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At least 271 records · Page 15Linked to original sources
PubMed2000

Development of a homogeneous time-resolved fluorescence assay for high throughput screening to identify Lck inhibitors: comparison with scintillation proximity assay and streptavidin-coated plate assay.

This study details the development of a homogeneous time-resolved fluorescence (HTRF) high throughput screening assay to identify inhibitors of Lck. HTRF was compared with scintillation proximity and streptavidin-coated plate assays. Because of the differences in the sensitivity of detection of phosphotyrosine among the three assays, different amounts of enzyme were used. However, the concentrations of the other assay components were standardized. When using similar assay conditions, the calculated IC(50) values of inhibitory compounds were independent of assay format. Furthermore, filtration experiments revealed that phosphorylation of a biotinyl poly-Glu,Ala, Tyr peptide substrate was less than autophosphorylation of the Lck enzyme; this was due to the low K(m) value for biotinyl poly-Glu,Ala,Tyr. In the HTRF assay, small amounts of enzyme and high concentrations of ATP could be used, thereby minimizing the effects of autophosphorylation. Higher ATP concentration would also minimize the effect of ATP competitors. Using this technology, it may be possible to find novel kinase inhibitors that do not act at the ATP binding site of protein tyrosine kinases.

Biotin↗
PubMed2005

[High throughput screening of pharmacokinetics and metabolism in drug discovery (I)--establishment of assessment system for absorption to compounds with a wide diversity of physical properties].

The application of combinatorial chemistry and high-throughput screening to biological targets has led to efficient identification of lead compounds in wide therapeutic areas. However, the physicochemical properties of some lead compounds are lipophilic with low water soluble. Since these parameters determine in vivo absorption, we established robust screening methods for solubility and Caco-2 membrane permeability which are applicable to our screening strategy based on the structure-pharmacokinetic parameter relationship (SPR). Of test compounds with different core structures, turbidimetric solubility and apparent solubility as determined by HPLC-UV analysis after dilution of aqueous media from DMSO stock solution was overestimated in comparison with the corresponding thermodynamic solubility obtained using a traditional shake-flask method. A new powder-dissolution method providing thermodynamic solubility similar to that in the traditional method was developed using 96-well plates for equilibrium dialysis. The throughput of the method was the almost the same as that using the apparent solubility method. In a conventional Caco-2 assay, membrane permeability (P(app)) of some lipophilic compounds was underestimated due to low solubility in the apical site and adhesion to the device, resulting in a poor relationship between the in vivo absorption fraction and the P(app) values. The addition of 0.1% Gelucire 44/14 into the apical site and 4% bovine serum albumin into the basolateral site improved the relationship. These newly developed methods are therefore useful to optimize lead compounds with less water solubility and high lipophilicity on the basis of SPR.

Animals↗
PubMed1997

Gene expression systems in the development of high-throughput screens.

Recent advances in the development of combinatorial automated chemical synthesis, robotic sample handling, and data collection and analysis have significantly increased the number of compounds available for screening against potential therapeutic targets. The implementation of highly sensitive in vitro biochemical and cell-based high-throughput screening assays is essential to facilitate the rapid identification of selective and potent lead molecules from compound libraries. The ability to easily produce functional proteins in sufficient quantities for in vitro biochemical assays and to devise useful cell-based systems is dependent on the successful application of a variety of gene expression systems.

Animals↗
PubMed2003

A modular, fully integrated ultra-high-throughput screening system based on confocal fluorescence analysis techniques.

The rapid increase in size of compound libraries, as well as new targets emerging from the Human Genome Project, require progress in ultra-high-throughput screening (uHTS) systems. In a joint effort with scientists and engineers from the biotech and the pharmaceutical industry, a modular, fully integrated system for miniaturized uHTS was developed. The goal was to achieve high data quality in small assay volumes (1-4 microL) combined with reliable and unattended operation. Two new confocal fluorescence readers have been designed. One of the instruments is a 4-channel confocal fluorescence reader, measuring with 4 objectives in parallel. The fluorescence readout is based on single-molecule detection methods, allowing high sensitivity at low tracer concentrations and delivering an information-rich output. The other instrument is a confocal fluorescence imaging reader, where the images are analyzed in terms of generic patterns and quantified in units of intensity per pixel. Both readers are spanning the application range from assays with isolated targets in homogenous solution or membrane vesicle-based assays (4-channel reader) to cell-based assays (imaging reader). Results from a comprehensive test on these assay types demonstrate the high quality and robustness of this screening system.

Anti-Bacterial Agents↗
PubMedDate not supplied

Strategic pooling of compounds for high-throughput screening.

Bringing new medicines to the market depends on the rapid discovery of new and effective drugs, often initiated through the biological testing of many thousands of compounds in high-throughput screening (HTS). Mixing compounds together into pools for screening is one way to accelerate this process and reduce costs. This paper contains both theoretical and experimental data which suggest that careful selection of compounds to be pooled together is necessary in order to reduce the risk of reactivity between compounds within the pools.

Chromatography, Liquid↗
PubMed2002

Development of a high throughput screening assay for mitochondrial membrane potential in living cells.

The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC(50) values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.

Animals↗
PubMed2006

High-throughput screening of RNA polymerase inhibitors using a fluorescent UTP analog.

RNA polymerase (RNAP) is a well-validated target for the development of antibacterial and antituberculosis agents. Because the purification of large quantities of native RNA polymerase from pathogenic mycobacteria is hazardous and cumbersome, the primary screening was carried out using Escherichia coli RNAP. The authors have developed a high-throughput screening (HTS) assay to screen for novel inhibitors of RNAP. In this assay, a fluorescent analog of UTP, gamma-amino naphthalene sulfonic acid (gamma-AmNS) UTP, was used as one of the nucleotide substrates. Incorporation of UMP in RNA results in the release of gamma-AmNS-PPi, which has higher intrinsic fluorescence than (gamma-AmNS) UTP. The assay was optimized in a 384-well format and used to screen 670,000 compounds at a concentration of 10 microM. About 0.1% of the compounds showed more than 60% inhibition in the primary HTS. All the primary actives tested for dose response using the same assay had an EC(50) below 100 microM. Eighty percent of the primary HTS actives obtained using E. coli RNAP showed comparable activity against Mycobacterium smegmatis RNAP in the conventional radioactive assay. Activity of hits selected for the hit-to-lead optimization was also confirmed against Mycobacterium bovis RNAP which has >99% sequence identity with Mycobacterium tuberculosis RNAP subunits.

DNA-Directed RNA Polymerases↗
PubMed2003

A fully integrated high-throughput screening methodology for the discovery of new polyolefin catalysts: discovery of a new class of high temperature single-site group (IV) copolymerization catalysts.

For the first time, new catalysts for olefin polymerization have been discovered through the application of fully integrated high-throughput primary and secondary screening techniques supported by rapid polymer characterization methods. Microscale 1-octene primary screening polymerization experiments combining arrays of ligands with reactive metal complexes M(CH(2)Ph)(4) (M = Zr, Hf) and multiple activation conditions represent a new high-throughput technique for discovering novel group (IV) polymerization catalysts. The primary screening methods described here have been validated using a commercially relevant polyolefin catalyst, and implemented rapidly to discover the new amide-ether based hafnium catalyst [eta(2)-(N,O)[bond](2-MeO[bond]C(6)H(4))(2,4,6-Me(3)C(6)H(2))N]Hf(CH(2)Ph)(3) (1), which is capable of polymerizing 1-octene to high conversion. The molecular structure of 1 has been determined by X-ray diffraction. Larger scale secondary screening experiments performed on a focused 96-member amine-ether library demonstrated the versatile high temperature ethylene-1-octene copolymerization capabilities of this catalyst class, and led to significant performance improvements over the initial primary screening discovery. Conventional one gallon batch reactor copolymerizations performed using selected amide-ether hafnium compounds confirmed the performance features of this new catalyst class, serving to fully validate the experimental results from the high-throughput approaches described herein.

Journal Article↗
PubMed2003

Beta galactosidase enzyme fragment complementation as a novel technology for high throughput screening.

In this review, the applications of beta galactosidase complementation are described. alpha Complementation is a naturally occurring process in bacteria and in engineered cells, and can also occur in eukaryotic cells. Two forms of alpha complementation have been used in high throughput screening (HTS), in which interacting fragments complement with either low or high affinity. Low affinity complementation is used to monitor protein protein interactions, such as those occurring in homodimerization of the epidermal growth factor receptor (EGFR), and provides a robust screen for detection of EGFR inhibitors. High affinity complementation provides the basis for several HTS assays, in which analytes, such as cAMP or IP(3), are detected in crude cell lysates. A development of the latter approach is protein labeling, providing for measurement of cell protein expression and trafficking. Collectively, the use of beta galactosidase complementation provides a novel and flexible technology for highly sensitive, homogeneous HTS assay development.

Animals↗
PubMed2006

High-throughput screen for poly-3-hydroxybutyrate in Escherichia coli and Synechocystis sp. strain PCC6803.

A novel, quantitative method for detecting poly-3-hydroxybutyrate (PHB) amounts in viable cells was developed to allow for high-throughput screening of mutant libraries. The staining technique was demonstrated and optimized for the cyanobacterium Synechocystis sp. strain PCC6803 and the eubacterium Escherichia coli to maximize the fluorescence difference between PHB-accumulating and control cells by flow cytometry. In Synechocystis, the level of nonspecific dye binding was reduced by using nonionic stain buffer that allowed quantitation of fluorescence levels. In E. coli, the use of a mild sucrose shock facilitated uptake of Nile red without significant loss of viability. The optimized staining protocols yielded a linear response for the mean fluorescence against (chemically measured) PHB. The staining protocols are novel methods useful in the high-throughput evaluation of combinatorial libraries of Synechocystis and E. coli using fluorescence-activated cell sorting to identify mutants with increased PHB-accumulating properties.

Escherichia coli↗
PubMed2004

Model system for high-throughput screening of novel human immunodeficiency virus protease inhibitors in Escherichia coli.

Novel human immunodeficiency virus (HIV) protease inhibitors are urgently needed for combating the drug-resistance problem in the fight against AIDS. To facilitate lead discovery of HIV protease inhibitors, we have developed a safe, convenient, and cost-effective Escherichia coli-based assay system. This E. coli-based system involves coexpression of an engineered beta-galactosidase as an HIV protease substrate and the HIV protease precursor comprising the transframe region and the protease domain. Autoprocessing of the HIV protease precursor releases the mature HIV protease. Subsequently, the HIV protease cleaves beta-galactosidase, resulting in a loss of the beta-galactosidase activity, which can be detected in high-throughput screens. Using Food and Drug Administration-approved HIV protease inhibitors, this E. coli-based system is validated as a surrogate screening system for identifying inhibitors that not only possess inhibitory activity against HIV protease but also have solubility and permeability for in vivo activity. The usefulness of the E. coli-based system was demonstrated with the identification of a novel HIV protease inhibitor from a library of compounds that were prepared by an amide-forming reaction with transition-state analog cores. A novel inhibitor with a sulfonamide core of amprenavir, E2, has shown good correlation with the in vitro enzymatic assay and in vivo E. coli-based system. This system can also be used to generate drug resistance profiles that could be used to suggest therapeutic uses of HIV protease inhibitors to treat the drug-resistant HIV strains. This simple yet efficient E. coli system not only represents a screening platform for high-throughput identification of leads targeting the HIV proteases but also can be adapted to all other classes of proteases.

Amides↗
PubMed2005

Development of a miniaturized assay for the high-throughput screening program for poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.

Biotinylation↗
PubMed2003

Agrosuppression: a bioassay for the hypersensitive response suited to high-throughput screening.

We describe a novel method, agrosuppression, that addresses the need for an assay of the hypersensitive response (HR) in intact plants that is rapid and adapted to high-throughput functional screening of plant and pathogen genes. The agrosuppression assay is based on inoculation of intact plants with a mixture of Agrobacterium tumefaciens strains carrying (i) a binary plasmid with one or more candidate HR-inducing genes and (ii) a tumor-inducing (oncogenic) T-DNA. In the absence of HR induction, tumor formation is initiated, resulting in a typical crown gall phenotype. However, upon induction of the HR, tumor formation by the oncogenic T-DNA is suppressed, resulting in a phenotype that can be readily scored. We tested and optimized agrosuppression in Nicotiana benthamiana using the inf1 elicitin gene from the oomycete pathogen Phytophthora infestans, which specifically induces the HR in Nicotiana spp., and the gene-for-gene pair Avr9/Cf-9 from the fungal pathogen Cladosporium fulvum and Lycopersicon pimpinellifolium (currant tomato), respectively. Agrosuppression protocols that can be rapidly performed using simple mechanical wounding of petioles of intact N. benthamiana plants were developed and appeared particularly adapted to intensive high-throughput screening. This assay promises to greatly facilitate the cloning of novel plant R genes and pathogen Avr genes and to accelerate functional analyses and structure-function studies of these genes.

Agrobacterium tumefaciens↗
PubMed2005

Soluble domains of telomerase reverse transcriptase identified by high-throughput screening.

Telomerase is a ribonucleoprotein complex responsible for extending the ends of eukaryotic chromosomes. Structural and biophysical studies of this enzyme have been limited by the inability to produce large amounts of recombinant protein. Here we perform a high-throughput screen to map regions of the Tetrahymena thermophila TERT (Telomerase Reverse Transcriptase) protein that are overexpressed in a soluble form in Escherichia coli using a GFP-fusion system. Many of the soluble protein domains identified do not coincide with domains inferred from multiple sequence alignment, so screening for fluorescent colonies provided information not otherwise readily obtained. The method revealed an essential, independently folded N-terminal domain that was expressed and purified with high yield and found to be suitable for structural analysis. These results provide a tool for future structural and biophysical studies of TERT.

Amino Acid Sequence↗
PubMed2001

High throughput screening: a rapid way to recombinant allergens.

Complex allergenic sources such as moulds, foods and mites contain large panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. Phage display of cDNA libraries allows selective enrichment of allergen-expressing clones using IgE from allergic patients. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, however, high-throughput screening technology is required. We have used a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries in order to characterize whole allergenic repertoires from complex allergenic sources. The strategy, based on a combination of phage display and high-density arrays, allowed fast discovery of panels of related structures from different allergenic sources. They cover secreted, cytoplasmic and structural proteins with or without enzymatic activity and offer a rational explanation for the IgE-mediated cross-reactivity frequently encountered in clinical practice.

Allergens↗
PubMed2002

An evaluation of fluorescence polarization and lifetime discriminated polarization for high throughput screening of serine/threonine kinases.

We used two kinases, c-jun N terminal kinase (JNK-1) and protein kinase C (PKC), as model enzymes to evaluate the potential of fluorescence polarization (FP) for high-throughput screening and the susceptibility of these assays to compound interference. For JNK-1 the enzyme kinetics in the FP assay were consistent with those found in a [gamma-33P]ATP filter wash assay. Determined pIC(50)s for nonfluorescent JNK-1 inhibitors were also consistent with those found in the filter wash assay. In contrast, fluorescent compounds were found to interfere with the JNK-1 FP assay, appearing as false positives, defined by their lack of activity in the filter wash assay. We also developed a second assay using a different kinase, protein kinase C, which was used to test a 5000 compound diversity set. As for JNK-1, interference from fluorescent compounds caused a high false positive rate. The Molecular Devices Corporation 'FLARe' instrument is capable of discriminating between fluorophores on the basis of their fluorescence (excited state) lifetime, and may assist in reducing compound interference in fluorescent assays. In both model FP kinase assays described here some, although not complete, reduction in interference from fluorescent compounds was achieved by the use of FLARe.

Activating Transcription Factor 2↗
PubMed2006

The high throughput screening of neuropeptide FF2 receptor ligands from Korean herbal plant extracts.

We have screened 356 libraries of Korean herbal plant extracts to find potential anti-obesity drugs. We employed the recently developed fluorescence polarization high throughput screening (FP HTS) assays of human neuropeptide FF (NPFF) receptors in 384-well microtiter plates. The primary hits were cherry-picked from the libraries and further analyzed by secondary displacement curve assays, in vitro GTPgammaS binding assays and cell-based CRE luciferase reporter assays. Agonists of NPFF receptors showed biphasic affinity curves while the antagonist, BIBP 3226, gave a monophasic affinity curve in competitive binding assays. We isolated and characterized two agonists of human NPFF2 receptor, PC 314 with K(i) of 1.42 microM, and PC 315 with K(i) of 2.17 microM from Schizandra chinensis. PC 314 and PC 315 have been characterized as benzoylgomisin Q (M.W. 552) and gomisin G (M.W. 536). We report that PC 314 and PC 315 are the first non-peptide, natural compounds, which bind to human NPFF2 receptors with good affinity. PC 314 and PC 315 inhibit forskolin-stimulated luciferase expression when CHO cells are co-transfected with NPFF2 receptor and CRE reporter vector. They possess the pharmacological and functional profiles of full agonists. The FP HTS system provides a specific, sensitive and reproducible methodology for studying and screening NPFF receptor ligands.

Combinatorial Chemistry Techniques↗
PubMed2002

High-throughput screening of soluble recombinant proteins.

The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure-function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well-expressed soluble fusion protein, total cell lysates of bacteria culture ( approximately 1.5 mL) were subjected to high-speed centrifugation in a 96-tube format and analyzed by multiwell denaturing SDS-PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis.

Animals↗