan, Tammy; Malik, Preeti; Singh, Rahul



The E-screen assay: a comparison of different MCF7 cell stocks.  

PubMed Central

MCF7 human breast cancer cells have been studied extensively as a model for hormonal effects on breast cancer cell growth and specific protein synthesis. Because the proliferative effect of natural estrogen is considered the hallmark of estrogen action, it was proposed that this property be used to determine whether a substance is an estrogen. The E-screen assay, developed for this purpose, is based on the ability of MCF7 cells to proliferate in the presence of estrogens. The aim of our study was to characterize the response of four MCF7 cell stocks (BUS, ATCC, BB, and BB104) and determine which of them performed best in the E-screen test. The four stocks assayed were distinguishable by their biological behavior. In the absence of estrogen, MCF7 BUS cells stopped proliferating and accumulated in the G0/G1 phase of the cell cycle; estrogen receptors increased, progesterone receptors decreased, and small amounts of pS2 protein were secreted. Of all the MCF7 stocks tested, MCF7 BUS cells showed the highest proliferative response to estradiol-17 beta: cell yields increased up to sixfold over those of nontreated cells in a 144-hr period. The differences between estrogen-supplemented and nonsupplemented MCF7 BUS cells were due mostly to G0/G1 proliferative arrest mediated by charcoal dextran-stripped serum. MCF7 BUS cell stocks and others showing a similar proliferative pattern should be chosen for use in the E-screen test, or whenever a proliferative effect of estrogen is to be demonstrated. Images Figure 1. A Figure 1. B Figure 1. C Figure 1. D Figure 2. A Figure 2. B Figure 2. C Figure 2. D Figure 3. A Figure 3. B Figure 4. A Figure 4. B Figure 5. A Figure 5. B Figure 5. C Figure 5. D

Villalobos, M; Olea, N; Brotons, J A; Olea-Serrano, M F; Ruiz de Almodovar, J M; Pedraza, V



Bioluminescence-based neuraminidase inhibition assay for monitoring influenza virus drug susceptibility in clinical specimens.  


The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n = 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50% inhibitory concentrations (IC50s) generated were compared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof of principle, clinical specimens (n = 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyrosequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens. PMID:23917311

Marjuki, Henju; Mishin, Vasiliy P; Sleeman, Katrina; Okomo-Adhiambo, Margaret; Sheu, Tiffany G; Guo, Lizheng; Xu, Xiyan; Gubareva, Larisa V



Drug susceptibility testing of Mycobacterium tuberculosis with nitrate reductase assay.  


The nitrate reductase assay (NRA) was evaluated for susceptibility testing of Mycobacterium tuberculosis using 80 clinical isolates of M. tuberculosis and H37Rv as a control strain. All isolates were tested by the proportion method and the NRA for isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (ETM). The proportion method was carried out according to NCCLS on Löwenstein-Jensen (LJ) medium and the NRA on LJ medium containing 1000 microg/ml potassium nitrate (KNO(3)). After incubation for 7, 10, 14 and 21 days, Griess reagent was added to each LJ medium and nitrate reduction was determined by a colour change. Comparing the NRA with the proportion method, sensitivities were 100, 100, 82.1 and 92.2% for INH, RIF, STR and ETM, respectively. Specificities were 100, 100, 92.3 and 100% for INH, RIF, STR and ETM, respectively. The results of 2, 22 and 56 isolates were obtained after 7, 10 and 14 days, respectively. The proportion method result were read at 21-28 days. The NRA is rapid, inexpensive and easy to perform. Our results indicated that the NRA is suitable for the early determination of INH and RIF resistance in countries where sophisticated procedures are not always available. PMID:15325439

Coban, Ahmet Yilmaz; Birinci, Asuman; Ekinci, Bora; Durupinar, Belma



Use of human neuroblastoma continuous cell lines for in vitro drug sensitivity screening  

Microsoft Academic Search

We have used three continuous human neuroblastoma cell lines to establish patterns of in vitro drug sensitivities, as judged by clonogenic assay. We evaluated 12 ‘standard’ antitumor drugs already in clinical usage, and tested four newer analogues, one of cisplatin and three of doxorubicin, and the investigational agent desferrioxamine. A certain heterogeneity of drug sensitivities was noted amongst these three

Bridget T. Hill; R. D. H. Whelan; Louise K. Hosking



A high-throughput screening assay of ascorbate in brain samples.  


Ascorbate is a vital reductant/free radical scavenger in the CNS, whose content defines - to a large extent - the redox status and the antioxidant reserves. Quick, reliable and specific methods for its measurement in brain samples are highly desirable. We have developed a new high-throughput screening assay for measurements of ascorbate using a fluorescence plate-reader. This assay is based on a direct reaction of ascorbate with a nitroxide radical conjugated with a fluorogenic acridine moiety, 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl radical (AC-TEMPO), yielding fluorescent hydroxylamine product (AC-TEMPO-H). The reaction was monitored over time using fluorescence and electron spin resonance techniques. The appearance of fluorescent AC-TEMPO-H was linear within the range of 3.75-75?M AscH(-) in the sample (0.5-10?M AscH(-) in the well). Assay was validated with high performance liquid chromatography method. The concentration of ascorbate in murine tissue samples, including brain samples after traumatic brain injury and hemorrhagic shock, was measured. PMID:21855575

Belikova, Natalia A; Glumac, Ashley L; Kapralova, Valentyna; Cheikhi, Amin; Tyurina, Yulia Y; Vagni, Vincent A; Kochanek, Patrick M; Kagan, Valerian E; Bayir, Hülya



Challenges of developing and validating immunogenicity assays to support comparability studies for biosimilar drug development.  


Imminent patent expiry for a number of biological products currently on the market (many of which are blockbusters) has created an increasing opportunity for the development of biosimilars in the biotechnology industry. The key for successful biosimilar development is to demonstrate biosimilarity to the originator drug. In addition to demonstrating the similarity of physical and chemical properties between biosimilar and originator compounds, regulatory agencies require that immunogenicity be evaluated in comparative studies between biosimilar and originator drugs. Immunogenicity assays are generally non-quantitative (qualitative) and proving similarity/comparability based on qualitative assays can be very challenging. This review will discuss the challenges of developing and validating immunogenicity assays to support preclinical and clinical comparative studies for biosimilar drug development as well as the challenges in association with the interpretation of the data. PMID:23013399

Cai, Xiao-Yan; Thomas, Jeff; Cullen, Constance; Gouty, Dominique



Direct Multiplex Assay of Lysosomal Enzymes in Dried Blood Spots for Newborn Screening  

PubMed Central

Background Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann–Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards containing dried blood spots as the enzyme source. Methods We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid ?-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. Results We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann–Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5–9 heterozygous carriers were approximately one-half those measured with 15–32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals. Conclusion For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newborn-screening centers.

Li, Yijun; Scott, C. Ronald; Chamoles, Nestor A.; Ghavami, Ahmad; Pinto, B. Mario; Turecek, Frantisek; Gelb, Michael H.



Are drunk-driving offenders referred for screening accurately reporting their drug use?  

Microsoft Academic Search

Several studies report that a substantial percentage of offenders arrested for impaired driving test positive for drugs of abuse besides alcohol. Current guidelines recommend screening offenders for both alcohol and other drug use, yet little is known about the accuracy of self-reports of drug use in this population. We compared drug abuse and dependence DSM-III-R diagnoses from an initial, court-ordered

Sandra C Lapham; Janet C'de Baca; Iyiin Chang; William C Hunt; Lawrence R Berger



Screening for the drug-phospholipid interaction: correlation to phospholipidosis.  


Phospholipid bilayers represent a complex, anisotropic environment fundamentally different from bulk oil or octanol, for instance. Even "simple" drug association to phospholipid bilayers can only be fully understood if the slab-of-hydrocarbon approach is abandoned and the complex, anisotropic properties of lipid bilayers reflecting the chemical structures and organization of the constituent phospholipids are considered. The interactions of drugs with phospholipids are important in various processes, such as drug absorption, tissue distribution, and subcellular distribution. In addition, drug-lipid interactions may lead to changes in lipid-dependent protein activities, and further, to functional and morphological changes in cells, a prominent example being the phospholipidosis (PLD) induced by cationic amphiphilic drugs. Herein we briefly review drug-lipid interactions in general and the significance of these interactions in PLD in particular. We also focus on a potential causal connection between drug-induced PLD and steatohepatitis, which is induced by some cationic amphiphilic drugs. PMID:19551800

Alakoskela, Juha-Matti; Vitovic, Pavol; Kinnunen, Paavo K J



Method for Treatment of Drug Addiction and for Screening of Pharmaceutical Agents Therefor.  

National Technical Information Service (NTIS)

The present invention is directed to a method for treatment of drug addiction and screening methods for identifying pharmaceutical agents that ameloriate or prevent the deleterious effects of addition. The invention is as well directed to a group of genes...

P. P. Sanna G. Koob S. Ahmed



Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts  

PubMed Central

Identification of factors that improve muscle function in boys with Duchenne muscular dystrophy (DMD) could lead to an improved quality of life. To establish a functional in vitro assay for muscle strength, mdx murine myoblasts, the genetic homologue of DMD, were tissue engineered in 96-microwell plates into 3-dimensional muscle constructs with parallel arrays of striated muscle fibers. When electrically stimulated, they generated tetanic forces measured with an automated motion tracking system. Thirty-one compounds of interest as potential treatments for patients with DMD were tested at 3 to 6 concentrations. Eleven of the compounds (insulin-like growth factor-1, creatine, ?-hydroxy-?-methylbutyrate, trichostatin A, lisinopril, and 6 from the glucocorticoid family) significantly increased tetanic force relative to placebo-treated controls. The glucocorticoids methylprednisolone, deflazacort, and prednisone increased tetanic forces at low doses (EC50 of 6, 19, and 56 nM, respectively), indicating a direct muscle mechanism by which they may be benefitting DMD patients. The tetanic force assay also identified beneficial compound interactions (arginine plus deflazacort and prednisone plus creatine) as well as deleterious interactions (prednisone plus creatine inhibited by pentoxifylline) of combinatorial therapies taken by some DMD patients. Since mdx muscle in vivo and DMD patients respond in a similar manner to many of these compounds, the in vitro assay will be a useful tool for the rapid identification of new potential treatments for muscle weakness in DMD and other muscle disorders.—Vandenburgh, H., Shansky, J., Benesch-Lee, F., Skelly, K., Spinazzola, J.M., Saponjian, Y., Tseng, B.S. Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts.

Vandenburgh, Herman; Shansky, Janet; Benesch-Lee, Frank; Skelly, Kirsten; Spinazzola, Janelle M.; Saponjian, Yero; Tseng, Brian S.



Development of an HTS-compatible assay for discovery of ROR? modulators using AlphaScreen® technology.  


The retinoid acid receptor-related orphan receptors (RORs) represent important targets for the treatment of metabolic and immune disorders. Here the authors describe the application of AlphaScreen(®) technology to develop a high-throughput screening (HTS)-compatible assay to facilitate the discovery of ROR? modulators. Using the ligand binding domain (LBD) of ROR? and a peptide derived from the NR1 box of the nuclear receptor coactivator PGC-1?, a 384-well format assay was developed exhibiting high sensitivity, requiring only low nanomolar concentration of reagents. Recently, it was shown that oxysterols such as 7?-hydroxycholesterol (7?-OHC) function as modulators of the RORs. In this assay, 7?-OHC produced a concentration-response curve with an EC(50) of 162 nM, a Z' factor of 0.6, and a signal-to-background (S/B) ratio of 4.2, demonstrating that the assay is HTS compatible. Validation of the assay was afforded by screening against the Sigma LOPAC1280™ library in a 384-well format. In summary, the results presented here demonstrate that this assay can be used to screen large chemical libraries to discover novel modulators of ROR?. PMID:21297105

Istrate, Monica A; Spicer, Timothy P; Wang, Yan; Bernard, Jerrold A; Helvering, Leah M; Bocchinfuso, Wayne P; Richardson, Timothy I; Zink, Richard; Kumar, Naresh; Montrose-Rafizadeh, Chahrzad; Dodge, Jeffrey; Hodder, Peter; Griffin, Patrick R



A Novel High-Throughput Screening Assay to Identify Inhibitors of HIV-1 gp120 Protein Interaction with DC-SIGN  

PubMed Central

The 2010 UNAIDS report states that approximately 34 million people are living with human immunodeficiency virus type 1 (HIV-1), despite highly active antiretroviral therapy (HAART). Despite being effective, ARV therapy has many disadvantages including a cost trajectory unsustainable for economically challenged countries, serious side effects, and the development of drug-resistant strains. Several measures are under way to develop alternatives for ARV therapy, particularly for the control of early HIV-1 infection, but lack of efficient drug targets and assays hinders the search of potential ARV molecules. The dendritic cells present in the mucosal tissue, together with CD4+ T lymphocytes and macrophages, are among the first cells to encounter HIV-1. The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) molecule plays a crucial role in binding HIV-1 through high affinity interaction with viral envelope glycoprotein gp120. DC-SIGN, a mannose-binding C-type lectin expressed on cells in the mucosal tissue of the rectum, uterus and cervix, facilitates early HIV-1 infection after sexual transmission. In this study we report a novel target-specific high-throughput screening (HTS) assay capable of quantifying the binding as well as the inhibition of DC-SIGN and gp120. The specificity of the assay was determined through competitive inhibition while optimization occurred for DMSO tolerance (0.5%), Z’ factor (0.51), signal-to-noise ratio (3.26), and coefficient of variation (5.1%). For assay validation previously recognized antagonists of DC-SIGN/gp120 binding were tested to detect inhibition demonstrating the suitability of the assay for future HTS screen of potential inhibitors that block the binding between DC-SIGN and gp120 which may prevent early HIV-1 infection.

Tran, Thuong H.; El Baz, Rasha; Cuconati, Andrea; Arthos, James; Jain, Pooja; Khan, Zafar K.



A Novel High-Throughput Screening Assay to Identify Inhibitors of HIV-1 gp120 Protein Interaction with DC-SIGN.  


The 2010 UNAIDS report states that approximately 34 million people are living with human immunodeficiency virus type 1 (HIV-1), despite highly active antiretroviral therapy (HAART). Despite being effective, ARV therapy has many disadvantages including a cost trajectory unsustainable for economically challenged countries, serious side effects, and the development of drug-resistant strains. Several measures are under way to develop alternatives for ARV therapy, particularly for the control of early HIV-1 infection, but lack of efficient drug targets and assays hinders the search of potential ARV molecules. The dendritic cells present in the mucosal tissue, together with CD4(+) T lymphocytes and macrophages, are among the first cells to encounter HIV-1. The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) molecule plays a crucial role in binding HIV-1 through high affinity interaction with viral envelope glycoprotein gp120. DC-SIGN, a mannose-binding C-type lectin expressed on cells in the mucosal tissue of the rectum, uterus and cervix, facilitates early HIV-1 infection after sexual transmission. In this study we report a novel target-specific high-throughput screening (HTS) assay capable of quantifying the binding as well as the inhibition of DC-SIGN and gp120. The specificity of the assay was determined through competitive inhibition while optimization occurred for DMSO tolerance (0.5%), Z' factor (0.51), signal-to-noise ratio (3.26), and coefficient of variation (5.1%). For assay validation previously recognized antagonists of DC-SIGN/gp120 binding were tested to detect inhibition demonstrating the suitability of the assay for future HTS screen of potential inhibitors that block the binding between DC-SIGN and gp120 which may prevent early HIV-1 infection. PMID:22102941

Tran, Thuong H; El Baz, Rasha; Cuconati, Andrea; Arthos, James; Jain, Pooja; Khan, Zafar K



Intracellular Mechanics-Based Drug Screening for Cancer Metastasis  

NASA Astrophysics Data System (ADS)

In 2007 alone, close to 1.5 million new cancer cases and over half of a million deaths from cancer are projected to occur in US. In general, cancer is much easier to be successfully treated before metastasis; the five-year survival rates for most of the cancers in the metastatic stage are lower than 10%. The origin of cancer is due to genomic instability; however, the genomics or proteomics studies focus on this phenomenon cannot thoroughly elucidate how cancer metastasis proceeds. During this process, cancer cells protrude and conquer their physical barriers, resist shear stress, establish anchorages and finally settle in a new environment. Each development in this process involves mechanical forces. Thus, whether force generation and cancer cells' mechanical properties can be integrated into the current mainstream of cancer research and offer new insight is worthy of being investigated. To measure the change of cell mechanics, specifically intracellular mechanics, a tool that least disrupts the probed cell's behavior and, simultaneously, can obtain real time quantitative measurement is necessary. To satisfy these criteria, we have developed a technique, ballistic intracellular nanorheology (BIN), in which we trace and analyze the trajectories of nanospheres that have been ballistically bombarded into the cytoplasm of individual cells. This technique allows us to probe the effects of chemical or mechanical stimuli on intracellular mechanics in various types of cells, on culture dishes or in a three-dimensional matrix. BIN is, currently, the first and only method available that can be applied to perform such tasks. Using this technique, we have gained detailed information about how the cytoskeletal remodeling pathways control the intracellular mechanics. We have also obtained information on the tempo-correlation between agonists and intracellular mechanics and how cells utilize their intracellular mechanics to react extracellular shear stress. These studies have set the framework for us to understand the mechanical mechanism of cancer cell metastasis on a molecular level. In this talk, I will describe the working principal using this technique to screen cancer drugs that prevent cancer metastasis.

Tseng, Yilder



Michigan Assessment Screening Test for Alcohol and Drugs (MAST=AD): Evaluation in a Clinical Sample  

Microsoft Academic Search

In this study, we sought to evaluate a modification of the Michigan Alcohol Screening Test designed to include problems associated with other drug abuse=dependence besides alcohol. Scores of the lifetime Michigan Assess- ment-Screening Test=Alcohol-Drug (MAST=AD) were compared to other lifetime measures of substance abuse and dependence and to psychiatric scales reflecting current or recent symptoms. Two university medical centers with

Joseph Westermeyer; Ilhan Yargic; Paul Thuras


Screening for drugs of abuse in hair with ion spray LC–MS–MS  

Microsoft Academic Search

Analyzing hair for many substances can be tedious and expensive, and a rapid screening method should prove helpful. Generally, screening has been performed using immunological tests, mainly in workplace drug testing, where the number of samples has been high.The aim of this study was to develop an LC–MS–MS method for the simultaneous analysis of several drugs of abuse in human

Robert Kronstrand; Ingrid Nyström; Joakim Strandberg; Henrik Druid



Study on microvisualizing assay of delivered drug infiltration using 2-color optical coherence dosigraphy  

NASA Astrophysics Data System (ADS)

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 ?m and accuracy plus-minus 13.0 ?M. 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.

Nakamichi, Yu; Saeki, Souichi; Saito, Takashi; Hiro, Takafumi; Matsuzaki, Masunori



Implementing mental health screening within a youth alcohol and other drugs service  

Microsoft Academic Search

Background: While clinical studies consistently demonstrate high rates of co-occurring mental health problems among young people with substance use disorders, mental health assessments are not routinely conducted within Alcohol and Other Drug (AOD) settings.Aims: To describe the implementation of a universal mental health screening program within a youth AOD service. We report on the adoption of screening by AOD staff

Dan I. Lubman; Leanne Hides; Antonietta Scaffidi; Kathryn Elkins; Maria Stevens; Richard Marks



Screening for antiradical efficiency of 21 semi-synthetic derivatives of quercetin in a DPPH assay  

PubMed Central

The group of 21 novel semi-synthetic derivatives of quercetin was screened for the antiradical efficiency in a DPPH assay. The initial fast absorbance decrease of DPPH, corresponding to the transfer of the most labile H atoms, was followed by a much slower absorbance decline representing the residual antiradical activity of the antioxidant degradation products. Initial velocity of DPPH decolorization determined for the first 75-s interval was used as a marker of the antiradical activity. Application of the kinetic parameter allowed good discrimination between the polyphenolic compounds studied. The most efficient chloronaphthoquinone derivative (compound Ia) was characterized by antiradical activity higher than that of quercetin and comparable with that of trolox. Under the experimental conditions used, one molecule of Ia was found to quench 2.6±0.1 DPPH radicals.

Milackova, Ivana; Kovacikova, Lucia; Veverka, Miroslav; Gallovic, Jan



A High-Throughput TNP-ATP Displacement Assay for Screening Inhibitors of ATP-Binding in Bacterial Histidine Kinases  

PubMed Central

Abstract Bacterial histidine kinases (HK) are members of the GHKL superfamily, which share a unique adenosine triphosphate (ATP)-binding Bergerat fold. Our previous studies have shown that Gyrase, Hsp90, MutL (GHL) inhibitors bind to the ATP-binding pocket of HK and may provide lead compounds for the design of novel antibiotics targeting these kinases. In this article, we developed a competition assay using the fluorescent ATP analog, 2?,3?-O-(2,4,6-trinitrophenyl) adenosine 5?-triphosphate. The method can be used for high-throughput screening of compound libraries targeting HKs or other ATP-binding proteins. We utilized the assay to screen a library of GHL inhibitors targeting the bacterial HK PhoQ, and discuss the applications of the 2?,3?-O-(2,4,6-trinitrophenyl) adenosine 5?-triphosphate competition assay beyond GHKL inhibitor screening.

Guarnieri, Michael T.; Blagg, Brian S. J.



Cell-Based Hepatitis C Virus Infection Fluorescence Resonance Energy Transfer (FRET) Assay for Antiviral Compound Screening  

PubMed Central

Hepatitis C virus (HCV) affects an estimated 3% of the population and is a leading cause of chronic liver disease worldwide. Since HCV therapeutic and preventative options are limited, the development of new HCV antivirals has become a global health care concern. This has spurred the development of cell-based infectious HCV high-throughput screening assays to test the ability of compounds to inhibit HCV infection. This unit describes methods that may be used to assess the in vitro efficacy of HCV antivirals using a cell-based high-throughput fluorescence resonance energy transfer (FRET) HCV infection screening assay, which allows for the identification of inhibitors that target HCV at any step in the viral life cycle. Basic protocols are provided for compound screening during HCV infection and analysis of compound efficacy using an HCV FRET assay. Support protocols are provided for propagation of infectious HCV and measurement of viral infectivity.

Yu, Xuemei; Uprichard, Susan L.



Comparing label-free biosensors for pharmacological screening with cell-based functional assays.  


The diversity and impact of label-free technologies continues to expand in drug discovery. Two classes of label-free instruments, using either an electrical impedance-based or an optical-based biosensor, are now available for investigating the effects of ligands on cellular targets. Studies of GPCR function have been especially prominent with these instruments due to the importance of this target class in drug discovery. Although both classes of biosensors share similar high sensitivity to changes in cell shape and structure, it is unknown whether these biosensors yield similar results when comparing the same GPCR response. Furthermore, since cell morphology changes induced by GPCRs differ depending on which G-protein is activated, there is potential for these instruments to have differential sensitivities to G-protein signaling. Here 1 impedance (CellKey)- and 2 optical-based instruments (BIND and Epic) are compared using Gi-coupled (ACh M2), Gq-coupled (ACh M1), and Gs-coupled (CRF1) receptors. All 3 instruments were robust in agonist and antagonist modes yielding comparable potencies and assay variance. Both the impedance and optical biosensors showed similar high sensitivity for detecting an endogenous D1/D5 receptor response and a melanocortin-4 receptor inverse agonist (agouti-related protein). The impedance-based biosensor was uniquely able to qualitatively distinguish G-protein coupling and reveal dual signaling by CRF1. Finally, responses with a ligand-gated ion channel, TRPV1, were similarly detectable in each instrument. Thus, despite some differences, both impedance- and optical-based platforms offer robust live-cell, label-free assays well suited to drug discovery and typically yield similar pharmacological profiles for GPCR ligands. PMID:20085460

Peters, Matthew F; Vaillancourt, François; Heroux, Madeleine; Valiquette, Manon; Scott, Clay W



A Novel High-Throughput Screening Assay for Putative Antidiabetic Agents through PPAR? Interactions  

PubMed Central

As natural peroxisome proliferator-activated receptor-? (PPAR?) ligands, high levels of fatty acids and glucose could lead to hyperactivation of PPAR?, like that seen in diabetes. Important diabetes research goals are to uncover new metabolic or signaling pathways involved in hyperglycemic cellular injury and to develop therapeutics for preventing or reversing this injury. Consequently, 1040 putative antidiabetic agents were screened for their ability to 1) affect PPAR? lipid binding, 2) directly bind PPAR?, and 3) alter PPAR? transactivation in the presence of high glucose. A high-throughput fluorescent binding assay was developed to examine each compound’s ability to restore fatty acyl-CoA binding to PPAR? in the presence of high glucose concentrations. Approximately 1% of the compounds restored acyl-CoA binding by 60% or more. These compounds directly interacted with PPAR ? with high affinity (nM Kds), validating the primary screen. Furthermore, these compounds altered PPAR? transactivation, and 1 strongly reversed the hyperactivation of PPAR? found in the presence of clofibrate and high glucose levels.




Cost-effectiveness of interferon-gamma release assay for entry tuberculosis screening in prisons.  


The incidence of active tuberculosis (TB) and latent tuberculosis infection (LTBI) in inmates and prison staff is higher than that in the general population. Mycobacterium tuberculosis-specific interferon-gamma release assays (IGRAs) provide more accurate diagnosis of M. tuberculosis infection with higher specificity than the tuberculin skin test (TST). To assess the cost effectiveness of QuantiFERON®-TB Gold In-Tube (QFT) compared to TST, TST followed by QFT and chest X-ray, we constructed Markov models using a societal perspective on the lifetime horizon. The main outcome measure of effectiveness was quality-adjusted life-years (QALYs) gained. The incremental cost-effectiveness was compared. The QFT-alone strategy was the most cost-effective for entry TB screening in prisons in developed countries. Cost-effectiveness was not sensitive to the rates of BCG vaccination, LTBI, TB, HIV infection and multidrug-resistant TB. Entry TB screening using an IGRA in prisons should be considered on the basis of its cost-effectiveness by public health intervention. PMID:23286364

Kowada, A



Implementation of an interferon-gamma release assay to screen for tuberculosis in refugees and immigrants.  


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

Simpson, Terri; Tomaro, Julie; Jobb, Cynthia



Development of a dimethylarginine dimethylaminohydrolase (DDAH) assay for high throughput chemical screening  

PubMed Central

Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/Dimethylarginine dimethylaminohydrolase (DDAH)/Asymmetric Dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by iNOS) could play a role in idiopathic pulmonary fibrosis (IPF), sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high throughput screen for DDAH modulators.

Ghebremariam, Yohannes T; Erlanson, Daniel; Yamada, Keisuke; Cooke, John P



Chemical Library Screening Using a SPR-Based Inhibition in Solution Assay: Simulations and Experimental Validation.  


We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 ?M) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 ?M) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 ?M, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with KD in the 10-50 ?M range. We propose that a medium (?M) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening. PMID:23931734

Choulier, Laurence; Nominé, Yves; Zeder-Lutz, Gabrielle; Charbonnier, Sebastian; Didier, Bruno; Jung, Marie-Louise; Altschuh, Danièle



High content screening analysis of phospholipidosis: validation of a 96-well assay with CHO-K1 and HepG2 cells for the prediction of in vivo based phospholipidosis.  


Drug-induced phospholipidosis is marked by an excessive accumulation of phospholipids in lysosomes which can occur after exposure to cationic amphiphilic drugs. Phospholipidosis is considered as an adverse side effect and may delay or negatively affect registration of drug candidates. Currently, the gold standard method of phospholipidosis detection is electron microscopy on tissue samples. This technique is time consuming and only performed relatively late in drug development. Therefore, in vitro screening methods for phospholipidosis are essential in early drug development. In this study, an in vitro phospholipidosis detection assay is developed with CHO-K1 and HepG2 cells by using the fluorescent marker NBD-PE and high content screening analysis. Lysosomal localization of NBD-PE was demonstrated by colocalization with Lysotracker and lamellar body formation by electron microscopy. Upon drug exposure, lysosomal NBD-PE accumulation can be visualized and quantified. Validation with 56 reference compounds, divided in 25 phospholipidosis inducers and 31 negative compounds, showed that this new in vitro assay has a high sensitivity (CHO-K1=92.0% and HepG2=88.0%) and specificity (CHO-K1=87.1% and HepG2=80.6%) for predicting phospholipidosis in vivo. Thus a selective screening tool has been developed for early selection of drug candidates with low probability for phospholipidosis. PMID:21651975

van de Water, F M; Havinga, J; Ravesloot, W T; Horbach, G J M J; Schoonen, W G E J



Preclinical Drug Safety and Cardiac Ion Channel Screening  

Microsoft Academic Search

\\u000a An effective approach to detect drug candidates that may exert unwanted effects on heart rate and rhythm is critical when\\u000a characterizing the cardiovascular safety profile of novel drugs. As all cardiac channels represent potential off-target liabilities,\\u000a numerous approaches have been developed to detect undesirable cardiac electrophysiologic effects during drug discovery. This\\u000a chapter will describe strategies to assess potential proarrhythmic liabilities

Zhi Su; Gary Gintant


Universal screening for alcohol and drug use and racial disparities in Child Protective Services reporting  

PubMed Central

This study examines racial disparities in Child Protective Services (CPS) reporting at delivery in a county with universal screening for alcohol/drug use in prenatal care. It also explores two mechanisms through which universal screening could reduce reporting disparities: Equitable Surveillance and Effective Treatment. Equitable Surveillance is premised on the assumptions that identification of drug use through screening in prenatal care leads to CPS reporting at delivery and that Black women are screened more than White women, which leads to disproportionate reporting of Black newborns. Universal screening would correct this by ensuring that prenatal providers screen and therefore also report White women to CPS, thereby reducing disparities. Effective Treatment is premised on the idea that identification of drug use through screening in prenatal care leads women to receive treatment during pregnancy, which thereby reduces CPS reporting at delivery. Universal screening would lead to prenatal providers screening more Black women and thereby to more Black women receiving treatment prenatally. The increase in treatment receipt during pregnancy would then decrease the number of Black newborns reported to CPS at delivery, thereby reducing disparities. County data were used to compare the racial/ethnic distribution of women and newborns in three points in the system (identification in prenatal care, treatment entry during pregnancy, and reporting to CPS at delivery related to maternal alcohol/drug use) and explore pathways to treatment. Despite Black women having alcohol/drug use identified by prenatal care providers at similar rates to White women and entering treatment more than expected, Black newborns were 4 times more likely than White newborns to be reported to CPS at delivery. This contradicts the premise of Effective Treatment. By default, findings were more consistent with Equitable Surveillance than Effective Treatment. Providers and policy makers should not assume that universal screening in prenatal

Roberts, Sarah C. M.; Nuru-Jeter, Amani



Universal screening for alcohol and drug use and racial disparities in child protective services reporting.  


This study examines racial disparities in Child Protective Services (CPS) reporting at delivery in a county with universal screening for alcohol/drug use in prenatal care. It also explores two mechanisms through which universal screening could reduce reporting disparities: Equitable Surveillance and Effective Treatment. Equitable Surveillance is premised on the assumptions that identification of drug use through screening in prenatal care leads to CPS reporting at delivery and that Black women are screened more than White women, which leads to disproportionate reporting of Black newborns. Universal screening would correct this by ensuring that prenatal providers screen and therefore also report White women to CPS, thereby reducing disparities. Effective Treatment is premised on the idea that identification of drug use through screening in prenatal care leads women to receive treatment during pregnancy, which thereby reduces CPS reporting at delivery. Universal screening would lead to prenatal providers screening more Black women and thereby to more Black women receiving treatment prenatally. The increase in treatment receipt during pregnancy would then decrease the number of Black newborns reported to CPS at delivery, thereby reducing disparities. County data were used to compare the racial/ethnic distribution of women and newborns in three points in the system (identification in prenatal care, treatment entry during pregnancy, and reporting to CPS at delivery related to maternal alcohol/drug use) and explore pathways to treatment. Despite Black women having alcohol/drug use identified by prenatal care providers at similar rates to White women and entering treatment more than expected, Black newborns were four times more likely than White newborns to be reported to CPS at delivery. This contradicts the premise of Effective Treatment. By default, findings were more consistent with Equitable Surveillance than Effective Treatment. Providers and policy makers should not assume that universal screening in prenatal care reduces CPS reporting disparities. PMID:21681593

Roberts, Sarah C M; Nuru-Jeter, Amani



New Drug Susceptibility Test forMycobacterium tuberculosis Using the Hybridization Protection Assay  

Microsoft Academic Search

We developed a novel method for early detection of drug-resistant strains ofMycobacterium tuberculosisby 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



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...


Screening-level assays for potentially human-infectious environmental Legionella spp.  


In spite of the fact that various Legionella species are isolated from nonclinical water settings, there is no standard method to determine whether environmental legionellae may be infectious to humans. Here we provide a screening-level approach based on an in vivo murine (A/J mouse) model and three in vitro proliferation assays using Acanthamoeba polyphaga, and THP-1 human and J774 murine macrophage cell lines to identify potentially human-infectious legionellae. As an initial demonstration the infectivity potential of three clinical (Legionella pneumophila, L, longbeacheae, and L. micdadei) and three environmental (L. dumoffii, L. maceachernii, and L. sainthelensi) legionellae were evaluated. A/J mice were intranasally infected and by 6 h post infection (p.L), there were significant bacterial titers in the lungs. L. pneumophila, L. dumoffii, and L. micdadei densities were higher than L. longbeacheae, L. maceacherni, and L. sainthelensi at 24 h p.i. However, only L. pneumophila and L. micdadei persisted in the lungs after 48 h, indicating that the other isolates were rapidly cleared. Results from the in vitro assays showed that only L. pneumophila significantly multiplied within A. polyphaga, THP-1 and J774 cells after 72 h, but lysis of any of the in vitro hosts also flagged the strains for potential concern (e.g. L. dumoffii and L. micdadei). The results demonstrate the value of using multiple approaches to assess the potential level of pathogenicity of Legionella strains isolated from different environmental matrices. PMID:21538239

Buse, Helen Y; Brehm, Abby; Santo Domingo, Jorge W; Ashbolt, Nicholas J



Selective fluorescent nonpeptidic antagonists for vasopressin V? GPCR: application to ligand screening and oligomerization assays.  


A series of fluorescent benzazepine ligands for the arginine-vasopressin V? receptor (AVP V?R) was synthesized using "Click" chemistry. Their in vitro pharmacological profile at AVP V?R, V(1a)R, V(1b)R, and oxytocin receptor was measured by binding assay and functional studies. Compound 9p, labeled with Lissamine Rhodamine B using novel solid-phase organic tagging (SPOrT) resin, exhibited a high affinity for V?R (4.0 nM), an excellent selectivity toward V?R and antagonist properties. By changing the nature of the dye, DY647 and Lumi4-Tb probes 44 and 47 still display a high affinity for V?R (5.6 and 5.8 nM, respectively). These antagonists constitute the first high-affinity selective nonpeptidic fluorescent ligands for V?R. They enabled the development of V?R time-resolved FRET-based assay readily amenable to high-throughput screening. Taking advantage of their selectivity, these compounds were also successfully involved in the study of V(1a)R-V?R dimerization on cell surface. PMID:22984902

Loison, Stéphanie; Cottet, Martin; Orcel, Hélène; Adihou, Hélène; Rahmeh, Rita; Lamarque, Laurent; Trinquet, Eric; Kellenberger, Esther; Hibert, Marcel; Durroux, Thierry; Mouillac, Bernard; Bonnet, Dominique



GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations  

SciTech Connect

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.

Cronn, M.T.; Miyada, C.G.; Fucini, R.V. [Affymetrix, Santa Clara, CA (United States)] [and others



Rapid and Simple Kinetics Screening Assay for Electrophilic Dermal Sensitizers using Nitrobenzenethiol  

PubMed Central

The need for alternatives to animal based skin sensitization testing has spurred research on the use of in-vitro, in silico and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped flow techniques and conventional UV spectrophotometric measurements enabled determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for 7 extreme, 5 strong, 7 moderate and 4 weak/non-sensitizers. 17 out of the 23 tested chemicals were pseudo-first order and 3 were second order. In 3 out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, diones and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid and inexpensive absorbance based method has great potential for use as a preliminary screening tool for skin allergens.

Chipinda, Itai; Ajibola, Risikat O.; Morakinyo, Moshood K.; Ruwona, Tinashe B.; Simoyi, Reuben H.; Siegel, Paul D.



A simple assay for 3-deoxy-d-manno-octulosonate cytidylyltransferase and its use as a pathway screen.  


This article describes the adaptation of a simple colorimetric assay for inorganic pyrophosphate to the enzyme 3-deoxy-d-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase, KdsB, EC, a key enzyme in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative organisms. This assay is particularly useful because it can be combined with the malachite green (MG) assay for inorganic phosphate to form an assay system capable of determining inorganic phosphate and inorganic pyrophosphate in the same solution (the MG/EK (eikonogen reagent) assay). This assay system has the potential for simultaneous screening of the 3-deoxy-d-manno-octulosonate (KDO) biosynthesis pathway. We tested this potential using two enzymes, KdsB and KdsC, involved in the biosynthesis and use of the key bacterial 8-carbon sugar, KDO. PMID:21669179

Yi, Li; Velasquez, Melvin S; Holler, Tod P; Woodard, Ronald W



Automated High Throughput Screening for Serine Kinase Inhibitors Using a LEADSeeker™ Scintillation Proximity Assay in the 1536Well Format  

Microsoft Academic Search

High-throughput screening in the 1536-well format has been largely restricted to solution-based and cell-based screens. In this article, we show the feasibility of a completely automated, robust scintillation proximity assay in the 1536-well format that is suitable to identify inhibitors for a serine\\/threonine kinase from a compound library. The introduction of [33P]phosphate into a biotinylated peptide substrate mirrors the activity

Gabriele Sorg; Hans-Dieter Schubert; Frank H. Büttner; Ralf Heilker



Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish  

Microsoft Academic Search

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition

Steven L. Walker; Junko Ariga; Jonathan R. Mathias; Veena Coothankandaswamy; Xiayang Xie; Martin Distel; Reinhard W. Köster; Michael J. Parsons; Kapil N. Bhalla; Meera T. Saxena; Jeff S. Mumm



Quantitative assessment of estrogenic activity in the water environment of Korea by the E-SCREEN assay  

Microsoft Academic Search

In this study, the E-SCREEN assay was optimized and validated for the sensitive quantitative determination of the total estrogenicity in river samples. River water and sediment samples were collected and analyzed with the E-SCREEN. River water (10 l) was extracted using combined solid-phase extraction in static adsorption mode with Soxhlet extraction. Estrogenic pollutants adsorbed to the XAD-4 resin were recovered

Seung-Min Oh; Se-Young Choung; Yhun-Yhong Sheen; Kyu-Hyuck Chung



Detection of thyroid system-disrupting chemicals using in vitro and in vivo screening assays in Xenopus laevis.  


We developed a thyroid hormone (TH) inducible primary screening assay for the identification and assessment of man-made chemicals that interfere with the TH-signalling pathway within target cells. The assay was developed in a Xenopus laevis cell line that was transduced with a self-inactivating (SIN) lentivirus vector (LV) containing a luciferase gene. The luciferase activation in this cell line was TH-specific: 3,3',5-L-triiodothyronine (T(3)) > 3,3'5-L-triiodothyroacetic acid (Triac) > 3,3',5-D-triiodothyronine (D-T(3)), > L-thyroxine (T(4)) > 3,3',5'-L-triiodothyronine (rT(3)). The application of the ligand-dependent luciferase assay for screening for thyroid system-disrupting chemicals revealed that three phthalates (dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate), two herbicides (ioxynil and pentachlorophenol) and a miticide (dicofol) had 3,3',5-L-triiodothyronine- T(3)- antagonist activity at concentrations ranging from 10(-6) to 10(-5) M. These chemicals also inhibited the expression of the endogenous primary T(3)-response TH nuclear receptor beta (TRbeta) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T(3)-dependent activation of TRbeta gene was more sensitive than the luciferase assay. These results indicate that the luciferase assay was a rapid method with a small intra-assay variation for the primary screening of thyroid system-disrupting chemicals. Of the six chemicals, only n-butylbenzyl phthalate and pentachlorophenol exhibited T(3)-antagonist activity in an in vivo metamorphosis-based assay. It should be noted that chemicals elicited thyroid system-disrupting activity in the luciferase assay did not always interfere with the thyroid system in vivo. PMID:16179385

Sugiyama, Shin-Ichiro; Shimada, Naoyuki; Miyoshi, Hiroyuki; Yamauchi, Kiyoshi



A GFP-tagged nucleoprotein-based aggregation assay for anti-influenza drug discovery and antibody development.  


Influenza is a viral pandemic that affects millions of people worldwide. Seasonal variations due to genetic shuffling and antigenic drifts in the influenza viruses have necessitated continual updating of therapeutics. The growing resistance to current influenza drugs has increased demand for new antivirals. The highly conserved nature of NP, a multi-functional viral protein that is serotypically distinct and abundantly expressed during infection, has led to its use in developing universal biotherapeutics and vaccines that could be effective against the virus, irrespective of its strain variations. Compounds causing aggregation of NP have recently been shown to be potent antivirals but require the development of new high-throughput assays capable of screening compounds with similar modes of action. Here, we describe the development of a new bioassay for the Influenza A nucleoprotein (NP). The assay was developed to quantify ligand-induced aggregation of a GFP-tagged NP and was validated with aggregation-inducing compounds such as nucleozin and a NP-specific antibody. The new NP-GFP aggregation assay can be performed with partially purified or mixtures of proteins and is amenable to a high-throughput format. Using this assay, we demonstrate the potential of a new anti-NP polyclonal antibody that we have obtained from chicken. This cost-effective high-yield source of anti-NP IgY has potential for large-scale production and development of therapeutic antibodies. The simplicity, speed and flexibility of this assay make it an invaluable tool for timely development of effective antivirals that can help to control future epidemics. PMID:23961535

Antony, Helma; Schaeffer, Patrick M



A new colorimetric assay for glutathione transferase-catalyzed halogen ion release for high-throughput screening.  


Glutathione transferases (GSTs; EC form a group of multifunctional enzymes catalyzing the conjugation of a broad range of toxicologically important halogenated compounds to the tripeptide glutathione (GSH) with concomitant halogen ion release. In the present work, a rapid quantitative screening method for GSTs based on colorimetric measurement of halogen ions released from halogenated xenobiotics was developed. The assay is based on the color formation resulting from the reaction of Hg(SCN)(2) with the released halogen ion of the substrate in the presence of Fe(3+). The color intensity is proportional to the extent of the catalytic reaction, allowing a quantitative measurement of the GST catalytic activity. The assay can be performed using purified recombinant enzyme (the isoenzyme GmGSTU4-4 from Glycine max) or crude recombinant Escherichia coli cell lysates in 96-well microtiter plates. The suitability of the colorimetric assay for screening mutant GST variants derived from a directed evolution library was successfully evaluated. In addition, the assay was also used for screening GST synthetic inhibitors. It was concluded that the proposed colorimetric assay is selective and sensitive and allows the screening of large numbers of samples within a few minutes. PMID:20529659

Skopelitou, Katholiki; Labrou, Nikolaos E



Efficacy of a Polyethylene Glycol Marker System in Urine Drug Screening in an Opiate Substitution Program  

Microsoft Academic Search

Aims: Screening for concomitant drug consumption is necessary in opiate substitution therapy of opiate-dependent patients. Adulteration of samples is a common problem in this setting. A recently developed polyethylene glycol marker system allows reliable identification of urine samples. In this study, we aimed to compare the rates of drug detection in conventional and marker urine samples. Design: This cross-sectional evaluation

Harald Jörn Schneider; Birgit Rühl; Kirsten Meyer; Ruprecht Keller; Markus Backmund



Organ donor screening using parallel nucleic acid testing allows assessment of transmission risk and assay results in real time.  


Expansion of the donor pool may lead to utilization of donors with risk factors for viral infections. Donor laboratory screening relies on serological and nucleic acid testing (NAT). The increased sensitivity of NAT in low prevalence populations may result in false-positive results (FPR) and may cause unnecessary discard of organs.We developed a screening algorithm to deal, in real time, with potential FPR. Three NAT assays: COBAS AmpliScreen assay (CAS), AmpliPrep Total Nucleic Acid Isolation/CAS, and AmpliPrep/TaqMan assays, were validated and used in parallel for prospective screening of increased-risk donors (IRD), and the probability of FPR was calculated. The lower limit of detection of this algorithm was 9.79, 21.02, and 4.31 IU/mL for human immunodeficiency virus-1, hepatitis C virus, and hepatitis B virus, respectively, with an average turn-around-time of 7.67 h from sample receipt to result reporting. The probability that a donor is potentially infectious with two NAT concordant results was >90%. NAT screening of 35 IRD within 18 months resulted in transplantation of 102 additional organs that without screening would either not be used or used with restrictions in Australia. Using a parallel testing algorithm, real-time confirmation of seropositive donors allows use of organs from IRD and safer expansion of the donor pool. PMID:22519518

Baleriola, C; Tu, E; Johal, H; Gillis, J; Ison, M G; Law, M; Coghlan, P; Rawlinson, W D



Defining Drug Targets in Yeast Haploinsufficiency Screens: Application to Human Translational Pharmacology  

NSDL National Science Digital Library

A major challenge in drug discovery is to identify the cellular targets responsible for the pharmacological activity of drug candidates. In the yeast Saccharomyces cerevisiae, a heterozygous diploid mutant collection of ~6000 strains, in each of which one copy of a single gene is deleted, is commercially available. With this collection, it is possible to evaluate the role of each gene product in the response of cells to a drug. Drug-induced haploinsufficiency refers to the situation where a heterozygous diploid mutant is more sensitive to a drug than is the wild-type strain. Drug-induced haploinsufficiency screening has the potential to reveal pharmacological targets of drugs and those that contribute to undesired side effects, as well as gene products involved in drug transport, metabolism, or resistance. Using published studies, I present advantages and limitations of this technique and discuss its value for predicting drug targets in human cells.

Michel Roberge (University of British Columbia;Department of Biochemistry and Molecular Biology REV)



A pair of new statistical parameters for quality control in RNA interference high-throughput screening assays  

Microsoft Academic Search

RNA interference (RNAi) high-throughput screening (HTS) enables massive parallel gene silencing and is increasingly being used to reveal novel connections between genes and disease-relevant phenotypes. The application of genome-scale RNAi relies on the development of high-quality RNAi HTS assays. To obtain high-quality HTS assays, there is a strong need for an easily interpretable and theoretically based quality control (QC) metric.

Xiaohua Douglas Zhang



High-Throughput Screening of Ototoxic and Otoprotective Pharmacological Drugs  

ERIC Educational Resources Information Center

|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…

Kalinec, Federico



A fluorescence lifetime-based assay for serine and threonine kinases that is suitable for high-throughput screening.  


We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing that the assay performs as well as, and in some cases better than, the "gold standard" radiometric kinase assays with respect to Z' values, demonstrating its utility in high-throughput screening applications. We also show that the assay gives nearly identical results in trial screening to those obtained by radiometric assays and that it is less prone to interference than simple fluorescence intensity measurements. PMID:20230774

Paterson, Michael J; Dunsmore, Colin J; Hurteaux, Reynald; Maltman, Beatrice A; Cotton, Graham J; Gray, Alexander



High Throughput Screening for Small Molecule Enhancers of the Interferon Signaling Pathway to Drive Next-Generation Antiviral Drug Discovery  

PubMed Central

Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z?>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit.

Patel, Dhara A.; Patel, Anand C.; Nolan, William C.; Zhang, Yong; Holtzman, Michael J.



New Approach for High-Throughput Screening of Drug Activity on Plasmodium Liver Stages  

PubMed Central

Plasmodium liver stages represent potential targets for antimalarial prophylactic drugs. Nevertheless, there is a lack of molecules active on these stages. We have now developed a new approach for the high-throughput screening of drug activity on Plasmodium liver stages in vitro, based on an infrared fluorescence scanning system. This method allowed us to count automatically and rapidly Plasmodium-infected hepatocytes, using different hepatic cells and different Plasmodium species, including Plasmodium falciparum. This new technique is well adapted for high-throughput drug screening and should facilitate the identification of new antimalarial compounds active on Plasmodium liver stages.

Gego, Audrey; Silvie, Olivier; Franetich, Jean-Francois; Farhati, Khemais; Hannoun, Laurent; Luty, Adrian J. F.; Sauerwein, Robert W.; Boucheix, Claude; Rubinstein, Eric; Mazier, Dominique



Screening for small molecule modulators of Hsp70 chaperone activity using protein aggregation suppression assays: inhibition of the plasmodial chaperone PfHsp70-1.  


Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-?-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC(50) values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function. PMID:21426241

Cockburn, Ingrid L; Pesce, Eva-Rachele; Pryzborski, Jude M; Davies-Coleman, Michael T; Clark, Peter G K; Keyzers, Robert A; Stephens, Linda L; Blatch, Gregory L



Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization  

PubMed Central

Targeting protein–protein interactions (PPI) is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV)-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of HIV-1 proteins with many host cellular components. In this study, a bimolecular fluorescence complementation (BiFC)-based screening assay was developed that can quantify changes in dimerization, using HIV-1 viral protein R (Vpr) dimerization as a “proof of concept.” Results demonstrated that Venus Vpr (generated by BiFC Vpr constructs) could be competed off in a dose-dependent manner using untagged, full-length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of small molecules for an effect on Vpr dimerization. Among the tested molecules, a few of the small molecules demonstrate an effect on Vpr dimerization in a dose-dependent manner.

Zych, Courtney; Domling, Alexander; Ayyavoo, Velpandi



The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants.  

PubMed Central

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.

Soto, A M; Sonnenschein, C; Chung,