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At least 919 records · Page 51Linked to original sources
PubMed1997

Preclinical drug metabolism in the age of high-throughput screening: an industrial perspective.

With the advent of genomics, combinatorial paradigms and high-throughput screen (HTS)-based pharmacological testing, the number of compounds flowing through the discovery pipeline is likely to escalate. At the same time, with increased knowledge of the human drug-metabolizing enzymes and the availability of in vitro absorption-metabolism (AM) models, Preclinical Drug Metabolism is poised to meet the challenges of HTS. In order to be successful, however, a rational HTS strategy (vs. serendipitous HTS) has to be employed. Such a strategy is based on automation, validation and integration of in vitro AM models and database management (AVID). A generalized strategy for rational (AVID-based) HTS in Preclinical Drug Metabolism is described briefly.

Animals↗
PubMed2004

Proteome Analyst: custom predictions with explanations in a web-based tool for high-throughput proteome annotations.

Proteome Analyst (PA) (http://www.cs.ualberta.ca/~bioinfo/PA/) is a publicly available, high-throughput, web-based system for predicting various properties of each protein in an entire proteome. Using machine-learned classifiers, PA can predict, for example, the GeneQuiz general function and Gene Ontology (GO) molecular function of a protein. In addition, PA is currently the most accurate and most comprehensive system for predicting subcellular localization, the location within a cell where a protein performs its main function. Two other capabilities of PA are notable. First, PA can create a custom classifier to predict a new property, without requiring any programming, based on labeled training data (i.e. a set of examples, each with the correct classification label) provided by a user. PA has been used to create custom classifiers for potassium-ion channel proteins and other general function ontologies. Second, PA provides a sophisticated explanation feature that shows why one prediction is chosen over another. The PA system produces a Naïve Bayes classifier, which is amenable to a graphical and interactive approach to explanations for its predictions; transparent predictions increase the user's confidence in, and understanding of, PA.

Internet↗
PubMed2025

Dosa: A method to covalently barcode proteins for high throughput biochemistry.

Deep mutational scanning couples a protein's activity to DNA sequencing for high throughput assessment of the effects of all single amino acid substitutions, but it largely uses indirect assays, like growth, as proxy for protein activity. Here, we covalently link variant proteins in vivo to an RNA barcode by fusing them to E. coli tRNA (m5U54) methyltransferase TrmA (E358Q), which forms a covalent bond with a tRNA stem-loop. Following cell lysis, variant proteins are separated in vitro according to their biochemical properties and identified by their barcodes. We use this method, Dosa, to analyze a large pool of FLAG epitope variants for binding to an anti-FLAG antibody, to profile the cleavage preferences of variants of enteropeptidase and human rhinovirus 3C protease, and to measure the solubility of several hundred Aβ(1-42) variants. This method should be amenable to numerous biochemical assays with proteins produced in E. coli or mammalian cells.

Protein display↗
PubMed2006

The open-access high-throughput crystallization facility at EMBL Hamburg.

Here, the establishment of Europe's largest high-throughput crystallization facility with open access to the general user community is reported. The facility covers every step in the crystallization process from the preparation of crystallization cocktails for initial or customized screens to the setup of hanging-drop vapour-diffusion experiments and their automatic imaging. In its first year of operation, 43 internal and 40 external users submitted over 500 samples for a total of 2985 crystallization plates. An electronic booking system for registration, the selection of experimental parameters (e.g. drop size, sample-to-reservoir ratio) and the reservation of time slots was developed. External users can choose from more than 1000 initial crystallization conditions. By default, experiments are kept for six months and are imaged 15 times during this time period. A remote viewing system is available to inspect experiments via the internet. Over 100 stock solutions are available for users wishing to design customized screens.

Crystallization↗
PubMed2004

Enrichment of extremely noisy high-throughput screening data using a naïve Bayes classifier.

The noise level of a high-throughput screening (HTS) experiment depends on various factors such as the quality and robustness of the assay itself and the quality of the robotic platform. Screening of compound mixtures is noisier than screening single compounds per well. A classification model based on naïve Bayes (NB) may be used to enrich such data. The authors studied the ability of the NB classifier to prioritize noisy primary HTS data of compound mixtures (5 compounds/well) in 4 campaigns in which the percentage of noise presumed to be inactive compounds ranged between 81% and 91%. The top 10% of the compounds suggested by the classifier captured between 26% and 45% of the active compounds. These results are reasonable and useful, considering the poor quality of the training set and the short computing time that is needed to build and deploy the classifier.

Bayes Theorem↗
PubMed2006

Large-scale, high-throughput validation of short hairpin RNA sequences for RNA interference.

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for position specific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNA interference (RNAi).

Base Sequence↗
PubMed2001

Flow cytometric platform for high-throughput single nucleotide polymorphism analysis.

We have developed a rapid, cost-effective, high-throughput readout for single nucleotide polymorphism (SNP) genotyping using flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs a PCR-derived target DNA containing the SNP, a synthetic SNP-complementary ZipCode-bearing capture probe, a fluorescent reporter molecule, and a thermophilic DNA polymerase. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to the reaction products and sequestered them for flow cytometric analysis. The single base chain extension (SBCE) reaction was used to assay 20 multiplexed SNPs for 633 patients in 96-well format. Comparison of the microsphere-based SBCE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.3% agreement in genotype assignments. Substitution of direct-labeled R6G dideoxynucleotide with indirect-labeled phycoerythrin dideoxynucleotide enhanced signal five- to tenfold while maintaining low noise levels. A new assay based on allele-specific primer extension (ASPE) was validated on a set of 15 multiplexed SNPs for 96 patients. ASPE offers both the advantage of streamlining the SNP analysis protocol and the ability to perform multiplex SNP analysis on any mixture of allelic variants.

Flow Cytometry↗
PubMed2006

Normalization regarding non-random missing values in high-throughput mass spectrometry data.

We propose a two-step normalization procedure for high-throughput mass spectrometry (MS) data, which is a necessary step in biomarker clustering or classification. First, a global normalization step is used to remove sources of systematic variation between MS profiles due to, for instance, varying amounts of sample degradation over time. A probability model is then used to investigate the intensity-dependent missing events and provides possible substitutions for the missing values. We illustrate the performance of the method with a LC-MS data set of synthetic protein mixtures.

Chromatography, Liquid↗
PubMed1993

High-throughput purification of M13 templates for DNA sequencing.

Two variants of methods for high-throughput preparation of single-stranded M13 DNA are described. Both variants are derived from previously described chemistry and are appropriate for purification of M13 templates in 96-deep well plates. In both variants, phenol extraction is replaced by treatment with sodium iodide to disrupt phage proteins prior to ethanol precipitation of M13 DNA. In one of the variants, nonderivatized paramagnetic particles are employed to collect aggregated M13 phage particles and DNA, thereby replacing the need for centrifugation. The other variant omits the magnetic particles and utilizes a centrifuge that can accommodate the 96-deep well plates. Although the purification scheme that uses magnetic separation results in a decreased yield of M13 DNA, it is amenable to robotic automation strategies and thus will be useful for genomic sequencing projects. Performed manually, either method can easily produce 192 templates in a few hours. Although both variants produce DNA of sufficient quantity for automated fluorescent DNA sequencing, the procedure that utilizes magnetic separation provides template DNA of higher quality.

Autoanalysis↗
PubMed2006

Fast gradient elution reversed-phase high-performance liquid chromatography with diode-array detection as a high-throughput screening method for drugs of abuse. I. Chromatographic conditions.

A new approach for the high-throughput screening of biological samples to detect the presence of regulated intoxicants has been developed by modifying a conventional gradient elution high-performance liquid chromatograph (HPLC). The goal of this work was to improve the speed of gradient elution screening methods over current approaches by optimizing the operational parameters of both the column and the instrument without compromising the reproducibility of the retention times, which is the basis for the identification of intoxicant compounds. Most importantly, the novel instrument configuration substantially reduces the time needed to re-equilibrate the column between consecutive gradient runs, thereby reducing the total time for each analysis. The total analysis time for each gradient elution run is only 2.80 min, including 0.30 min for column re-equilibration between analyses. Retention times of standard calibration solutes are reproducible to better than 0.002 min in consecutive runs. A corrected retention index was adopted to account for day-to-day and column-to-column variations in retention time. For a set of forty-seven target compounds, the discriminating power and mean list length were found to be 0.95 and 3.26, respectively. In comparison to previous work with similar numbers of target compounds, the current approach provides an order of magnitude improvement in analysis time, and a four-fold decrease in mean list length.

Chromatography, High Pressure Liquid↗
PubMed2004

Novel neuroprotective K+ channel inhibitor identified by high-throughput screening in yeast.

Discovery of K+ channel modulators is limited by low-throughput capacity of existing K+ channel assays. To enable high-throughput screening for novel pharmacological modulators of K+ channels, we developed an assay based on growth of yeast that functionally expresses mammalian Kir2.1 channels. Screening of 10,000 small molecules from a combinatorial chemical library yielded 42 potential Kir2.1 inhibitors. One compound, 3-bicyclo[2.2.1]hept-2-yl-benzene-1,2-diol, was confirmed to inhibit K+ channels in patch-clamp measurements in mammalian cells with EC50 values of 60 and 1 microM for Kir2.1 and Kv2.1 channels, respectively. Inhibition of Kv2.1 channels decreased in the presence of the external pore blocker tetraethylammonium (TEA) and depended on a residue required for extracellular TEA action, suggesting that the identified compound targets the external mouth of the channel. Furthermore, at the nontoxic concentration of 3 microM, the identified compound completely abolished in vitro neuronal apoptosis mediated by Kv2.1 channels. Therefore, yeast-based screening has identified a novel uncharged neuroprotective mammalian K+ channel inhibitor.

Animals↗
PubMed2004

A periplasmic fluorescent reporter protein and its application in high-throughput membrane protein topology analysis.

We have developed a periplasmic fluorescent reporter protein suitable for high-throughput membrane protein topology analysis in Escherichia coli. The reporter protein consists of a single chain (scFv) antibody fragment that binds to a fluorescent hapten conjugate with high affinity. Fusion of the scFv to membrane protein sites that are normally exposed in the periplasmic space tethers the scFv onto the inner membrane. Following permealization of the outer membrane to allow diffusion of the fluorescent hapten into the periplasm, binding to the anchored scFv renders the cells fluorescent. We show that cell fluorescence is an accurate and sensitive reporter of the location of residues within periplasmic loops. For topological analysis, a set of nested deletions in the membrane protein gene is employed to construct two libraries of gene fusions, one to the scFvand one to the cytoplasmic reporter green fluorescent protein (GFP). Fluorescent clones are isolated by flow cytometry and the sequence of the fusion junctions is determined to identify amino acid residues within periplasmic and cytoplasmic loops, respectively. We applied this methodology to the topology analysis of E. coli TatC protein for which previous studies had led to conflicting results. The ease of screening libraries of fusions by flow cytometry enabled the rapid identification of almost 90 highly fluorescent scFv and GFP fusions, which, in turn, allowed the fine mapping of TatC membrane topology.

Amino Acid Sequence↗
PubMed2005

Drug-excipient compatibility testing using a high-throughput approach and statistical design.

The aim of our research was to develop a miniaturized high throughput drug-excipient compatibility test. Experiments were planned and evaluated using statistical experimental design. Binary mixtures of a drug, acetylsalicylic acid, or fluoxetine hydrochloride, and of excipients commonly used in solid dosage forms were prepared at a ratio of approximately 1:100 in 96-well microtiter plates. Samples were exposed to different temperature (40 degrees C/ 50 degrees C) and humidity (10%/75%) for different time (1 week/4 weeks), and chemical drug degradation was analyzed using a fast gradient high pressure liquid chromatography (HPLC). Categorical statistical design was applied to identify the effects and interactions of time, temperature, humidity, and excipient on drug degradation. Acetylsalicylic acid was least stable in the presence of magnesium stearate, dibasic calcium phosphate, or sodium starch glycolate. Fluoxetine hydrochloride exhibited a marked degradation only with lactose. Factor-interaction plots revealed that the relative humidity had the strongest effect on the drug excipient blends tested. In conclusion, the developed technique enables fast drug-excipient compatibility testing and identification of interactions. Since only 0.1 mg of drug is needed per data point, fast rational preselection of the pharmaceutical additives can be performed early in solid dosage form development.

Aspirin↗
PubMed2006

High-throughput axial MALDI-TOF MS using a 2-kHz repetition rate laser.

A high-throughput axial MALDI-TOF mass spectrometer utilizing a laser with a 2-kHz pulse repetition-rate was constructed and tested. This fast mass spectrometer provided a data acquisition rate 10 times faster than a commercially available (200 Hz) axial mass spectrometer, while maintaining comparable limits of detection (200 amol of Glu fib peptide). Mass resolution, only slightly less than the commercial instrument (10 000 vs 14 000), was sufficient for baseline resolution of isotopic clusters of peptides with m/z <2700. A new method of mass calibration, which combined a limited number of internal and external standards, provided the same 15 ppm mass accuracy over the entire sample plate on either instrument. Implementing the 2-kHz laser required a faster data acquisition system and high-voltage pulse electronics, together with a novel strategy for rapid sample plate movements during acquisition, to achieve a sample analysis rate of up to 2 spots/s (with 800 shots/spot). The overall performance of the fast MALDI-TOF MS instrument was demonstrated by the acquisition, in 12 min, of an LC-MS data set from a plate of 625 fractions collected during LC separation of an 16O/18O differentially labeled proteomic sample of a tryptic digest of an E. coli lysate.

Bacterial Proteins↗
PubMed2006

Parallel, high-throughput purification of recombinant antibodies for in vivo cell assays.

We describe a method for high-throughput, parallel purification of secreted proteins to analyse large numbers of protein samples in cell-based assays for the discovery of protein therapeutics. The procedure is generic and capable of 96 parallel purifications and compatible, in both yield and purity, with a wide assay range. By optimising expression and purification steps as well as using novel hardware, in particular a chromatography press capable to purify target proteins from viscous media, we exemplify the process for the generation of single-chain Fv antibody fragments (scFv) and the purification of full-length IgG. The described process can operate robustly with a throughput of over 2,000 samples per month.

Antibodies, Monoclonal↗
PubMed2000

Ternary-column system for high-throughput direct-injection bioanalysis by liquid chromatography/tandem mass spectrometry.

As a continuation of our efforts to improve our high-flow on-line bioanalytical approach for high-throughput quantitation of drugs and metabolites in biological matrices by high-performance liquid chromatography (LC) and tandem mass spectrometry (MS/MS), we have developed a ternary-column on-line LC/MS/MS system with dual extraction columns used in parallel for purification and an analytical column for analysis. The advantage of the dual extraction column system is that sample analysis can take place in one of the extraction columns while the other column is being equilibrated. Thus, the equilibration time does not add to the run time, hence shortening the injection cycle time and increasing the sample throughput. Moreover, the use of two extraction columns in parallel increases the number of samples that can be injected before the system fails due to an overused extraction column. Such a system has successfully been used to develop and validate a positive ion electrospray LC/MS/MS bioanalytical method for the quantitative determination of a guanidine-containing drug candidate in rat plasma. The system used for this work utilized two Oasis HLB extraction columns (1 x 50 mm, 30 microm), one C18 analytical column (3.9 x 50 mm, 5 microm), a ten-port switching value and a tandem mass spectrometer. The on-line analysis was accomplished by the direct injection of 10 microL of the sample, obtained by mixing a rat plasma sample 1:1 with an aqueous internal standard solution. Selected reaction monitoring (SRM) was utilized for the detection of the analyte and internal standard. The standard curve range was 1.00-200 ng/mL. The intra- and inter-day precision and accuracy were within 6.6%. The on-line purification step lasted for only 0.3 min and total run time was only 1.6 min.

Animals↗