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At least 1,117 records · Page 62Linked to original sources
PubMed2004

Polyplexes and lipoplexes for mammalian gene delivery: from traditional to microarray screening.

Gene therapy requires the development of non-toxic and highly efficient delivery systems for DNA and RNAi. Polycations, especially dendrimers, have shown enormous potential as gene transfer vehicles, displaying minimal toxicity with a broad range of cell lines. In this paper, a total of 13 dendrimers, up to G3.0, were constructed from AB(3) type isocyanate monomers using solid phase methodology and evaluated for transfection activity. Among the library of compounds prepared, a G3.0 dendrimer displayed comparable activity to Superfect. Gel retardation assays demonstrated that all of the compounds completely bound plasmid DNA, indicating the efficient formation of complexes between DNA and the dendrimers. A "transfection microarray" approach was developed for screening these compounds as well as a panel of lipoplexes (complexes of DNA with cationic lipids) and polyplexes (complexes of DNA with synthetic polycationic polymers), in 3D solution like micro-assay). Five cationic lipids with a cholesterol tail showed stronger or comparable transfection activity relative to Effectene. The new, micro-array screening method was rapid and miniaturized, offering the potential of high throughput screening of large libraries of transfection candidates, with thousands of library members per array, and the ability to rapidly screen a broad range of cell types.

Cell Line↗
PubMed2006

[Directed evolution of Thermophilic esterase from the archaeon Aeropyrum pemix K1].

Thermophilic esterase (APE1547) from Aeropyrum pernix K1 was subjected to error-prone PCR (epPCR) to enhance activity. For the screening of mutants, an efficient and reliable assay suitable for high throughput screening was developed based on the enzyme thermostability. Two successive rounds of random mutagenesis by epPCR resulted in a four amino acid substitution variant M020 with significantly increased activity (six-fold under the screening condition. Further assay for the purified enzymes showed that the mutant possess 1.5-fold higher specific activity and nearly 4-fold higher expressed level than the wild-type. The mutant has an optimal activity at pH 8.5, corresponding to an alkaline shift of 0.5 pH unit compared to the wild type. The structure analysis suggests that R526S may contribute to the enhanced activity and the shift of pKi.

Aeropyrum↗
PubMed2003

Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity.

alpha-Amylases, in particular, microbial alpha-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for alpha-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4- and 1.5-fold, respectively. Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial alpha-amylases.

Amino Acid Sequence↗
PubMed2001

New fluorogenic probes for oxygen and carbene transfer: a sensitive assay for single bead-supported catalysts.

A high-throughput screening assay for atom transfer catalysis has been developed. This assay is based on two probes, developed herein, which generate highly fluorescent products upon carbene or oxygen atom transfer. The emission wavelength of probes 1 and 5 shift significantly (up to 90 nm) upon epoxidation, allowing detection of product at 3% conversion. Probe 7 is not fluorescent, while fluorescence emission by carbene insertion/rearrangement product 8 allows detection at less than 1% conversion. Such sensitivity allows for examination of single-bead reactions in a high throughput array format (1536 wells per plate), and provides a broad detection window ranging from single to high turnover numbers. Thousands of metal complexes are evaluated in a single screening experiment. Preliminary screening of a diverse ligand library with probe 7 in the presence of Rh(II) uncovered new catalysts capable of cyclopropanation and C-H insertion.

Journal Article↗
PubMed2006

High-throughput measurements of biochemical responses using the plate::vision multimode 96 minilens array reader.

The plate::vision is a high-throughput multimode reader capable of reading absorbance, fluorescence, fluorescence polarization, time-resolved fluorescence, and luminescence. Its performance has been shown to be quite comparable with other readers. When the reader is integrated into the plate::explorer, an ultrahigh-throughput screening system with event-driven software and parallel plate-handling devices, it becomes possible to run complicated assays with kinetic readouts in high-density microtiter plate formats for high-throughput screening. For the past 5 years, we have used the plate::vision and the plate::explorer to run screens and have generated more than 30 million data points. Their throughput, performance, and robustness have speeded up our drug discovery process greatly.

Absorption↗
PubMed1998

Genetic recombination as a reporter for screening steroid receptor agonists and antagonists.

Reporter cell lines are often used for high throughput screening of chemical libraries to identify new receptor ligands. Here we show how Cre recombinase can be used in mammalian cells to screen for steroid receptor ligands. A translational fusion of Cre recombinase and the ligand binding domain of the human glucocorticoid receptor was transfected into mammalian cells with a loxP/luciferase reporter gene. The recombinase function of the fusion is dependent on ligand binding to the receptor, and Cre-mediated recombination results in constitutive expression of luciferase from the reporter gene. A stable transfected clone was isolated and used to characterize the kinetics, ligand specificity, and dose response to various receptor ligands. The Cre fusion system, unlike a transcriptional reporter using the mouse mammary tumor virus promoter, can detect binding of the receptor antagonist RU486. We also studied the Cre reporter in a sensitive, miniaturized, assay format using an 864-well plate and show that as few as 560 cells per assay well was sufficient to measure a dose response to ligand.

Amino Acid Sequence↗
PubMed2000

Protein arrays for gene expression and molecular interaction screening.

The array format has revolutionised biomedical experimentation and diagnostics, enabling ordered high-throughput analysis. During the past decade, classic solid phase substrates, such as microtitre plates, membrane filters and microscopic slides, were turned into high-density, chip-like structures. The concept of the arrayed library was central to this development which now extends from DNA to protein. The new and versatile protein array technology allows high-throughput screening for gene expression and molecular interactions. As a major platform for functional genomics, it is already on its way into medical diagnostics.

Bacterial Proteins↗
PubMed2004

The bud scar-based screening system for hunting human genes extending life span.

We developed a high-throughput screening system that allows identification of genes prolonging life span in the budding yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with an extended number of cell divisions as indicated by the increased number of bud scars on their surface. Fluorescently labeled wheat germ agglutinin (WGA) was used for specific staining of bud scars. Screening of a human HepG2 cDNA expression library in yeast resulted in the isolation of several yeast transformants with a potentially prolonged life span. The budding yeast S. cerevisiae, one of the favorite models used to study aging, has been studied extensively for the better understanding of the mechanisms of human aging. Because human disease genes often have yeast counterparts, they can be studied efficiently in this organism. One interesting example is the WRN gene, the human DNA helicase, which participates in the DNA repair pathway. The mutation of the WRN gene causes Werner syndrome showing premature-aging phenotype. Budding yeast contains WRN homologue, SGS1, and its mutation results in shortening yeast life span. The knowledge gained from the studies of budding yeast will benefit studies in humans for better understanding of aging and aging-related disease.

Aging↗
PubMed2001

Development of a K562 cell-based assay for screening anticancer agents.

AIM: To develop a leukemia cell line K562-based assay for high-throughput screening. METHODS: The screening was carried out on 96-well plates with monitoring cell proliferation by a combined 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTS)/phenazine methosulfate (PMS) method. Conditions for evaluating effects on the proliferation of K562 cells by individual compounds on the 96-well plates were optimized. RESULTS: A set of 800 small organic compounds was screened for anticancer activity by this cell-based assay, with consumption of each compound at 500 ng. Eleven compounds were identified with >80 % inhibitory activity at 5 mg/L, among which 9 compounds were confirmed by subsequent testing at multiple concentrations. The most potent compound showed an IC50 at 170 nmol/L, and there were total of 7 compounds showed IC50 less than 10 micromol/L. CONCLUSION: The high-throughput method using K562 cell line is fast, economical, effective, and practical in identifying inhibitors as potential therapeutic agents for cancer.

Cell Division↗
PubMed1998

Novel screen methodologies for identification of new microbial metabolites with pharmacological activity.

Micro-organisms continue to provide an important source of chemical diversity for the discovery of compounds with new biological activities. Microbial metabolites discovered recently using assays to detect compounds with potential pharmacological utility are surveyed and found to represent an extensive range of structural types produced by a wide variety of organisms. Assays used for screening samples produced by microbial processes must be robust, sensitive and specific and able to operate above a background of potential interferences from a number of sources. Discovery assays currently in use fall into three main categories cell-based, receptor-ligand interaction and enzyme inhibition assays. Trends in the use of these assays and new developments in assay technology applicable to the screening of microbial samples are examined with particular reference to the high throughput screening environment. For microbial screening to be a competitive route to new drug leads, the disciplines involved must be engineered into a seamlessly integrated process to deliver novel compounds with the required biological properties rapidly.

Animals↗
PubMedDate not supplied

The continuing evolution of the drug discovery process in the pharmaceutical industry.

Many early discoveries in the pharmaceutical industry were through serendipity. Later, targets were mainly identified in animals and systematically exploited through the identification of potent and selective molecules. A disease association was normally obtained through the clinical testing of candidate molecules in patients. The technological advances in the last few years offer the possibility of knowing more about the disease, and this is driving the industry towards a disease-based approach where understanding the disease becomes central to the process. This is now possible thanks to the recent explosion in molecular and cellular biology, together with the application of genetics and genomics. New screening technologies have also revolutionized the identification of chemical leads. Now, high throughput screening allows a wide chemical diversity to be applied in order to obtain tractable leads, which can then be optimized by the medicinal chemist. It is envisaged that these trends of continuously searching for process improvement will continue, being driven by the need to find medicines that add value in treating unmet medical need.

Drug Industry↗
PubMed1998

Production of G-protein-coupled receptors in yeast.

Yeasts combine the advantages of fast and easy handling with the potential to perform eukaryotic post-translational modifications and are for this reason interesting hosts for heterologous production of G-protein-coupled receptors. The possibility to connect foreign receptors to a yeast internal MAP kinase pathway was used to establish yeast-based systems for high-throughput screening of compound libraries. In addition, yeasts have the potential for high level production of G-protein-coupled receptors. In this field, non-Saccharomyces yeasts seems to be interesting alternatives to S. cerevisiae, as well as to systems based on higher eukaryotic cells.

Animals↗
PubMed2000

2-(Alkylthio)pyrimidin-4-ones as novel, reversible inhibitors of lipoprotein-associated phospholipase A2.

Starting from two weakly active hits from high throughput screening, a novel series of 2-(alkylthio)-pyrimidin-4-ones with high potency and selectivity for lipoprotein-associated phospholipase A2 has been designed. In contrast to previously known inhibitors, these have been shown to act by a non-covalent and substrate competitive mechanism.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2006

Transgenic Drosophila models of Noonan syndrome causing PTPN11 gain-of-function mutations.

Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, causes Noonan syndrome (NS), an autosomal dominant disorder with pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker than the leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, we generated transgenic fruitflies with GAL4-inducible expression of wild-type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles were lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous expression of the weaker N308D allele caused ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses established that csw(N308D)'s ectopic wing vein phenotype required intact EGF ligand and receptor, and that this transgene interacted genetically with Notch, DPP and JAK/STAT signaling. Expression of the mutant csw transgenes increased RAS-MAP kinase activation, which was necessary but not sufficient for transducing their phenotypes. The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers.

Animals↗
PubMed2003

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP.

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.

Adipocytes↗
PubMed2005

Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes.

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.

Cytochrome P-450 Enzyme Inhibitors↗
PubMed2005

TargetTalk--IBC Conference. Kinase and G protein-coupled receptor targets.

TargetTalk combined IBC's Fifth International Protein Kinases and Phosphatases Conference with an in-depth track on G protein-coupled receptors (GPCRs), and this conference ran in conjunction with IBC's 14th Annual ScreenTech Meeting, which comprised separate tracks on high-throughput screening applications and technologies, and assay development for multiplexing and high-content screening. There were also pre-conference symposia that explored: (i) the current and future outlook for high-thoughput screening; (ii) technologies and research advances for the study of protein-protein interaction; and (iii) mechanisms of receptor signaling. This report will focus on research presented for the pre-conference symposium on mechanisms of receptor signaling and on the GPCR track.

Animals↗
PubMed2001

HTS Technologies--IBC Informa Conference.

In 1997, approximately US $6 billion were spent on worldwide screening technologies. The estimate for the total global market for high-throughput screening (HTS) products and services was about US $1.5 billion (source: Medical and Healthcare Marketplace Guide, 15th Edition, 1999-2000). Genomics and combinatorial chemistry demand a technological shift away from present-day laboratory to fully automated HTS and ultra HTS (uHTS), with over 100,000 compounds screened per day. The potential of new technology in miniaturization and automation will ensure faster and cheaper solutions to discovery. The conference explored the rapidly advancing field of HTS and examined numerous areas of HTS application. Identification and prioritization of viable targets, and the development of 'information rich' screens without reducing screening capacity, are important topics. Furthermore, the essential integration of all these screening, analytical and parallel synthesis systems in one coherent process is considered to be of significant interest. Focused sessions addressed the rapidly advancing areas of ADME (absorption, distribution, metabolism, elimination), toxicity and formulation screening, including virtual 'in silico' screening.

Journal Article↗