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PubMedDate not supplied

High-throughput study of poly(ethylene glycol)/ibuprofen formulations under controlled environment using FTIR imaging.

Simultaneous analysis of many samples under identical conditions improves the effectiveness of research and accelerates product design. A novel spectroscopic imaging approach using a multichannel detector has been developed for parallel analysis of pharmaceutical formulations under controlled environments. Samples of formulations of ibuprofen in poly(ethylene glycol) have been prepared with ibuprofen concentrations ranging from 0 to 100% using a microdroplet deposition approach. The concentration of ibuprofen in PEG at which dimerization of ibuprofen molecules can be avoided has been determined via simultaneous measurement of all samples using in situ FTIR spectroscopic imaging. FTIR spectra from all samples have been analyzed to assess the molecular state of the drug and the degree of polymer swelling as a function of drug concentration. The effect of elevated temperature on the stability of all formulations was also studied. This high-throughput approach identified the concentration range for stable formulations and provided evidence that hydrogen bonding between ibuprofen and the polymer is responsible for enhanced stability at higher temperatures. This high-throughput imaging approach, based on a miniature sampling system, significantly reduces the experimental time by allowing many (potentially a few thousand) experiments to be run in parallel and increases the accuracy by minimizing variations between experiments.

Drug Stability↗
PubMed2005

Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets.

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

Animals↗
PubMed2000

A high-throughput AFLP-based method for constructing integrated genetic and physical maps: progress toward a sorghum genome map.

Sorghum is an important target for plant genomic mapping because of its adaptation to harsh environments, diverse germplasm collection, and value for comparing the genomes of grass species such as corn and rice. The construction of an integrated genetic and physical map of the sorghum genome (750 Mbp) is a primary goal of our sorghum genome project. To help accomplish this task, we have developed a new high-throughput PCR-based method for building BAC contigs and locating BAC clones on the sorghum genetic map. This task involved pooling 24,576 sorghum BAC clones ( approximately 4x genome equivalents) in six different matrices to create 184 pools of BAC DNA. DNA fragments from each pool were amplified using amplified fragment length polymorphism (AFLP) technology, resolved on a LI-COR dual-dye DNA sequencing system, and analyzed using Bionumerics software. On average, each set of AFLP primers amplified 28 single-copy DNA markers that were useful for identifying overlapping BAC clones. Data from 32 different AFLP primer combinations identified approximately 2400 BACs and ordered approximately 700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located approximately 200 of the BAC contigs on the sorghum genetic map. Restriction endonuclease fingerprinting of the entire collection of sorghum BAC clones was applied to test and extend the contigs constructed using this PCR-based methodology. Analysis of the fingerprint data allowed for the identification of 3366 contigs each containing an average of 5 BACs. BACs in approximately 65% of the contigs aligned by AFLP analysis had sufficient overlap to be confirmed by DNA fingerprint analysis. In addition, 30% of the overlapping BACs aligned by AFLP analysis provided information for merging contigs and singletons that could not be joined using fingerprint data alone. Thus, the combination of fingerprinting and AFLP-based contig assembly and mapping provides a reliable, high-throughput method for building an integrated genetic and physical map of the sorghum genome.

Chromosomes, Bacterial↗
PubMed2004

Development and implementation of a highly miniaturized confocal 2D-FIDA-based high-throughput screening assay to search for active site modulators of the human heat shock protein 90beta.

The beta isoform of the heat shock protein 90 (Hsp90beta) is a cellular chaperone required for the maturation of key proteins involved in growth response to extracellular factors as well as oncogenic transformation of various cell types. Compounds that inhibit the function of Hsp90beta are thus believed to have potential as novel anticancer drugs. To date, 2 fungal metabolites are known to inhibit Hsp90beta. However, insolubility and liver toxicity restrict the clinical use of these molecules. The limitation to identify novel and safe Hsp90beta inhibitors is that presently no suitable high-throughput screening assay is available. Here, the authors present the development of a homogenous assay based on 2-dimensional fluorescence intensity distribution analysis of tetramethyl-rhodamine (TAMRA)-labeled radicicol bound to Hsp90beta. Furthermore, the assay has been shown to be compatible with the confocal nanoscreening platform Mark II from Evotec-Technologies and can therefore be used for miniaturized high-throughput screening. The applied detection technology provides critical information about the nature of biomolecular interaction at the thermodynamic equilibrium, such as affinity constants and stoichiometric parameters of the binding. The assay is used to identify small molecular weight compounds displacing TAMRA-radicicol. Such compounds are believed to be important molecules in the discovery of novel anticancer drugs.

Biological Assay↗
PubMed2004

A nonradiometric, high-throughput assay for poly(ADP-ribose) glycohydrolase (PARG): application to inhibitor identification and evaluation.

The enzyme poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of glycosidic bonds of ADP-ribose polymers, producing monomeric ADP-ribose units. Thus, in conjunction with poly(ADP-ribose) polymerase (PARP), PARG activity regulates the extent of in vivo poly(ADP-ribosyl)ation. Small molecule inhibitors of PARP and PARG have shown considerable promise in cellular models of ischemia-reperfusion injury and oxidative neuronal cell death. However, currently available PARG inhibitors are not ideal due to cell permeability, size, and/or toxicity concerns; therefore, new small molecule inhibitors of this important enzyme are sorely needed. Existing methodologies for in vitro assessment of PARG enzymatic activity do not lend themselves to high-throughput screening applications, as they typically use a radiolabeled substrate and determine product quantities through TLC analysis. This article describes a method whereby the ADP-ribose product of the PARG-catalyzed reaction is converted into a fluorescent dye. This highly sensitive and reproducible method is demonstrated by identifying two known PARG inhibitors in a 384-well plate assay and by subsequently determining IC(50) values for these compounds. Thus, this high-throughput, nonradioactive PARG assay should find widespread use in experiments directed toward identification of novel PARG inhibitors.

Animals↗
PubMed2001

High-throughput fluorogenic assay for determination of botulinum type B neurotoxin protease activity.

Botulinum neurotoxins are responsible for botulism, a flaccid muscular paralysis caused by inhibition of acetylcholine release at the neuromuscular junction. This occurs by cleavage of conserved proteins involved in exocytosis such as synaptobrevin by the zinc metallopeptidase activity of the light chain of some botulinum neurotoxins. Botulism, for which there is presently no therapy available, is a relatively widespread disease that may result in death. Consequently, the development of drugs able to inhibit the hydrolytic activity of these neurotoxins is of great interest. Design and screening of such inhibitors could be largely facilitated by using high-throughput assays. With this aim, a novel in vitro test for quantifying the proteolytic activity of botulinum type B neurotoxin was developed. The substrate is the 60--94 fragment of human synaptobrevin-1 which was modified by introduction of the fluorescent amino acid l-pyrenylalanine in position 74 and a p-nitrophenylalanyl residue as quenching group in position 77. The cleavage of Syb 60-94 [Pya(74), Nop(77)] by the toxin active chain occurs selectively between residues 76 and 77 as in the case of the unmodified synaptobrevin and is directly quantified by measuring the strong fluorescence of the formed metabolite Syb 60-76 [Pya(74)]. This is the easiest, quickest, and cheapest assay described to date for measuring the proteolytic activity of botulinum type B neurotoxin. It can be easily automated for high-throughput screening. Moreover, amounts of about 3.5 pg/ml of botulinum type B neurotoxin could be detected by this method.

Biological Assay↗
PubMed2006

A high-throughput microfluidic assay for SH2 domain-containing inositol 5-phosphatase 2.

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) is a potential drug target for the treatment of type 2 diabetes. This enzyme serves as a negative regulator of insulin-mediated signal transduction by catalyzing the dephosphorylation of the second messenger lipid molecule phosphatidylinositol 3,4,5-triphosphate. Traditionally, assays for phosphoinositide phosphatases such as SHIP2 have relied on radiolabeled phosphatidylinositol-containing lipid membranes and chromatographic separation of labeled phospholipid substrate from product by thin-layer chromatography. We have expressed and purified catalytically active phosphatase domain constructs of SHIP2 from Escherichia coli and developed a sensitive and antibody- or binding protein-independent assay for SHIP2 amenable to high-throughput screening of phosphoinositide phosphatases or phosphoinositide kinases. This microfluidic assay, with Z' values approximately 0.8, is based upon the difference in mobility within an electric field between a fluorophore-labeled phosphatidylinositol 3,4,5-triphosphate substrate and the corresponding 3,4-bisphosphate product. High-throughput screening of a 91,060-member compound library in 384-well format resulted in the identification of SHIP2 inhibitors.

Chromatography, Thin Layer↗
PubMed2005

Development of a miniaturized assay for the high-throughput screening program for poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.

Biotinylation↗
PubMed2005

Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry.

Methylation is one of the major epigenetic processes pivotal to our understanding of carcinogenesis. It is now widely accepted that there is a relationship between DNA methylation, chromatin structure, and human malignancies. DNA methylation is potentially an important clinical marker in cancer molecular diagnostics. Understanding epigenetic modifications in their biological context involves several aspects of DNA methylation analysis. These aspects include the de novo discovery of differentially methylated genes, the analysis of methylation patterns, and the determination of differences in the degree of methylation. Here we present a previously uncharacterized method for high-throughput DNA methylation analysis that utilizes MALDI-TOF mass spectrometry (MS) analysis of base-specifically cleaved amplification products. We use the IGF2/H19 region to show that a single base-specific cleavage reaction is sufficient to discover methylation sites and to determine methylation ratios within a selected target region. A combination of cleavage reactions enables the complete evaluation of all relevant aspects of DNA methylation, with most CpGs represented in multiple reactions. We successfully applied this technology under high-throughput conditions to quantitatively assess methylation differences between normal and neoplastic lung cancer tissue samples from 48 patients in 47 genes and demonstrate that the quantitative methylation results allow accurate classification of samples according to their histopathology.

Binding Sites↗
PubMed2006

High-throughput single-nucleotide polymorphisms genotyping: TaqMan assay and pyrosequencing assay.

Single-nucleotide polymorphisms (SNPs) are DNA sequence variations that occur at a single base in the genome sequence. SNPs are valuable markers for identifying genes responsible for susceptibility to common diseases, and in some cases, they are the causes of human diseases. A genetic study of a complex disease usually involves a case-control association study that requires genotyping of a large number of SNPs in hundreds of patients (cases) and matched controls. A significant difference of the allele frequency or genotypic frequency of a SNP between the two populations is considered to be the evidence for the association between the SNP and disease. A key to a fast and effective case-control association study requires high-throughput genotyping of SNPs. Two assays-the TaqMan SNP genotyping assay and the pyrosequencing assay-have been developed for this purpose and proven to be particularly useful. Here, we present the operative protocol, clarify the key technical issues, and highlight certain cautionary notes for high throughput SNP genotyping using TaqMan and pyrosequencing assays.

Gene Frequency↗
PubMed2004

The role of atelocollagen-based cell transfection array in high-throughput screening of gene functions and in drug discovery.

The human genome project has been completed, but the function of many genes is unknown. It is, therefore, necessary to elucidate the function of a large number of genes within a short time. To achieve this goal, materials are needed that condense or package DNA into nano-particles that can easily be taken up by cells and would allow DNA to be retained without degradation. Atelocollagen is a reliable carrier for gene delivery because it is considered safe and appropriate for practical use. We developed a basic technique for high-throughput gene transfer and expression screening by pre-coating a multi-well plate with an Atelocollagen/DNA complex in which cells are then seeded. Complexes with a nano-particle form were efficiently transduced into cells without the use of additional transfection reagents, and they allowed for long-term gene expression. The complex spotted onto the well of a plate was stable for a long period and allowed the cells to transduce and express reporter genes. We also showed that the present method with Atelocollagen-based gene transfer is applicable to gene medicines, such as antisense ODNs, siRNA, and adenovirus vectors. These results suggest that an Atelocollagen-based cell transfection array may be appropriate for general use in the high-throughput screening of large sets of gene medicines with functions in mammalian cells.

Collagen↗
PubMed2005

High-throughput two-hybrid analysis. The promise and the peril.

The two-hybrid method detects the interaction of two proteins by their ability to reconstitute the activity of a split transcription factor, thus allowing the use of a simple growth selection in yeast to identify new interactions. Since its introduction about 15 years ago, the assay largely has been applied to single proteins, successfully uncovering thousands of novel protein partners. In the last few years, however, two-hybrid experiments have been scaled up to focus on the entire complement of proteins found in an organism. Although a single such effort can itself result in thousands of interactions, the validity of these high-throughput approaches has been questioned as a result of the prevalence of numerous false positives in these large data sets. Such artifacts may not be an obstacle to continued scale-up of the method, because the classification of true and false positives has proven to be a computational challenge that can be met by a growing number of creative strategies. Two examples are provided of this combination of high-throughput experimentation and computational analysis, focused on the interaction of Plasmodium falciparum proteins and of Saccharomyces cerevisiae membrane proteins.

Animals↗
PubMed2002

Identification of oncogenes collaborating with p27Kip1 loss by insertional mutagenesis and high-throughput insertion site analysis.

The p27(Kip1) protein is a cyclin-dependent kinase inhibitor that blocks cell division in response to antimitogenic cues. p27 expression is reduced in many human cancers, and p27 functions as a tumor suppressor that exhibits haploinsufficiency in mice. Despite the well characterized role of p27 as a cyclin-dependent kinase inhibitor, its mechanism of tumor suppression is unknown. We used Moloney murine leukemia virus to induce lymphomas in p27+/+ and p27-/- mice and observed that lymphomagenesis was accelerated in the p27-/- animals. To identify candidate oncogenes that collaborate with p27 loss, we used a high-throughput strategy to sequence 277 viral insertion sites derived from two distinct sets of p27-/- lymphomas and determined their chromosomal location by comparison with the Celera and public (Ensembl) mouse genome databases. This analysis identified a remarkable number of putative protooncogenes in these lymphomas, which included loci that were novel as well as those that were overrepresented in p27-/- tumors. We found that Myc activations occurred more frequently in p27-/- lymphomas than in p27+/+ tumors. We also characterized insertions within two novel loci: (i) the Jun dimerization protein 2 gene (Jundp2), and (ii) an X-linked locus termed Xpcl1. Each of the loci that we found to be frequently involved in p27-/- lymphomas represents a candidate oncogene collaborating with p27 loss. This study illustrates the power of high-throughput insertion site analysis in cancer gene discovery.

Animals↗
PubMed2002

Protein crystallization for genomics: towards high-throughput optimization techniques.

Protein crystallization has gained a new strategic and commercial relevance in the next phase of the genome projects, in which X-ray crystallography will play a major role. Considerable advances have been made in the automation of protein preparation and also in the X-ray analysis and bioinformatics stages once diffraction-quality crystals are available. These advances have not yet been matched by equally good methods for the crystallization process itself. In the area of crystallization, the main effort and resources are currently being invested into the automation of screening procedures to identify potential crystallization conditions. However, in spite of the ability to generate numerous trials, so far only a small percentage of the proteins produced have led to structure determinations. This is because screening in itself is not usually enough; it has to be complemented by an equally important procedure in crystal production, namely crystal optimization. In the rush towards structural genomics, optimization techniques have been somewhat neglected, mainly because it was hoped that large-scale screening alone would produce the desired results. In addition, optimization has relied on particular individual methods that are often difficult to automate and to adapt to high throughput. This article addresses a major gap in the field of structural genomics by describing practical ways of automating individual optimization methods in order to adapt them to high-throughput techniques.

Automation↗
PubMed2004

Mining the structural genomics pipeline: identification of protein properties that affect high-throughput experimental analysis.

Structural genomics projects represent major undertakings that will change our understanding of proteins. They generate unique datasets that, for the first time, present a standardized view of proteins in terms of their physical and chemical properties. By analyzing these datasets here, we are able to discover correlations between a protein's characteristics and its progress through each stage of the structural genomics pipeline, from cloning, expression, purification, and ultimately to structural determination. First, we use tree-based analyses (decision trees and random forest algorithms) to discover the most significant protein features that influence a protein's amenability to high-throughput experimentation. Based on this, we identify potential bottlenecks in various stages of the structural genomics process through specialized "pipeline schematics". We find that the properties of a protein that are most significant are: (i.) whether it is conserved across many organisms; (ii). the percentage composition of charged residues; (iii). the occurrence of hydrophobic patches; (iv). the number of binding partners it has; and (v). its length. Conversely, a number of other properties that might have been thought to be important, such as nuclear localization signals, are not significant. Thus, using our tree-based analyses, we are able to identify combinations of features that best differentiate the small group of proteins for which a structure has been determined from all the currently selected targets. This information may prove useful in optimizing high-throughput experimentation. Further information is available from http://mining.nesg.org/.

Algorithms↗
PubMed2005

Development of a time-resolved fluorometric assay for the high throughput screening of melanin concentrating hormone receptor antagonists.

INTRODUCTION: Melanin concentrating hormone is an orexigenic hypothalamic neuropeptide, which plays an important role in the complex regulation of energy balance and body weight mediated by the melanin concentrating hormone receptor subtype 1 (MCH1). Compelling pharmacological evidence implicating MCH1 signaling in the regulation of food intake and energy expenditure has generated a great deal of interest by pharmaceutical companies as MCH1 antagonists may have potential therapeutic benefit in the treatment of obesity and metabolic syndrome. METHODS: Although radioligand receptor binding assay has been one of the most powerful tools for receptor research and drug discovery, the limitations of radioisotopes and the problems related to safety and waste disposal limits their application in high throughput screening and has led to a growing interest in alternative, nonradioactive technologies. To develop a sensitive and reproducible assay system for MCH1, the time-resolved fluorescence (TRF) receptor binding assay with AcroWell filter plates was tested and validated. RESULTS: Comparing to the radioligand receptor binding assay for MCH1, the TRF assay presented higher Z/Z' factors with the lower signal-to-noise ratio. The known high-affinity MCH1 receptor antagonist, SNAP-7941, exhibited an IC50 value of 1.66+/-0.10 nM that is very similar to the IC50 value of MCH in a radioligand binding assay with an excellent correlation coefficient (0.9884). DISCUSSION: These results suggest that our TRF receptor binding assay for MCH1 can achieve the desired sensitivity and reproducibility to replace the radioligand receptor assay in a fluorometric system that can be developed for high throughput screening.

Animals↗
PubMed2005

High-throughput inference of protein-protein interfaces from unassigned NMR data.

SUMMARY: We cast the problem of identifying protein-protein interfaces, using only unassigned NMR spectra, into a geometric clustering problem. Identifying protein-protein interfaces is critical to understanding inter- and intra-cellular communication, and NMR allows the study of protein interaction in solution. However it is often the case that NMR studies of a protein complex are very time-consuming, mainly due to the bottleneck in assigning the chemical shifts, even if the apo structures of the constituent proteins are known. We study whether it is possible, in a high-throughput manner, to identify the interface region of a protein complex using only unassigned chemical shifts and residual dipolar coupling (RDC) data. We introduce a geometric optimization problem where we must cluster the cells in an arrangement on the boundary of a 3-manifold, where the arrangement is induced by a spherical quadratic form [corrected] The arrangement is induced by a spherical quadratic form, which in turn is parameterized by a SO(3)xR2. We show that this formalism derives directly from the physics of RDCs. We present an optimal algorithm for this problem that runs in O(n3 log n) time for an n-residue protein. We then use this clustering algorithm as a subroutine in a practical algorithm for identifying the interface region of a protein complex from unassigned NMR data. We present the results of our algorithm on NMR data for seven proteins from five protein complexes, and show that our approach is useful for high-throughput applications in which we seek to rapidly identify the interface region of a protein complex. AVAILABILITY: Contact authors for source code.

Algorithms↗
PubMed2006

High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1.

The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening.

Antineoplastic Agents↗