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At least 865 records · Page 48Linked to original sources
PubMed2004

Reliable and high-throughput mutation screening for beta-thalassemia by a single-base extension/fluorescence polarization assay.

beta-thalassemia is one of the most common inherited diseases with incidence varying between 3% and 10% in the high-prevalence regions of South China. The molecular defects are mostly due to single-nucleotide substitutions, minor insertions, and deletions in the beta-globin gene. Large-scale population genetic screening combined with prenatal diagnosis is necessary for the effective prevention of this disease. We present a single base extension (SBE) method based on homogenous fluorescence polarization (FP) for simultaneous detection of the eight most common causative mutations [CDs 41-42 (-TCTT), IVS-2-654 (C-->T), -28 (A-->G), CD17 (A-->T), CD 71/72 (+A), CD26 (G-->A), -29 (A-->G), and CD43 (G-->T)] in the beta-globin gene in a Chinese population. This assay has been validated by a blind experiment with 100 clinical samples previously characterized by reverse dot-blot and direct sequencing. The results demonstrate that this high-throughput method is simple, reliable, and cost effective. We expect this approach can be used in large-scale genetic screening for beta-thalassemia.

Asian People↗
PubMed2005

TaqMan genotyping of insertion/deletion polymorphisms.

The 5' fluorogenic (TaqMan) assay has been successfully used in screening for single-nucleotide polymorphisms; the very few steps required and the ability to automate each step allow for high-throughput screening. Insertion/deletion polymorphisms are an important class of markers that can be studied for different applications, such as diagnostics, genome variation, and species identification. Polymerase chain reaction (PCR) and post-PCR analysis are required to score the insertion or the deletion allele. In this chapter, we describe an expansion of the TaqMan technology for a rapid, high-throughput, screening for insertion/deletion polymorphisms in which the exact endpoints are known. The method requires minimal post-PCR analysis and can be applied to polymorphisms of any size.

Alleles↗
PubMed2003

Fluorescence imaging of electrically stimulated cells.

Designing high-throughput screens for voltage-gated ion channels has been a tremendous challenge for the pharmaceutical industry because channel activity is dependent on the transmembrane voltage gradient, a stimulus unlike ligand binding to G-protein-coupled receptors or ligand-gated ion channels. To achieve an acceptable throughput, assays to screen for voltage-gated ion channel modulators that are employed today rely on pharmacological intervention to activate these channels. These interventions can introduce artifacts. Ideally, a high-throughput screen should not compromise physiological relevance. Hence, a more appropriate method would activate voltage-gated ion channels by altering plasma membrane potential directly, via electrical stimulation, while simultaneously recording the operation of the channel in populations of cells. The authors present preliminary results obtained from a device that is designed to supply precise and reproducible electrical stimuli to populations of cells. Changes in voltage-gated ion channel activity were monitored using a digital fluorescent microscope. The prototype electric field stimulation (EFS) device provided real-time analysis of cellular responsiveness to physiological and pharmacological stimuli. Voltage stimuli applied to SK-N-SH neuroblastoma cells cultured on the EFS device evoked membrane potential changes that were dependent on activation of voltage-gated sodium channels. Data obtained using digital fluorescence microscopy suggests suitability of this system for HTS.

Calcium↗
PubMed2000

Maximizing synthetic efficiency: multi-component transformations lead the way

With the emergence of high-throughput screening in the pharmaceutical industry in the early 1990's, organic chemists were faced with a new challenge: how to prepare large collections of molecules (the libraries) to "feed" the high-throughput screen? The unique exploratory power of some reactions (such as the 40 year-old Ugi four-component condensation) was soon recognized to be extremely valuable to produce libraries in a time- and cost-effective manner. Over the last five years, industrial and academic researchers have made these powerful transformations into one of the most efficient and cost-effective tools for combinatorial and parallel synthesis.

Journal Article↗
PubMed2006

Chemical database preparation for compound acquisition or virtual screening.

Virtual and high-throughput screening are time-saving techniques that have been successfully applied to identify novel chemotypes in biologically active molecules. Both methods require the ability to aptly handle large numbers of chemicals prior to an experiment or acquisition. We describe a step-by-step preparation procedure for handling large collections of existing or virtual compounds prior to virtual screening or acquisition.

Algorithms↗
PubMed2006

Derivation of a novel SARS-coronavirus replicon cell line and its application for anti-SARS drug screening.

The severe acute respiratory syndrome (SARS) outbreak in 2002, which had a high morbidity rate and caused worldwide alarm, remains untreated today even though SARS was eventually isolated and controlled. Development and high-throughput screening of efficacious drugs is therefore critical. However, currently there remains a lack of such a safe system. Here, the generation and characterization of the first selectable, SARS-coronavirus (SARS-CoV)-based replicon cell line which can be used for screening is described. Partial SARS-CoV cDNAs and antibiotic resistance/reporter gene DNA were generated and assembled in vitro to produce the replicon transcription template, which was then transcribed in vitro to generate the replicon RNA. The latter was introduced into a mammalian cell line and the transfected cells were selected for by antibiotic application. For the antibiotic-resistant cell lines thus generated, the expression of reporter gene was ensured by continued monitoring using fluorescent microscopy and flow cytometry. The suitability of this replicon cell line in drug screening was demonstrated by testing the inhibitory effect of several existing drugs and the results demonstrate that the SARS-CoV replicon cell lines provide a safe tool for the identification of SARS-CoV replicase inhibitors. The replicon cell lines thus developed can be applied to high-throughput screening for anti-SARS drugs without the need to grow infectious SARS-CoV.

Animals↗
PubMed2006

A high-throughput drug screening system for HIV-1 transcription inhibitors.

Identification of HIV-1 transcription inhibitors was previously performed using infectivity assays. As de novo HIV-1 infection is highly sensitive to even minor compound toxicities, these assays are plagued by extremely high levels of false-positive hits. Hit identification is further complicated because infectivity assays lack target specificity. The authors demonstrate that it is possible to overcome these limitations by establishing a stable, chronically actively HIV-1-infected reporter cell line that exclusively identifies HIV-1 transcription inhibitors. In the reporter cell line, 2 spectrally separated fluorescence proteins serve as simultaneously accessible quantitative markers of HIV-1 expression and drug toxicity. The combined analysis of these markers drastically reduces the level of false-positive hits. As determination of fluorescence intensity in a plate-based format can be performed in a noninvasive manner, repeated measurements of fluorescence levels over several days after compound addition can be used to define the kinetic and dynamic characteristics of inhibitory drug effects. In addition, because of the stable nature of the reporter cell line, the assay requires no cell manipulation during assay preparation or assay analysis, rendering the system extremely cost-effective and reliable.

Animals↗
PubMed1998

Radioligand-binding assay employing P-glycoprotein-overexpressing cells: testing drug affinities to the secretory intestinal multidrug transporter.

PURPOSE: To develop a rapid and reliable system for affinity determination of conventional as well as newly synthesized compounds to P-gp. METHODS: The principles of radioligand-binding assay were adapted to the human intestinal P-gp. Acceptor protein was obtained from the human carcinoma cell line Caco-2, where overexpression of P-gp was induced by growing cells in the presence of the cytostatic drug vinblastine. 3H-Verapamil was chosen as radioligand. RESULTS: The saturability and specificity of 3H-verapamil as the radioligand for the binding to P-gp was demonstrated. From concentration dependence of displacement of the radioligand by various non-labeled ligands for P-gp, affinity constants to P-gp binding sites were calculated. The binding results obtained were in agreement with those published earlier where influx and efflux experiments with cell monolayers had been conducted in order to functionally characterize the P-gp -drug interaction. CONCLUSIONS: A radioligand-binding assay on the basis of P-gp overexpressing Caco-2 cells has been developed. The method might be suitable for high-throughput screening of drug interaction with human P-gp. It will allow modeling of the interaction of drugs with the human multidrug transporter and has also the potential to serve as a high-throughput screening tool to detect compounds prone to P-gp mediated intestinal secretion and potential P-gp related drug/drug interactions in drug discovery and early development.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Hepatitis B virus e antigen production is dependent upon covalently closed circular (ccc) DNA in HepAD38 cell cultures and may serve as a cccDNA surrogate in antiviral screening assays.

Currently available antiviral nucleoside analogs for the treatment of chronic hepatitis B virus (HBV) infections profoundly reduce virus load, but rarely cure the virus infection. This is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) DNA from the nuclei of infected hepatocytes. To screen compound libraries for antiviral drugs targeting cccDNA, we set out to develop a cell-based assay suitable for high throughput screening. Since cccDNA is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccDNA level. We predicted that the secretion of HBV e antigen (HBeAg) by HepAD38 cells, a tetracycline inducible HBV expression cell line, would be cccDNA-dependent. This is because a large portion of pre-core mRNA leader sequence in the 5' terminus of integrated viral genome was deleted, preventing HBeAg expression from transgene, but could be restored from the 3' terminal redundancy of pre-genomic RNA during viral DNA replication and subsequent cccDNA formation. Our experimental results showed that following induction, HepAD38 produced and accumulated cccDNA, which became detectable between 7 and 8 days. HBeAg synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccDNA detected. Therefore, the secretion of HBeAg by HepAD38 cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting HBV cccDNA.

5' Untranslated Regions↗
PubMed2000

High-throughput and virtual screening: core lead discovery technologies move towards integration.

In addition to high-throughput screening (HTS), the main lead discovery technology employed by most pharmaceutical companies today is virtual screening (VS). Although the two techniques have somewhat different philosophical origins, they contain many synergies that can potentially enhance the lead discovery process. Here, we describe many of the latest developments in VS technology with particular emphasis on their potential impact on HTS in, for example, focussed screening and data mining. In addition, we highlight key issues that need to be addressed before the potential of such efforts can be fully realized.

Journal Article↗
PubMed2006

Obtaining and screening compound collections: a user's guide and a call to chemists.

Advances in genetics, proteomics and cell biology over the past 20 years have unearthed a multitude of potential macromolecular targets for the selective treatment of disease. The challenge remains to find appropriate small molecule ligands for these proteins (or nucleic acids), and to use these ligands to validate novel disease targets. The advent of low-cost instrumentation has made industrial-style high-throughput screening possible in academic settings. Unfortunately for many, access to large collections of compounds is still limited and limiting. This article is aimed at the user who has an interest in compound screening but does not have ready access to large collections of small molecules. High-throughput screening need not be the exclusive domain of institutions and centers with vast resources and NIH Roadmap-funded compound repositories. As it turns out, many of the most interesting compounds are probably within arm's reach, in our laboratory freezers and in those of our colleagues.

Combinatorial Chemistry Techniques↗
PubMed2007

Discovering potassium channel blockers from synthetic compound database by using structure-based virtual screening in conjunction with electrophysiological assay.

Potassium ion (K+) channels are attractive targets for drug discovery because of the essential roles played in biological systems. However, high-throughput screening (HTS) cannot be used to screen K+ channel blockers. To overcome this disadvantage of HTS, we have developed a virtual screening approach for discovering novel blockers of K+ channels. On the basis of a three-dimensional model of the eukaryotic K+ channels, molecular docking-based virtual screening was employed to search the chemical database MDL Available Chemicals Directory (ACD). Compounds were ranked according to their relative binding energy, favorable shape complementarity, and potential to form hydrogen bonds with the outer mouth of the K+ channel model. Twenty candidate compounds selected from the virtual screening were examined using the whole-cell voltage-clamp recording in rat dissociated hippocampal neurons. Among them, six compounds (5, 6, 8, 18-20) potently blocked both the delayed rectifier (IK) and fast transient K+ currents (IA). When applied externally, these six compounds preferentially blocked IK with potencies 2- to 500-fold higher than that of tetraethylammonium chloride. Intracellular application of the six compounds had no effect on both K+ currents. In addition, the interaction models and binding free energy calculations demonstrated that hydrophobic interaction and solvent effects play important roles in the inhibitory activities of these compounds. The results demonstrated that structure-based computer screening strategy could be used to identify novel, structurally diverse compounds targeting the pore binding pocket of the outer mouth of voltage-gated K+ channels. This study provides an alternative way of finding new blockers of voltage-gated K+ channels, while the techniques for high-throughput screening of K+ channel drugs remain in development.

Animals↗
PubMed2004

Shared components of protein complexes--versatile building blocks or biochemical artefacts?

Protein complexes perform many important functions in the cell. Large-scale studies of protein-protein interactions have not only revealed new complexes but have also placed many proteins into multiple complexes. Whilst the advocates of hypothesis-free research touted the discovery of these shared components as new links between diverse cellular processes, critical commentators denounced many of the findings as artefacts, thus questioning the usefulness of large-scale approaches. Here, we survey proteins known to be shared between complexes, as established in the literature, and compare them to shared components found in high-throughput screens. We discuss the various challenges to the identification and functional interpretation of bona fide shared components, namely contaminants, variant and megacomplexes, and transient interactions, and suggest that many of the novel shared components found in high-throughput screens are neither the results of contamination nor central components, but appear to be primarily regulatory links in cellular processes.

Algorithms↗
PubMed2005

Use of Leishmania donovani field isolates expressing the luciferase reporter gene in in vitro drug screening.

Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

Animals↗
PubMed2006

Configuration of a scintillation proximity assay for the activity assessment of recombinant human adenine phosphoribosyltransferase.

Adenine phosphoribosyltransferase plays a role in purine salvage by catalyzing the direct conversion of adenine to adenosine monophosphate. The involvement of the purine salvage pathway in tumor proliferation and angiogenesis makes adenine phosphoribosyltransferase a potential target for oncology drug discovery. We have expressed and characterized recombinant, N-terminally His-tagged human adenine phosphoribosyltransferase. Two assay formats were assessed for use in a high throughput screen: a spectrophotometric-based enzyme-coupled assay system and a radiometric ionic capture scintillation proximity bead assay format. Ultimately, the scintillation proximity assay format was chosen because of automated screening compatibility limitations of the coupled assay. We describe here the biochemical characterization of adenine phosphoribosyltransferase and the development of a robust, homogeneous, 384-well assay suitable for high throughput screening.

Adenine↗
PubMed2002

High throughput mutation screening of the factor VIII gene (F8C) in hemophilia A: 37 novel mutations and genotype-phenotype correlation.

Hemophilia A (HEMA) is an X-linked bleeding disorder caused by mutations in the factor VIII gene (F8C). Molecular genetic testing for the factor VIII gene is challenging due to its large size. Here we present results of high throughput mutation scanning based on Southern blot analysis and direct sequencing of all PCR amplified coding exons and the exon-intron boundaries of the factor VIII gene. The results of mutation analysis on 89 hemophiliac males showed presence of a disease-causing mutation in 80 individuals (90%, 95% CI of 82%-95%). Seven out of nine mutation-negative individuals were severe cases of hemophilia A with < 1% factor VIII protein in the blood. The correlation of phenotype with genotype as observed in this study was not absolute. This finding is supported by similar observations in the international database for hemophilia A mutations (HAMSTeRS). This issue raises the importance of genotypes at other loci that can act as modifiers for the phenotype. Thirty-four novel mutations and three novel substitutions for previously reported amino acid residues were identified in this series of 80 mutations. The mutations cover the full spectrum including rearrangements, deletions, frameshift, and point mutations. The novel missense mutations require careful evaluation. Prediction of a mutation as the disease-causing allele was made from the nature of the substitution and the degree of conservation of the mutated amino acid among species that have diverged in evolution. In some cases segregation analysis of the mutation with disease condition was performed when other family members were available.

Child↗
PubMed2002

High-throughput genetic screening using matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a powerful and widespread analytical tool in all fields of life science. Compared with other techniques, its high accuracy and sensitivity makes it a superior method, especially for the analysis of nucleic acids. Recent problems in the analysis of nucleic acids by MALDI-TOF MS can be solved using an automated MALDI-compatible sample-preparation system. Together with the reliable minisequencing assay, high-throughput genotyping of single nucleotide polymorphisms by MALDI-TOF MS is able to become a routine method in research, clinical genetics and diagnostics.

Automation↗
PubMed2004

Screening of some anti-androgenic endocrine disruptors using a recombinant cell-based in vitro bioassay.

The present work describes the development and optimization of a cell-based androgen reporter assay using the Chinese hamster ovarian cell line (CHO K1) in the 96-well format. The recent reports on increasing exposure of humans and wild-life to environmental endocrine disrupting chemicals (ED) prompt the need for high throughput screening systems for such compounds in environmental and biological samples. To this end, CHO cells were cotransfected with plasmids encoding mouse mammary tumour virus-neomycin-luciferase and human androgen receptor (hAR), and a stable cell line was established. After selection with neomycin, a highly active clone was obtained which stably expressed both the hAR and the androgen-responsive luciferase reporter. Stimulation of the cells with androgens for 24 h resulted in about 15-fold stimulation of luciferase activity, with the minimum effective dose of testosterone being 0.1 nmol/l. Potent steroidal and non-steroidal anti-androgens, such as hydroxyflutamide and cyproterone acetate, significantly inhibited the androgen-induced transactivation. Non-androgenic steroids like estradiol, progesterone, dexamethasone and cortisol showed weak activity at high concentrations. RT-PCR and western blot confirmed proper transcription and translation as well as stable expression of the AR gene in the cells. About 60 different chemicals (mostly pesticides or their metabolites, and common industrial chemicals) were screened with the cell line for their ability to stimulate luciferase activity or inhibit that evoked by 0.1 nmol/l R1881, used as a positive androgenic control. About 10 highly potent anti-androgenic chemicals were identified. The most potent anti-androgenic compounds identified in our assay included bisphenol A, alpha-hexachlorocyclohexane, vinclozolin and 4,4-DDE. These compounds had alone either no effect or were weak agonists (with cytotoxic effects at very high concentrations), but none showed any significant agonistic activity. In conclusion, we demonstrate that the bioassay based on this cell line provides a reliable test for detecting androgenic and anti-androgenic compounds. The 96-well plate format makes the assay suitable for high throughput screening.

Androgen Antagonists↗