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At least 937 records · Page 52Linked to original sources
PubMed2000

High-throughput and virtual screening: core lead discovery technologies move towards integration.

In addition to high-throughput screening (HTS), the main lead discovery technology employed by most pharmaceutical companies today is virtual screening (VS). Although the two techniques have somewhat different philosophical origins, they contain many synergies that can potentially enhance the lead discovery process. Here, we describe many of the latest developments in VS technology with particular emphasis on their potential impact on HTS in, for example, focussed screening and data mining. In addition, we highlight key issues that need to be addressed before the potential of such efforts can be fully realized.

Journal Article↗
PubMed2005

High-throughput method for N-terminal sequencing of proteins by MALDI mass spectrometry.

A high-throughput method for sequencing of N termini of proteins by using postsource decay (PSD) of matrix-assisted laser desorption/ionization mass spectrometry has been developed. After a protein blotted on the PVDF membrane was successively reduced, S-alkylated, and guanidinated, its N-amino group was coupled to biotinylcysteic acid. The protein was then extracted from the membrane and digested with trypsin. The derivatized N-terminal fragment was then specifically isolated from the tryptic digest with avidin resins, and its de novo sequencing was successfully performed by PSD utilizing a sulfonic acid group introduced to the N terminus.

Animals↗
PubMedDate not supplied

Colorimetric diffusion-reflection imaging high-throughput analysis of organic or inorganic compounds.

A colorimetric diffusion-reflection imaging (CDRI) high-throughput analytical technology was developed for library analysis. In the investigation, quartz sands were employed as light diffusion-reflection media. Inorganic and organic compounds with characteristic absorption bands in visible light could be quantified by this method. In the current investigation, compounds such as CrCl3, KMnO4, methylene blue, and acrolein were employed as substrates, and the UV spectrometer and traditional GC (with thermal conductivity detector) were employed to check the reliability of our CDRI technology. The current technology is capable of analyzing more then 100 samples simultaneously. Relative errors below 10% were achieved.

Acrolein↗
PubMed2002

Conditionally immortalized cell lines as model systems for high-throughput biology in drug discovery.

There is an increasing emphasis on the need for high-quality biological data much earlier in the drug-discovery process. This has led to the development of high-throughput approaches to biology, many of which rely on the use of cell-culture models. Unfortunately, available cell-culture models often reflect poorly the characteristics of the tissue they are supposed to represent. However, the conditional-immortalization approach as applied by Xcellsyz offers the possibility of producing human cell lines on demand, which are truly representative of the tissue from which they derive.

Animals↗
PubMed2003

Elucidation of crystal form diversity of the HIV protease inhibitor ritonavir by high-throughput crystallization.

Pharmaceutical compounds are molecular solids that frequently exhibit polymorphism of crystal form. One high profile case of polymorphism was ritonavir, a peptidomimetic drug used to treat HIV-1 infection and introduced in 1996. In 1998, a lower energy, more stable polymorph (form II) appeared, causing slowed dissolution of the marketed dosage form and compromising the oral bioavailability of the drug. This event forced the removal of the oral capsule formulation from the market. We have carried out high-throughput crystallization experiments to comprehensively explore ritonavir form diversity. A total of five forms were found: both known forms and three previously unknown forms. The novel forms include a metastable polymorph, a hydrate phase, and a formamide solvate. The solvate was converted to form I via the hydrate phase by using a simple washing procedure, providing an unusual route to prepare the form I "disappearing polymorph" [Dunitz, J. D. & Bernstein, J. (1995) Acc. Chem. Res. 28, 193-200]. Crystals of form I prepared by using this method retained the small needle morphology of the solvate and thus offer a potential strategy for particle size and morphology control.

Crystallography, X-Ray↗
PubMed1996

High-throughput plasmid mini preparations facilitated by micro-mixing.

We have developed a reliable high-throughput plasmid isolation system using a 96-well plate format. This system combines a novel glass bead micro-mixing method with modified alkaline lysis and Sephacryl S-500 DNA purification procedures. Mechanical forces generated by vortexing glass beads inside each well of the 96-well plates ensure that the bacterial pellets are homogeneously resuspended, the cells are completely lyzed, and the resulting bacterial lysates are thoroughly mixed with the potassium acetate solution. The vortexing speed and duration for glass bead mixing have been standardized to facilitate plasmid DNA yields without significant adjustments.

Cells, Cultured↗
PubMed1998

High-throughput asymmetric double-crystal monochromator of the SAXS beamline at ELETTRA.

A new high-flux wiggler beamline for fast time-resolved small-angle X-ray scattering (SAXS) based on double-focusing optics has recently commenced operation at the 2 GeV third-generation storage ring ELETTRA at Trieste, Italy. Its non-dispersive double-crystal monochromator contains three pairs of interchangeable asymmetrically cut flat Si(111)-crystal pairs, each of which is optimized for high throughput at one of the three fixed energies 5.4, 8 and 16 keV. To cope with the severe thermal power load produced by a 57-pole wiggler on the first crystal of each pair (up to 5.4 W mm(-2) and 700 W under normal incidence, for 400 mA), grazing angles of 2 degrees and optimized back-cooling have been chosen. This solution allows simultaneously a gain of 2.5-3.0 in throughput and, accordingly, in flux density. Finite-element analysis as well as commissioning tests showed that the cooling layout functions very satisfactorily, and that up to 5 x 10(12) photons s(-1) are available at the sample (at 8 keV and 250 mA), as predicted.

Journal Article↗
PubMed2002

An automated computer system to support ultra high throughput SNP genotyping.

Celera Genomics has constructed an automated computer system to support ultra high-throughput SNP genotyping that satisfies the increasing demand that disease association studies are placing on current genotyping facilities. This system consists of the seamless integration of target SNP selection, automated oligo design, in silico assay quality validation, laboratory management of samples, reagents and plates, automated allele calling, optional manual review of autocalls, regular status reports, and linkage disequilibrium analysis. Celera has proven the system by generating over 2.5 million genotypes from more than 10,000 SNPs, and is approaching the target capacity of over 10,000 genotypes per machine per hour using limited human intervention with state of the art laboratory hardware.

Automation↗
PubMed2005

Evaluation of division-arrested cells for cell-based high-throughput screening and profiling.

Just-in-time cell supply for cell-based high-throughput screening (HTS) is frequently problematic. In addition to scheduling and logistical issues, quality issues and variability due to passage effect, cell cycle, or confluency contribute to day-to-day signal variability in the course of cell-based HTS campaigns. Cell division-arrest and cryopreservation technologies permit the use of cells as assay-ready reagents for HTS and other cell-based profiling and structure-activity studies. In this report, the authors compare division-arrested and dividing cells in 2 assay types that are dependent on movement of proteins within or through cell membranes: a receptor tyrosine kinase assay involving A431 cells responsive to epidermal growth factor, and a secretion reporter assay, which measures secretion of a reporter gene, secreted alkaline phosphatase. In both assays, dividing and division-arrested cells yielded similar basal and maximal signals at a given cell density. Similar IC50s were obtained for reference inhibitors in each assay, type in both dividing and division-arrested cells. In addition, for the secretion reporter assay, when comparing IC50s obtained from 44 compounds randomly chosen from a primary screening hit list, the rank order of potency obtained from dividing cells and division-arrested cells was essentially identical. Furthermore, the results show that, under certain assay conditions, data generated using division-arrested cells are less variable than those generated using dividing cells. In summary, the results suggest that, in many cases, division-arrested cells can substitute for dividing cells and offer certain advantages for cell-based assays.

Alkaline Phosphatase↗
PubMed2003

High-throughput screening assays for CYP2B6 metabolism and inhibition using fluorogenic vivid substrates.

CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo- and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme's involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (K(i), K(d), or K(m) values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.

Aryl Hydrocarbon Hydroxylases↗
PubMed2006

High-throughput multi-antigen microfluidic fluorescence immunoassays.

Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.

Antigens↗
PubMed2004

High-throughput microarray detection of olfactory receptor gene expression in the mouse.

The large number of olfactory receptor genes necessitates high throughput methods to analyze their expression patterns. We have therefore designed a high-density oligonucleotide array containing all known mouse olfactory receptor (OR) and V1R vomeronasal receptor genes. This custom array detected a large number of receptor genes, demonstrating specific expression in the olfactory sensory epithelium for approximately 800 OR genes previously designated as ORs based solely on genomic sequences. The array also enabled us to monitor the spatial and temporal distribution of gene expression for the entire OR family. Interestingly, OR genes showing spatially segregated expression patterns were also segregated on the chromosomes. This correlation between genomic location and spatial expression provides unique insights about the regulation of this large family of genes.

Age Factors↗
PubMedDate not supplied

Development of combinatorial chemistry methods for coatings: high-throughput adhesion evaluation and scale-up of combinatorial leads.

Coupling of combinatorial chemistry methods with high-throughput (HT) performance testing and measurements of resulting properties has provided a powerful set of tools for the 10-fold accelerated discovery of new high-performance coating materials for automotive applications. Our approach replaces labor-intensive steps with automated systems for evaluation of adhesion of 8 x 6 arrays of coating elements that are discretely deposited on a single 9 x 12 cm plastic substrate. Performance of coatings is evaluated with respect to their resistance to adhesion loss, because this parameter is one of the primary considerations in end-use automotive applications. Our HT adhesion evaluation provides previously unavailable capabilities of high speed and reproducibility of testing by using a robotic automation, an expanded range of types of tested coatings by using the coating tagging strategy, and an improved quantitation by using high signal-to-noise automatic imaging. Upon testing, the coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Using our HT methodology, we have developed several coatings leads. These HT screening results for the best coating compositions have been validated on the traditional scales of coating formulation and adhesion loss testing. These validation results have confirmed the superb performance of combinatorially developed coatings over conventional coatings on the traditional scale.

Journal Article↗
PubMedDate not supplied

Further development of a robust workup process for solution-phase high-throughput library synthesis to address environmental and sample tracking issues.

During further improvement of a high-throughput, solution-phase synthesis system, new workup tools and apparatus for parallel liquid-liquid extraction and evaporation have been developed. A combination of in-house design and collaboration with external manufacturers has been used to address (1) environmental issues concerning solvent emissions and (2) sample tracking errors arising from manual intervention. A parallel liquid-liquid extraction unit, containing miniature high-speed magnetic stirrers for efficient mixing of organic and aqueous phases, has been developed for use on a multichannel liquid handler. Separation of the phases is achieved by dispensing them into a newly patented filter tube containing a vertical hydrophobic porous membrane, which allows only the organic phase to pass into collection vials positioned below. The vertical positioning of the membrane overcomes the hitherto dependence on the use of heavier-than-water, bottom-phase, organic solvents such as dichloromethane, which are restricted due to environmental concerns. Both small (6-mL) and large (60-mL) filter tubes were developed for parallel phase separation in library and template synthesis, respectively. In addition, an apparatus for parallel solvent evaporation was developed to (1) remove solvent from the above samples with highly efficient recovery and (2) avoid the movement of individual samples between their collection on a liquid handler and registration to prevent sample identification errors. The apparatus uses a diaphragm pump to achieve a dynamic circulating closed system with a heating block for the rack of 96 sample vials and an efficient condenser to trap the solvents. Solvent recovery is typically >98%, and convenient operation and monitoring has made the apparatus the first choice for removal of volatile solvents.

Amides↗
PubMed1998

High throughput fluorescence-based conformation-sensitive gel electrophoresis (F-CSGE) identifies six unique BRCA2 mutations and an overall low incidence of BRCA2 mutations in high-risk BRCA1-negative breast cancer families.

Mutational analysis of cancer susceptibility genes has opened up a new era in clinical genetics. In this report we present the results of mutational analysis of the BRCA2 coding sequences in 105 high-risk individuals affected with breast cancer and/or ovarian cancer and previously found to be negative for mutations of the BRCA1 coding sequence in our laboratory. These individuals have a positive family history with three or more cases of breast cancer and/or ovarian cancer at any age from the same side of the family tree. In order to perform a high throughput and reliable mutational analysis of the BRCA genes, we have adapted the conformation-sensitive gel electrophoresis mutation-scanning assay to a fluorescent platform. The advantages are speed, reproducibility and enhanced resolving power of the scanning method. Four unique mutations, including one missense and three frameshift mutations, were identified in the pool of 60 non-Jewish patients (7%). Two cases of the 6174delT mutation were identified in the 45 Ashkenazi Jewish individuals studied (5%). In addition, two novel frameshift mutations, not characteristic of the Jewish subgroup, were identified. Thus there were four mutations in total in this ethnic subgroup (9%). The six mutations identified in this combined patient pool, excluding the 6174delT mutations, are novel and have not been previously reported in the Breast Cancer Information Core (BIC) database. The results indicate that BRCA2 mutations account for the disease in less than 10% of this patient population. In addition, there is no significant difference in frequency of BRCA2 mutations between the Ashkenazi Jewish and non-Jewish families in our clinical patient pool.

Adult↗
PubMed2002

Adaptation of aequorin functional assay to high throughput screening.

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.

Aequorin↗
PubMed1999

96-Well plates providing high optical resolution for high-throughput, immunofluorescence-based screening of monoclonal antibodies against Toxoplasma gondii.

We have developed a method for high resolution, high magnification immunofluorescence-based screening in a multi-well format, using a recently introduced 96-well plate specifically developed for fluorescence microscopy. We report here on the use of these plates to screen hybridoma supernatants for reactivity with specific subcellular compartments of the protozoan parasite Toxoplasma gondii. This has proven to be a powerful screening strategy, particularly when combined with high-throughput immunoblotting, and has enabled us to generate nine different monoclonal antibodies (MAbs) against either the periphery or structures within the apical end of T. gondii. The availability of a disposable, inexpensive, 96-well plate with optical properties suitable for high magnification imaging could lead to applications in a variety of fluorescence-based screening protocols.

Animals↗
PubMed2005

High-throughput single-strand conformation polymorphism analysis on a microfabricated capillary array electrophoresis device.

A high-density 384-lane microfabricated capillary array electrophoresis device is evaluated for high-throughput single-strand conformation polymorphism (SSCP) analysis. A delayed back bias direct electrokinetic injection scheme is used to provide better than 10-bp resolution with an 8.0-cm effective separation length. Separation of a HaeIII digest of PhiX174 yielded theoretical plate numbers of 4.0 x 10(6). Using 5% PDMA containing 10% glycerol and 15% urea, 21 single-nucleotide polymorphisms (SNPs) from HFE, MYL2, MYL3, and MYH7 genes associated with hereditary hemochromatosis (HHC) and hereditary hypertrophic cardiomyopathy (HCM) are discriminated at two running temperatures (25 degrees C and 40 degrees C), providing 100% sensitivity. The data in this study demonstrate that the 384-lane microCAE device provides the resolution and detection sensitivity required for SSCP analysis, showing its potential for ultrahigh-throughput mutation detection.

Cardiomyopathies↗