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Results for “High Throughput Screening”

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PubMed2002

High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. In order to provide proteins for the 'protein structure factory', a structural genomics initiative, we are working on the high-throughput expression of human proteins. Therefore, cDNAs are cloned for intracellular expression. The resulting fusion proteins carry affinity tags (6*HIS and StrepII, respectively) at the N- and C-terminus for the immunological detection and chromatographic purification of full-length proteins. Expression is controlled by the tightly regulated and highly inducible alcoholoxidase 1 (AOX1) promoter. We have developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression. The screening procedure is based on a culture volume of 2 ml in a 24-well format. Lysis of the cells occurs via a chemical lysis without mechanical disruption. Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones which produce heterologous protein with a yield of 5 mg l(-1) culture volume or higher.

Base Sequence↗
PubMed1999

Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening.

DNA helicases are responsible for the unwinding of double-stranded DNA, facilitated by the binding and hydrolysis of 5'-nucleoside triphosphates. These enzymes represent an important class of targets for the development of novel anti-infective agents particularly because opportunity exists for synergy with existing therapies targeted at other enzymes involved in DNA replication. Unwinding reactions are conventionally monitored by low throughput, gel-based radiochemical assays; to overcome the limitations of low throughput to achieve comprehensive characterization of adenosine triphosphate (ATP)-dependent unwinding by viral and bacterial helicases and the screening for unwinding inhibitors, we have developed and validated homogeneous time-resolved fluorescence energy transfer (TRET) assays. Rapid characterization and screening of DNA helicase has been performed in 96- and 384-well plate densities, and the ability to assay in 1536-well format also demonstrated. We have successfully validated and are running full high throughput runs using 384-well TRET helicase assays, culminating in the identification of a range of chemically diverse inhibitors of viral and bacterial helicases. For screening in mixtures, we used a combination of quench correction routines and confirmatory scintillation proximity (SP) assays to eliminate false-positives due to the relatively high levels of compound quenching (unlike other Ln(3+)-based assays). This strategy was successful yet emphasised the need for further improvements in helicase assays.

Journal Article↗
PubMed2000

Microbiological high throughput screening: an opportunity for the lead discovery process.

Microbial HTS has been implemented at Rhône-Poulenc Rorer through the development of a dedicated robotic platform. This robot (Turbo) has been designed with the aim of fully integrating microbial HTS into the lead discovery processes. Innovative solutions have been found to reach high throughput as well as flexibility. This opens up new prospects for solid-phase microbial screening, taking advantage of the easy implementation and the very low costs of such screens. The different types of microbial screens done in our laboratory, as well as the throughputs and outputs obtained, are described. Some of the specific aspects of microbial HTS, as compared to biochemical and cell-based assays, are also discussed.

Bacteria↗
PubMed2004

High-throughput screen for inhibitors of transglycosylase and/or transpeptidase activities of Escherichia coli penicillin binding protein 1b.

Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC(50)s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other beta-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC(50)s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.

Anti-Bacterial Agents↗
PubMed2006

Novel Pseudomonas aeruginosa quorum-sensing inhibitors identified in an ultra-high-throughput screen.

The opportunistic pathogen Pseudomonas aeruginosa has two complete acyl-homoserine lactone (acyl-HSL) signaling systems, LasR-LasI and RhlR-RhlI. LasI catalyzes the synthesis of N-3-oxododecanoyl homoserine lactone (3OC12-HSL), and LasR is a transcription factor that requires 3OC12-HSL as a ligand. RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (C4), and RhlR is a transcription factor that responds to C4. LasR and RhlR control the transcription of hundreds of P. aeruginosa genes, many of which are critical virulence determinants, and LasR is required for RhlR function. We developed an ultra-high-throughput cell-based assay to screen a library of approximately 200,000 compounds for inhibitors of LasR-dependent gene expression. Although the library contained a large variety of chemical structures, the two best inhibitors resembled the acyl-homoserine lactone molecule that normally binds to LasR. One compound, a tetrazole with a 12-carbon alkyl tail designated PD12, had a 50% inhibitory concentration (IC50) of 30 nM. The second compound, V-06-018, had an IC50 of 10 microM and is a phenyl ring with a 12-carbon alkyl tail. A microarray analysis showed that both compounds were general inhibitors of quorum sensing, i.e., the expression levels of most LasR-dependent genes were affected. Both compounds also inhibited the production of two quorum-sensing-dependent virulence factors, elastase and pyocyanin. These compounds should be useful for studies of LasR-dependent gene regulation and might serve as scaffolds for the identification of new quorum-sensing modulators.

Anti-Bacterial Agents↗
PubMed2003

Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery.

Effective screening of large compound libraries in ion channel drug discovery requires the development of new electrophysiological techniques with substantially increased throughputs compared to the conventional patch clamp technique. Sophion Bioscience is aiming to meet this challenge by developing two lines of automated patch clamp products, a traditional pipette-based system called Apatchi-1, and a silicon chip-based system QPatch. The degree of automation spans from semi-automation (Apatchi-1) where a trained technician interacts with the system in a limited way, to a complete automation (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs.

Animals↗
PubMed2005

Integrity profiling of high throughput screening hits using LC-MS and related techniques.

Integrity profiling of HTS hits is valuable for verification of the hit identity and purity. This provides early discovery researchers with more confident SAR theories. Methodology for integrity profiling of HTS hits must be high throughput, consume little material, and selectively provide structure-based data. Analytical techniques that can be utilized for integrity profiling methods are reviewed for their appropriateness in sample preparation, component separation, detection, purity quantitation, identity confirmation, and follow-up.

Chromatography, Liquid↗
PubMed2000

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

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

Animals↗
PubMed2001

High-throughput screening of surface displayed gene products.

With the human genome project approaching completion, there is a growing interest in functional analysis of gene products. The characterization of large numbers of proteins, their expression patterns and in vivo localisations, demands the use of automated technology that maintains a logistic link to the encoding genes. As a complementary approach, phage display is used for recombinant protein expression and the selection of interacting (binding) molecules. Cloning of libraries in filamentous bacteriophage or phage mid vectors provides a physical link between the expressed protein and its encoding DNA sequence. High-throughput technology for automated library handling and phage display selection has been developed using picking-spotting robots and a module for pin-based magnetic particle handling. This system enables simultaneous interaction screening of libraries and the selection of binders to different target molecules at high throughput. Target molecules are either displayed on high-density filter membranes (protein filters) or tag-bound to magnetic particles and can be handled as native ligands. Binding activity is confirmed by magnetic particle ELISA in the microtitre format. The whole procedure from immobilisation of target molecules to confirmed clones of binders is automatable. Using this technology, we have selected human scFv antibody fragments against expression products of human cDNA libraries.

Bacteriophages↗
PubMed2000

High throughput screening of gene expression signatures.

This paper focuses on microarray image analysis and discusses a completely automated approach to image processing, which eliminates human intervention. A system for automated image processing is described, which is capable of processing image files in a batch-mode thus allowing high-throughput of microarray image analysis. Grid-placement and spot finding are achieved without operator's help. The software eliminates noise signals from the data analysis process and minimizes operator's involvement in the procedure.

Algorithms↗
PubMed2003

A novel high throughput screening assay for HCV NS3 serine protease inhibitors.

Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors.

Amino Acid Sequence↗
PubMed2000

A quantitative, high-throughput screen for protein stability.

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric lambda repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.

Bacterial Proteins↗
PubMed2000

Challenges and solutions to ultra-high-throughput screening assay miniaturization: submicroliter fluid handling.

The miniaturization of HTS assays is an important objective in the pharmaceutical industry. The ability to perform primary screening assays in high-density micro-well plates at volumes of 1-2 ml will accelerate the early stages of drug discovery and reduce costs. Ultra-HTS (uHTS) assays require an accurate and reliable means of fluid handling in the submicroliter volume range. This relates to the design of instrumentation for dispensing fluids, as well as assay plates. Fluid handling has been a major obstacle to the full implementation of miniaturized assays. This report focusses on current approaches to submicroliter fluid handling in high-density multi-well plates.

Journal Article↗
PubMed2002

High-throughput screening for evidence of association by using mass spectrometry genotyping on DNA pools.

To facilitate positional cloning of complex trait susceptibility loci, we are investigating methods to reduce the effort required to identify trait-associated alleles. We examined primer extension analysis by matrix-assisted laser desorptionionization time-of-flight mass spectrometry to screen single-nucleotide polymorphisms (SNPs) for association by using DNA pools. We tested whether this method can accurately estimate allele frequency differences between pools while maintaining the high-throughput nature of assay design, sample handling, and scoring. We follow up interesting allele frequency differences in pools by genotyping individuals. We tested DNA pools of 182, 228, and 499 individuals using 16 SNPs with minor allele frequencies 0.026-0.486 and allele frequency differences 0.001-0.108 that we had genotyped previously on individuals and 381 SNPs that we had not. Precision, as measured by the average standard deviation among 16 semidependent replicates, was 0.021 +/- 0.011 for the 16 SNPs and 0.018 +/- 0.008 for the 291381 SNPs used in further analysis. For the 16 SNPs, the average absolute error in predicting allele frequency differences between pools was 0.009; the largest errors were 0.031, 0.028, and 0.027. We determined that compensating for unequal peak heights in heterozygotes improved precision of allele frequency estimates but had only a very minor effect on accuracy of allele frequency differences between pools. Based on these data and assuming pools of 500 individuals, we conclude that at significance level 0.05 we would have 95% (82%) power to detect population allele frequency differences of 0.07 for control allele frequencies of 0.10 (0.50).

Alleles↗
PubMed2005

Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets.

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

Animals↗
PubMed2002

Microsomal triglyceride transfer protein (MTP) inhibitors: discovery of clinically active inhibitors using high-throughput screening and parallel synthesis paradigms.

The inhibition of microsomal triglyceride transfer protein (MTP) blocks the hepatic secretion of very low density lipoproteins (VLDL) and the intestinal secretion of chylomicrons. Consequently, this mechanism provides a highly efficacious pharmacological target for the lowering of low density lipoprotein (LDL) cholesterol and reduction of postprandial lipemia. The combination of these effects could afford unprecedented benefit in the treatment of atherosclerosis and consequent cardiovascular disease. The promise of this therapeutic target has attracted widespread interest in the pharmaceutical industry. Independent efforts have yielded strikingly similar series of lipophilic amide inhibitors. The way in which the evolutionary paths of distinct inhibitor series have tended to converge through the course of robotics-assisted synthesis efforts is illustrated with candidates from Bristol-Myers Squibb and Pfizer. Hanging in the balance with the exceptional potency of the compounds presented are the potential adverse effects due to blockage of intestinal fat absorption and hepatic lipid secretion. Finding a degree of efficacy that can be safely tolerated defines the dilemma surrounding the advancement of these compounds to clinical practice.

Animals↗
PubMed2003

Genomic profiling of iron-responsive genes in Salmonella enterica serovar typhimurium by high-throughput screening of a random promoter library.

The importance of iron to bacteria is shown by the presence of numerous iron-scavenging and transport systems and by many genes whose expression is tightly regulated by iron availability. We have taken a global approach to gene expression analysis of Salmonella enterica serovar Typhimurium in response to iron by combining efficient, high-throughput methods with sensitive, luminescent reporting of gene expression using a random promoter library. Real-time expression profiles of the library were generated under low- and high-iron conditions to identify iron-regulated promoters, including a number of previously identified genes. Our results indicate that approximately 7% of the genome may be regulated directly or indirectly by iron. Further analysis of these clones using a Fur titration assay revealed three separate classes of genes; two of these classes consist of Fur-regulated genes. A third class was Fur independent and included both negatively and positively iron-responsive genes. These may reflect new iron-dependent regulons. Iron-responsive genes included iron transporters, iron storage and mobility proteins, iron-containing proteins (redox proteins, oxidoreductases, and cytochromes), transcriptional regulators, and the energy transducer tonB. By identifying a wide variety of iron-responsive genes, we extend our understanding of the global effect of iron availability on gene expression in the bacterial cell.

Bacterial Proteins↗