Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “High Throughput Screening”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources
PubMed2002

Cell-Surface display of heterologous proteins: From high-throughput screening to environmental applications.

A variety of expression systems for the display of either short peptides or fully folded proteins on E.coli and, to a lesser extent, on Gram-positive bacteria have been developed. The expression of proteins on the surface of microbial cells has proved extremely important for numerous applications ranging from combinatorial library screening and protein engineering, to whole cell biocatalysts and adsorbants for bioremediation purposes.

Bacterial Proteins↗
PubMed1999

A scintillation proximity assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors.

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed in Escherichia coli as glutathione S-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [gamma-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.

Amino Acid Sequence↗
PubMed1999

Viral protease assay based on GAL4 inactivation is applicable to high-throughput screening in mammalian cells.

We present an assay for viral proteases that relies on the proteolytic cleavage of substrate leading to the dissociation of the yeast transcription factor GAL4. A consensus substrate for the cytomegalovirus protease is fused between the DNA binding and transactivating domains of GAL4. Proteolysis inactivates the transcription factor which drives a luciferase reporter system. The assay is performed in mammalian cells, has a robust signal-to-noise ratio, and assesses proteolysis in a physiologic context. A unique feature of the assay is its ability to detect inhibitors of viral replication that act on viral targets other than the protease.

Amino Acid Sequence↗
PubMed2000

High-throughput screening of enzyme inhibitors: simultaneous determination of tight-binding inhibition constants and enzyme concentration.

Active site titration by a reversible tight-binding inhibitor normally depends on prior knowledge of the inhibition constant. Conversely, the determination of tight-binding inhibition constants normally requires prior knowledge of the active enzyme concentration. Often, neither of these quantities is known with sufficient accuracy. This paper describes experimental conditions under which both the enzyme active site concentration and the tight-binding inhibition constant can be determined simultaneously from a single dose-response curve. Representative experimental data are shown for the inhibition of human kallikrein.

Binding Sites↗
PubMed2002

Oligonucleotide scanning arrays: application to high-throughput screening for effective antisense reagents and the study of nucleic acid interactions.

Oligonudeotide scanning arrays are useful tools in the study of nucleic acid interaction. Such arrays of oligonucleotides, corresponding to a full set of complements of a known sequence, can be readily made in a single series of coupling reactions, adding each nudeotide in the complement of the target sequence in turn. The synthesis is carried out on the surface of a solid substrate such as glass or polypropylene that has been modified to allow nucleotide coupling. A mask is used to apply the DNA synthesis reagents in a defined area and is moved by a fixed step size after each coupling reaction so that consecutive couplings overlap a portion of the previous one. The size of the mask and the displacement at each coupling determine the length of the oligonucleotides. A radiolabeled or fluorescently tagged target sequence is hybridised to a scanning array and its interaction with the complementary oligonudeotides is displayed as a hybridisation signal. It is thus possible to determine the exact sequence and lengths of large numbers of interacting sequences in a single hybridisation experiment. The array image is analysed using a computer program (xvseq) that calculates quantitative measurements of the binding strengths. We have found scanning arrays a useful tool not only to find effective antisense reagents, but also to study RNA folding and the mechanisms of heteroduplex formation. In this article, we discuss the format of these arrays, the technology used to fabricate and to read them, and their applications.

Algorithms↗
PubMed2002

Potassium channels: gene family, therapeutic relevance, high-throughput screening technologies and drug discovery.

Existing drugs that modulate ion channels represent a key class of pharmaceutical agents across many therapeutic areas and there is considerable further potential for potassium channel drug discovery. Potassium channels represent the largest and most diverse sub-group of ion channels and they play a central role in regulating the membrane potential of cells. Recent advances in genomics have greatly added to the number of these potential drug targets, but selecting a suitable potassium channel for drug discovery research is a key step. In particular, the potential therapeutic relevance of a potassium channel should be taken into account when selecting a target for screening. Potassium channel drug discovery is being driven by a need to identify lead compounds that can provide tractable starting points for medicinal chemistry. Furthermore, advances in laboratory automation have brought significant opportunities to increase screening throughput for potassium channel assays, but careful assay configuration to model drug-target interactions in a physiological manner is an essential consideration. Several potassium channel screening platforms are described in this review in order to provide some insight into the variety of formats available for screening, together with some of their inherent advantages and limitations. Particular emphasis is placed on the mechanistic basis of drug-target interaction and those aspects of structure/function that are of prime importance in potassium channel drug discovery.

Animals↗
PubMed2004

Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators.

Kinesins are a group of related molecular motor proteins that have great potential as targets for antimitotic drug development. We have developed two novel assays, one end-point and one kinetic, that are useful for the discovery and optimization of kinesin modulators. Both assays measure inorganic phosphate (Pi) generated by microtubule-activated kinesin adenosine triphosphatase activity. The assays were validated using the mitotic Eg5 kinesin-specific inhibitor, monastrol. A panel of nine kinesin motor domain proteins, representing 8 of the 14 classes of kinesins, was screened. The coefficient of variation for both assays was determined to be 4-14% depending on the panel member. Using the Eg5 kinetic assay with monastrol the IC50 value was 12 microM, which agrees well with previously published results. Two other closely related mitotic kinesins (AnBimC and MKLP1) were found to have IC50 values in the millimolar range. The other panel members (kinesin heavy chain, chromokinesin KIF4A, KIF3C, CENP-E, MCAK, and KIFC3) were not significantly inhibited by millimolar levels of monastrol. It is anticipated that screening of the nine-member panel of kinesins in these assays will serve as a platform for the discovery and development of specific kinesin modulators.

Adenosine Triphosphate↗
PubMed2005

Development of a mechanism-based assay for tissue transglutaminase--results of a high-throughput screen and discovery of inhibitors.

Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate in these disorders. Thus, this enzyme represents a viable target for drug discovery. We now report the development of a mechanism-based assay for TGase and the results of a screen using this assay in which we tested 56,500 drug-like molecules for their ability to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), respectively. Through a combination of steady state kinetic experiments and reaction progress curve simulations, we were able to calculate values for the initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state situation in which all thermodynamically significant forms of substrate-bound TGase exist in equal concentration. Under these conditions, the assay is sensitive to both competitive and mixed active-site inhibitors and to inhibitors that bind to the GTP site. The assay was optimized for automated screening in 384-well format and was then used to test our compound library. From among these compounds, 104 authentic hits that represent several mechanistic classes were identified.

Caseins↗