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At least 361 records · Page 20Linked to original sources
PubMed1997

G-protein-coupled receptors in Saccharomyces cerevisiae: high-throughput screening assays for drug discovery.

G-protein-coupled receptors are an important class of therapeutic drug targets by virtue of their roles in the regulation of diverse cellular functions. Recent advances in the expression of heterologous G-protein-coupled receptors in the yeast Saccharomyces cerevisiae have led to the development of sensitive and selective assays of their ligand-induced activation. Implementation of this new technology in the high-throughput screening of compound libraries has enabled the discovery of novel ligands for the G-protein-coupled somatostatin receptor. This article describes the broad applicability of the technology and its use in drug discovery.

Drug Industry↗
PubMed1999

Selective enrichment and high-throughput screening of phage surface-displayed cDNA libraries from complex allergenic systems.

Phage surface display technology, first described in 1985, enables the construction of large combinatorial peptide and antibody libraries. The basic concept of linking the phenotype, expressed as gene product displayed on the phage surface, to its genetic information integrated into the phage genome, allows the survey of large libraries for the presence of specific clones using the discriminative power of affinity purification. The selection procedure involves the enrichment of phage by binding to an immobilized target molecule. As a consequence of the physical linkage between genotype and phenotype, sequencing the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of a displayed gene product. Phage surface display technology has revolutionized our ability to select agonist and antagonist peptides, antibodies with desired specificities, and DNA-binding molecules. We have extended phage surface display to access cDNA libraries with this powerful screening technology based on affinity selection of desired clones. Here we discuss construction of cDNA libraries displayed on the phage surface, selective enrichment of clones and robotics-based high-throughput screening of enriched libraries.

Allergens↗
PubMed1999

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.

BACKGROUND: Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. RESULTS: We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. CONCLUSIONS: We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.

Acetylation↗
PubMed2006

A high-throughput screen for compounds that inhibit aggregation of the Alzheimer's peptide.

Aggregation of the Alzheimer's peptide Abeta produces toxic multimeric species that play a key role in the development of Alzheimer's disease. Compounds that inhibit this aggregation may prove useful as therapeutic agents for the prevention or treatment of Alzheimer's disease. Although aggregation inhibitors may already exist in combinatorial libraries, finding these compounds in a cost-effective high-throughput manner poses an enormous challenge. To meet this challenge, we have developed a novel high-throughput screen capable of isolating inhibitors of Abeta aggregation from large libraries of inactive candidates. The screen uses a fusion of Abeta42 to GFP. In the absence of inhibition, the rapid misfolding and aggregation of Abeta42 causes the entire fusion protein to misfold, thereby preventing fluorescence. Compounds that inhibit Abeta42 aggregation enable GFP to fold into its native structure and be identified by the resulting fluorescent signal. By implementing the screen on a pilot library of triazine derivatives, we have identified several putative inhibitors. One of the selected compounds was studied in detail by a series of biochemical and biophysical methods. These studies confirmed that the selected compound inhibits aggregation of synthetic Abeta42 peptide. The fluorescence-based method described here is rapid and inexpensive and can be used to screen large libraries for inhibitors of Abeta42 aggregation and/or amyloidogenesis.

Alzheimer Disease↗
PubMed2002

A high-throughput screening microplate test for the interaction of drugs with P-glycoprotein.

P-glycoprotein (P-gp) is a multidrug transporter responsible for resistance to anticancer chemotherapy and physiologically involved in absorption, distribution, and excretion of a large number of hydrophobic xenobiotics. P-gp exhibits both an ATPase activity correlated with its drug transport function and a basal ATPase activity in the absence of any drug. We have developed a high-throughput screening test to detect specific interactions between drugs and P-gp. We took into account the existence of multiple binding sites on P-gp to propose and validate an optimized strategy, based on the modulation of the basal ATPase activity of P-gp and of the ATPase activity stimulated by three reference substrates (verapamil, vinblastine, and progesterone). The ATPase activity measurements were performed on P-gp-containing membrane vesicles from actinomycin-D-selected hamster DC-3F lung fibroblasts by a spectrophotometric method based on continuous monitoring of ADP formation, regenerated in ATP by a coupled enzyme system. This assay may be performed on 96- or 384-well microtiter plates. When applying this ATPase assay to 41 compounds known from the literature for their interaction with P-gp, 95% of them were found to be positive, whereas only 78% were positive when considering solely the modulation of the basal activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay.

The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two receptors in this assay were found to be in good agreement with those from electrophysiology studies of the receptors expressed in Xenopus oocytes or mammalian cell lines. Hence, this high throughput screening assay will be of great use in future pharmacological studies of glycine receptors, particular in the search for novel compound structures acting at them.

Androstanes↗
PubMed2005

A novel high-throughput screening format to identify inhibitors of secreted acid sphingomyelinase.

Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z' factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC(50) values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.

Drug Evaluation, Preclinical↗
PubMed1997

Monoclonal antibodies as surrogate receptors in a high throughput screen for compounds that enhance insulin sensitivity.

Monoclonal antibodies (MoAbs) were made to a known insulin sensitivity enhancer (ISE) compound, CS-045. The MoAbs were characterized with respect to binding other known thiazolidinedione ISE compounds using a CS-045 labeled with b-phycoerythrin in a competitive particle concentration fluorescence immunoassay (PCFIA). By comparing the rank order of IC50 values for each compound to its respective potency as an ISE, one MoAb (13E3) was selected for further characterization. This MoAb was also used as a surrogate receptor in a high throughput screen to identify novel compounds that compete for binding to CS-045. Some of the hits were found to have efficacy in reducing blood glucose. Subsequently, another group reported that several compounds with the core thiazolidinedione structure of the ISE compounds bound with high affinity to peroxisome proliferator-activating receptors (PPAR). Therefore, we used the MoAb assay to test these and other compounds that are known to bind to PPARgamma and noted crossreactivity with some of the compounds.

Animals↗
PubMed2002

A high throughput screen for inhibitors of fungal cell wall synthesis.

Fungal cell wall synthesis is essential for viability, requiring the activity of genes involved in environmental sensing, precursor synthesis, transport, secretion, and assembly. This multitude of potential targets, the availability of known agents targeting this pathway, and the unique nature of fungal cell wall synthesis make this pathway an appealing target for drug discovery. Here we describe the adaptation of an assay monitoring cell wall synthesis for high-throughput screening. The assay requires fungal cell growth, in the presence of the test compound, for 3 h before the cells are subjected to osmotic shock in the presence of a dye that stains DNA. Miniaturization of the assay to a 384-well plate format and removing a mechanical transfer led to subtle changes in the assay characteristics. Validation of the assay with a library of known pharmacologically active agents has identified a number of different classes of compounds that are active in this assay, causing aberrant cell wall morphology and in many cases the inhibition of fungal cell growth.

Antifungal Agents↗
PubMed2003

Immobilized nicotinic receptor stationary phases: going with the flow in high-throughput screening and pharmacological studies.

The nicotinic acetylcholine receptor (nAChR) subtypes alpha3beta4-nAChR and alpha4beta2-nAChR have been immobilized and the resulting stationary phases used to determine binding affinities. The alpha3beta4-nAChR column was coupled to a C(18) column and a mixture of 18 compounds was sorted into ligands and non-ligands for the alpha3beta4-nAChR. The results demonstrate that the nAChR stationary phases can be used for on-line high-throughput screening (HTS).

Animals↗
PubMed2002

High-throughput screening for ion channel modulators.

Ion channels present a group of targets for major clinical indications, which have been difficult to address due to the lack of suitable rapid but biologically significant methodologies. To address the need for increased throughput in primary screening, the authors have set up a Beckman/Sagian core system to fully automate functional fluorescence-based assays that measure ion channel function. They apply voltage-sensitive fluorescent probes, and the activity of channels is monitored using Aurora's Voltage/Ion Probe Reader (VIPR). The system provides a platform for fully automated high-throughput screening as well as pharmacological characterization of ion channel modulators. The application of voltage-sensitive fluorescence dyes coupled with fluorescence resonance energy transfer is the basis of robust assays, which can be adapted to the study of a variety of ion channels to screen for both inhibitors and activators of voltage-gated and other ion channels.

Automation↗
PubMed2003

High-throughput screen for inhibitors of 1-deoxy-d-xylulose 5-phosphate reductoisomerase by surrogate ligand competition.

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is a key enzyme in a biosynthetic pathway for isoprenoids that is unique to eubacteria and plants. Dxr catalyzes the rearrangement and NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate. The authors have purified Escherichia coli Dxr and devised a high-throughput screen (HTS) for compounds that bind to this enzyme at a functional site. Evidence is presented that the surrogate ligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites. Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitive measurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89 potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligands may be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function.

Aldose-Ketose Isomerases↗
PubMed2002

An ATPase assay using scintillation proximity beads for high-throughput screening or kinetic analysis.

A new procedure for measuring ATPase activity in which gamma-(33)P-labeled inorganic orthophoshate is detected by addition of ammonium molybdate followed by selective adsorption of the resulting phosphomolybdate to scintillation proximity beads in the presence of cesium chloride is described. This method is shown to give accurate and reproducible results over a wide range of detection conditions and product concentrations. It requires no separation or filtration steps and is highly compatible with automated high-throughput screening. Rates of hydrolysis are easily and accurately determined over a wide range, and thus the method is useful for kinetic studies also. We show that this scintillation proximity assay is useful for the study of the E1 helicase of human papillomavirus, but it is a general procedure which could also be applied to any ATPase or other nucleotide triphosphate-hydrolyzing enzyme or any other enzyme which generates orthophosphate as a reaction product.

Adenosine Triphosphatases↗
PubMedDate not supplied

Retrospective analysis of an experimental high-throughput screening data set by recursive partitioning.

With the emergence of combinatorial chemistry, whether based on parallel, mixture, solution, or solid phase chemistry, it is now possible to generate large numbers of diverse or focused compound libraries. In this paper we aim to demonstrate that it is possible to design targeted libraries by applying nonparametric statistical methods, recursive partitioning in particular, to large data sets containing thousands of compounds and their associated biological data. Moreover, when applied to an experimental high-throughput screening (HTS) data set, our data strongly suggest that this method can improve the hit rate of our primary screens (about 4- to 5-fold) while increasing screening efficiency: less than one-fifth of the complete selection needs to be screened in order to identify about 75% of all actives present.

Combinatorial Chemistry Techniques↗
PubMed2006

Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries.

We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established a cell-based assay in which expression of a luciferase (luc) reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter approximately 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA self-cleavage.

Acridine Orange↗
PubMed2005

Homogeneous high-throughput screening assays for HIV-1 integrase 3beta-processing and strand transfer activities.

HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3beta processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.

Antiviral Agents↗
PubMed2004

Synthesis and high-throughput screening of N-acetyl-beta-hexosaminidase inhibitor libraries targeting osteoarthritis.

C1 Nitrogen iminocyclitols are potent inhibitors of N-acetyl-beta-hexosaminidases. Given hexosaminidases' important roles in osteoarthritis, we developed two straightforward and efficient syntheses of C1 nitrogen iminocyclitols from two readily available starting materials, D-mannosamine hydrochloride and the microbial oxidation product of fructose. A diversity-oriented synthetic strategy was then performed by coupling these core structures with various aldehydes, carboxylic acids, and alkynes to generate three separate libraries. High-throughput screening of the generated libraries with human N-acetyl-beta-hexosaminidases produced only moderate inhibitory activities. However, the synthetic approach and screening strategy for these compounds will be applied to develop new potent inhibitors of human N-acetyl-beta-hexosaminidases, particularly when combined with the structural information of these enzymes.

Enzyme Inhibitors↗
PubMed2005

High-throughput screening of 11beta-hydroxysteroid dehydrogenase type 1 in scintillation proximity assay format.

11beta-Hydroxysteroid dehydrogenase type-1 (11beta-HSD1) is a potential target for the treatment of diabetes, obesity, and hyperlipidemia. This enzyme is mainly responsible for reactivating glucocorticoid hormone inside cells such as adipose cells and liver cells by converting the inactive cortisone to active cortisol. Enzyme assays for 11beta-HSD1 involve either a thin-layer chromatography or high-performance liquid chromatography step to separate cortisol from the substrate cortisone. This additional step is labor intensive and increases the assay time, which limits assay throughput. A homogenous scintillation proximity assay-based method has been recently developed that enables high-throughput screening of 11beta-HSD1 inhibitors. We have applied this novel 11beta-HSD1 assay to screening a large-size compound collection and identified several structural classes of lead compounds that selectively inhibit the activity of 11beta-HSD1.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗