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High-throughput Screening (high-throughput + screening)
Selected AbstractsNew Monofunctionalized Fluorescein Derivatives for the Efficient High-Throughput Screening of Lipases and Esterases in Aqueous MediaHELVETICA CHIMICA ACTA, Issue 3 2006Yongzheng Yang Abstract Monoalkylation or acylation of fluorescein (1) with various acyloxymethyl or acyl halides afforded, respectively, a series of ether- (2) and ester-functionalized (3) fluorogenic probes. The highly reactive and water-soluble substrates release fluorescein (1) upon reaction with lipases and esterases within seconds or minutes, both under fully aqueous conditions or in the presence of DMSO (20%) as a co-solvent. The most-reactive substrates in the two series were the octanoic acid derivatives 2f (=,2-{6-[(octanoyloxy)methoxy]-3-oxo-3H -xanthen-9-yl}benzoic acid) and 3a (=,2-[6-(octanoyloxy)-3-oxo-3H -xanthen-9-yl]benzoic acid). Esterases were found to generally react faster under aqueous conditions, while lipases were more reactive in the presence of DMSO as a co-solvent. [source] Challenges and Progress in High-Throughput Screening of Polymer Mechanical Properties by IndentationADVANCED MATERIALS, Issue 35 2009Johannes M. Kranenburg Abstract Depth-sensing or instrumented indentation is an experimental characterization approach well-suited for high-throughput investigation of mechanical properties of polymeric materials. This is due to both the precision of force and displacement, and to the small material volumes required for quantitative analysis. Recently, considerable progress in the throughput (number of distinct material samples analyzed per unit time) of indentation experiments has been achieved, particularly for studies of elastic properties. Future challenges include improving the agreement between various macroscopic properties (elastic modulus, creep compliance, loss tangent, onset of nonlinear elasticity, energy dissipation, etc.) and their counterpart properties obtained by indentation. Sample preparation constitutes a major factor for both the accuracy of the results and the speed and efficiency of experimental throughput. It is important to appreciate how this processing step may influence the mechanical properties, in particular the onset of nonlinear elastic or plastic deformation, and how the processing may affect the agreement between the indentation results and their macroscopic analogues. [source] High-Throughput Screening of the Influence of Thermal Treatment on the Mechanical Properties of Semicrystalline Polymers: A Case Study for iPPMACROMOLECULAR RAPID COMMUNICATIONS, Issue 1 2004Konrad Schneider Abstract Summary: High-throughput screening is a convenient tool to determine the influence of parameters, such as composition or processing conditions on certain materials' properties. In the present study, iPP was used to construct a combinatorial library made from a processing temperature gradient requiring only a minimised amount of material. Clear changes in the crystalline modification and crystal morphology of the iPP, and their impact on mechanical properties have been identified. The construction of a combinatorial library to study the effect of thermal history upon the properties of polymers. [source] Fast Liquid Chromatography for High-Throughput Screening of PolymersMACROMOLECULAR RAPID COMMUNICATIONS, Issue 1 2003Harald Pasch Abstract Liquid chromatography of polymers is traditionally a slow technique with analysis times of typically 30 min per sample. For the application of liquid chromatographic techniques to combinatorial materials research the analysis time per sample must be reduced considerably. Analysis time in SEC can be reduced to about 2 min per sample when high-throughput columns are used. For HPLC small columns with improved separation efficiencies can be used. As compared to conventional technology, time savings of more than 80% are achieved. Chromatogram from conventional SEC column compared to high-speed SEC column tested on an identical instrument with polystyrene standards in THF. [source] Enzyme Assays: High-Throughput Screening, Genetic Selection and Fingerprinting.CHEMBIOCHEM, Issue 9 2006Edited by Jean-Louis Reymond. No abstract is available for this article. [source] A pH-Based High-Throughput Screening of Sucrose-Utilizing Transglucosidases for the Development of Enzymatic Glucosylation ToolsCHEMCATCHEM, Issue 8 2010Elise Champion Dr. Abstract Sucrose-utilizing transglucosidases are valuable enzymatic tools for the diversification of carbohydrate-based molecules. Among them, recombinant amylosucrase from Neisseria polysaccharea is a glucansucrase that naturally catalyzes the synthesis of an amylose-like polymer as well as the transglucosylation of exogenous hydroxylated acceptors. A semirational engineering approach was recently undertaken to redesign the enzyme active site and adapt it to the glucosylation of a nonnatural acceptor, allyl 2- N -acetyl-2-deoxy-,- D -glucopyranoside (,- D -GlcpNAcOAll), to produce a key building block in the chemoenzymatic synthesis of Shigella flexneri 1b,serotype O-antigen repeating unit. This prior work shows the beneficial effect of single amino acid mutations at two positions (228 and 290) on the recognition of the acceptor by amylosucrase. On the basis of these first results, a library of about 8000 amylosucrase variants combining mutations at these two positions is constructed by saturation mutagenesis. The library is prescreened using a novel pH-sensitive colorimetric screening method for the detection of sucrose-utilizing amylosucrase variants, thereby reducing by about 95,% the size of the library to be subsequently screened for acceptor glucosylation. Active clones (5,% of the initial library) are then screened for acceptor recognition, leading to the isolation of 20 variants of potential interest for the production of the target disaccharide ,- D -Glcp-(1,4)-,- D -GlcpNAc. [source] ChemInform Abstract: New Methods for the High-Throughput Screening of Enantioselective Catalysts and Biocatalysts.CHEMINFORM, Issue 32 2002Manfred T. Reetz Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] High-throughput screening of chemical exchange saturation transfer MR contrast agentsCONTRAST MEDIA & MOLECULAR IMAGING, Issue 3 2010Guanshu Liu Abstract A new high-throughput MRI method for screening chemical exchange saturation transfer (CEST) agents is demonstrated, allowing simultaneous testing of multiple samples with minimal attention to sample configuration and shimming of the main magnetic field (B0). This approach, which is applicable to diamagnetic, paramagnetic and liposome CEST agents, employs a set of inexpensive glass or plastic capillary tubes containing CEST agents put together in a cheap plastic tube holder, without the need for liquid between the tubes to reduce magnetic susceptibility effects. In this setup, a reference image of direct water saturation spectra is acquired in order to map the absolute water frequency for each volume element (voxel) in the sample image, followed by an image of saturation transfer spectra to determine the CEST properties. Even though the field over the total sample is very inhomogeneous due to air,tube interfaces, the shape of the direct saturation spectra is not affected, allowing removal of susceptibility shift effects from the CEST data by using the absolute water frequencies from the reference map. As a result, quantitative information such as the mean CEST intensity for each sample can be extracted for multiple CEST agents at once. As an initial application, we demonstrate rapid screening of a library of 16 polypeptides for their CEST properties, but in principle the number of tubes is limited only by the available signal-noise-ratio, field of view and gradient strength for imaging. Copyright © 2010 John Wiley & Sons, Ltd. [source] High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detectionELECTROPHORESIS, Issue 1-2 2003Yan He Abstract Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC50 value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases. [source] Duchenne's muscular dystrophy: animal models used to investigate pathogenesis and develop therapeutic strategiesINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2003C.A. Collins Summary., Duchenne's muscular dystrophy (DMD) is a lethal childhood disease caused by mutations of the dystrophin gene, the protein product of which, dystrophin, has a vital role in maintaining muscle structure and function. Homologues of DMD have been identified in several animals including dogs, cats, mice, fish and invertebrates. The most notable of these are the extensively studied mdx mouse, a genetic and biochemical model of the human disease, and the muscular dystrophic Golden Retriever dog, which is the nearest pathological counterpart of DMD. These models have been used to explore potential therapeutic approaches along a number of avenues including gene replacement and cell transplantation strategies. High-throughput screening of pharmacological and genetic therapies could potentially be carried out in recently available smaller models such as zebrafish and Caenorhabditis elegans. It is possible that a successful treatment will eventually be identified through the integration of studies in multiple species differentially suited to addressing particular questions. [source] High-throughput screening technologies for drug glucuronidation profilingJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 8 2008Olga Trubetskoy A significant number of endogenous and exogenous compounds, including many therapeutic agents, are metabolized in humans via glucuronidation, catalysed by uridine diphosphoglucurono-syltransferases (UGTs). The study of the UGTs is a growing field of research, with constantly accumulated and updated information regarding UGT structure, purification, substrate specificity and inhibition, including clinically relevant drug interactions. Development of reliable UGT assays for the assessment of individual isoform substrate specificity and for the discovery of novel isoform-specific substrates and inhibitors is crucial for understanding the function and regulation of the UGT enzyme family and its clinical and pharmacological relevance. High-throughput screening (HTS) is a powerful technology used to search for novel substrates and inhibitors for a wide variety of targets. However, application of HTS in the context of UGTs is complicated because of the poor stability, low levels of expression, low affinity and broad substrate specificity of the enzymes, combined with difficulties in obtaining individual UGT isoforms in purified format, and insufficient information regarding isoform-specific substrates and inhibitors. This review examines the current status of HTS assays used in the search for novel UGT substrates and inhibitors, emphasizing advancements and challenges in HTS technologies for drug glucuronidation profiling, and discusses possible avenues for future advancement of the field. [source] High-Throughput Screening of the Influence of Thermal Treatment on the Mechanical Properties of Semicrystalline Polymers: A Case Study for iPPMACROMOLECULAR RAPID COMMUNICATIONS, Issue 1 2004Konrad Schneider Abstract Summary: High-throughput screening is a convenient tool to determine the influence of parameters, such as composition or processing conditions on certain materials' properties. In the present study, iPP was used to construct a combinatorial library made from a processing temperature gradient requiring only a minimised amount of material. Clear changes in the crystalline modification and crystal morphology of the iPP, and their impact on mechanical properties have been identified. The construction of a combinatorial library to study the effect of thermal history upon the properties of polymers. [source] Status of HTS Data Mining ApproachesMOLECULAR INFORMATICS, Issue 4 2004Alexander Böcker Abstract High-throughput screening of large compound collections results in large sets of data. This review gives an overview of the most frequently employed computational techniques for the analysis of such data and the establishment of first QSAR models. Various methods for descriptor selection, classification and data mining are discussed. Recent trends include the application of kernel-based machine learning methods for the design of focused libraries and compilation of target-family biased compound collections. [source] Plant models for animal pathogenesisCELLULAR MICROBIOLOGY, Issue 3 2005B. Prithiviraj Summary Several bacteria that are pathogenic to animals also infect plants. Mechanistic studies have proven that some human/animal pathogenic bacteria employ a similar subset of virulence determinants to elicit disease in animals, invertebrates and plants. Therefore, the results of plant infection studies are relevant to animal pathogenesis. This discovery has resulted in the development of convenient, cost-effective, and reliable plant infection models to study the molecular basis of infection by animal pathogens. Plant infection models provide a number of advantages in the study of animal pathogenesis. Using a plant model, mutations in animal pathogenic bacteria can easily be screened for putative virulence factors, a process which if done using existing animal infection models would be time-consuming and tedious. High-throughput screening of plants also provides the potential for unravelling the mechanisms by which plants resist animal pathogenic bacteria, and provides a means to discover novel therapeutic agents such as antibiotics and anti-infective compounds. In this review, we describe the developing technique of using plants as a model system to study Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus pathogenesis, and discuss ways to use this new technology against disease warfare and other types of bioterrorism. [source] Using small molecules to study big questions in cellular microbiologyCELLULAR MICROBIOLOGY, Issue 8 2002Gary E. Ward Summary High-throughput screening of small molecules is used extensively in pharmaceutical settings for the purpose of drug discovery. In the case of antimicrobials, this involves the identification of small molecules that are significantly more toxic to the microbe than to the host. Only a small percentage of the small molecules identified in these screens have been studied in sufficient detail to explain the molecular basis of their antimicrobial effect. Rarer still are small molecule screens undertaken with the explicit goal of learning more about the biology of a particular microbe or the mechanism of its interaction with its host. Recent technological advances in small molecule synthesis and high-throughput screening have made such mechanism-directed small molecule approaches a powerful and accessible experimental option. In this article, we provide an overview of the methods and technical requirements and we dis-cuss the potential of small molecule approaches to address important and often otherwise experimentally intractable problems in cellular microbiology. [source] The Use of Biochemical and Biophysical Tools for Triage of High-Throughput Screening Hits , A Case Study with Escherichia coli Phosphopantetheine AdenylyltransferaseCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2010J. Richard Miller High-throughput screening is utilized by pharmaceutical researchers and, increasingly, academic investigators to identify agents that act upon enzymes, receptors, and cellular processes. Screening hits include molecules that specifically bind the target and a greater number of non-specific compounds. It is necessary to ,triage' these hits to identify the subset worthy of further exploration. As part of our antibacterial drug discovery effort, we applied a suite of biochemical and biophysical tools to accelerate the triage process. We describe application of these tools to a series of 9-oxo-4,9-dihydropyrazolo[5,1-b]quinazoline-2-carboxylic acids (PQ) hits from a screen of Escherichia coli phosphopantetheine adenylyltransferase (PPAT). Initial confirmation of specific binding to phosphopantetheine adenylyltransferase was obtained using biochemical and biophysical tools, including a novel orthogonal assay, isothermal titration calorimetry, and saturation transfer difference NMR. To identify the phosphopantetheine adenylyltransferase sub-site bound by these inhibitors, two techniques were utilized: steady-state enzyme kinetics and a novel 19F NMR method in which fluorine-containing fragments that bind the ATP and/or phosphopantetheine sites serve as competitive reporter probes. These data are consistent with PQs binding the ATP sub-site. In addition to identification of a series of PPAT inhibitors, the described hit triage process is broadly applicable to other enzyme targets in which milligram quantities of purified target protein are available. [source] Novel estrogen receptor ligands and their structure,activity relationship evaluated by scintillation proximity assay for high-throughput screeningDRUG DEVELOPMENT RESEARCH, Issue 4 2005Ling He Abstract The estrogen receptor (ER) is an important drug target with allosteric characteristics that binds orthotopic hormones and other ligands. A recently developed scintillation proximity (SPA)-based assay for high-throughput screening (HTS) of compound libraries was used to identify novel estrogen receptor ligands that might have ER subtype selective binding activity. Radioligand binding was determined in a multi-detector scintillation counter designed for microtitration plates. Equilibrium binding experiments and kinetic competition tests were performed with [3H]-estradiol and human ER, and ER, receptors. A library of 6,000 structurally diverse compounds was screened. From this, several novel ligands were identified that showed pronounced subtype-selective differences in ligand binding for ER, and ER,. The observed equilibrium dissociation constant (Kd) for the binding of [3H]estradiol to ER, and ER, receptors were approximately 0.25 and 0.64 nM, respectively. When 17,-estradiol, raloxifene and daidzein were tested for binding affinity to ER, in a competition assay, the IC50 values were 0.34, 1.31, and 75.6 nM, respectively. When tested for binding affinity to ER,, the IC50 values were 1.05, 11.4, and 10.6 nM, respectively. The results obtained show that the methodology is valid in comparison to previously published data regarding estradiol and other standard compounds (raloxifene and daidzein) binding characteristics of estrogen receptors. The assay is also well suited to applied research as a tool in HTS of compound libraries in the search of ER ligands. Several novel active compounds were identified and selected as potent ER subtype ligands. Drug Dev Res 64:203,212, 2005. © 2005 Wiley-Liss, Inc. [source] Development of microreactor array chip-based measurement system for massively parallel analysis of enzymatic activityELECTRONICS & COMMUNICATIONS IN JAPAN, Issue 4 2009Yosuke Hosoi Abstract Microarray chip technology such as DNA chips, peptide chips, and protein chips is one of the promising approaches for achieving high-throughput screening (HTS) of biomolecule function since it has great advantages in feasibility of automated information processing due to one-to-one indexing between array position and molecular function as well as massively parallel sample analysis as a benefit of downsizing and large-scale integration. Mostly, however, the function that can be evaluated by such microarray chips is limited to affinity of target molecules. In this paper, we propose a new HTS system and enzymatic activity based on microreactor array chip technology. A prototype of the automated and massively parallel measurement system for fluorometric assay of enzymatic reactions was developed by the combination of microreactor array chips and a highly sensitive fluorescence microscope. Design strategy of microreactor array chips and an optical measurement platform for the high-throughput enzyme assay are discussed. © 2009 Wiley Periodicals, Inc. Electron Comm Jpn, 92(4): 35,41, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecj.10056 [source] High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detectionELECTROPHORESIS, Issue 1-2 2003Yan He Abstract Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC50 value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases. [source] High-Throughput-Screening Systems for Hydrolases,ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 6 2004U.T. Bornscheuer Abstract In the past few years, several high-throughput screening (HTS) formats have been developed, driven by the increasing number of biocatalysts available and the development of directed evolution methods as a novel tool in the design of enzymes. This article focusses on HTS-methods to determine the activity and enantioselectivity of lipases and esterases. In addition, assays for amidase and racemase activity are included. [source] Development of a solvent-free, solid-phase in vitro bioassay using vertebrate cellsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2006Stephanie K. Bopp Abstract Miniaturized bioassays offer many advantages in exploring the toxic potential of chemicals, including small sample volumes and compatibility with high-throughput screening. One problem common to miniaturized systems, however, is the loss of test chemicals because of sorption. The idea of the current study was to use the sorption phenomenon in a positive way. It was found that contaminants sorbed to the growth surface in wells of tissue-culture plates or to the surface of selected sorbent bead materials are available to vertebrate cells growing in direct contact with the contaminant-coated surface. The use of beads provided more flexibility with regard to surface area, materials, and assay format. Biosilon, a bead cell-culture carrier made of polystyrene, was found to be most suitable. It supported cell adherence and allowed the detection of reproducible dose-response curves of an increase in cytochrome CYP1A enzyme activity by sorbed polycyclic aromatic hydrocarbons in the rainbow trout (Oncorhynchus mykiss) liver cell line, RTL-W1. The resulting bead assay provides a miniaturized, solvent-free exposure system. Potential future applications include the coupling to environmental sampling, in which the bead material is used as solid receiving phase before serving as a surface for vertebrate cells to attach and respond. [source] Real-Time Liquid Crystal pH Sensor for Monitoring Enzymatic Activities of PenicillinaseADVANCED FUNCTIONAL MATERIALS, Issue 23 2009Xinyan Bi Abstract A liquid crystal (LC)-based pH sensor for real-time monitoring of changes in localized pH values near a solid surface is reported, along with its application for the detection of enzymatic activities. It is found that 4-cyano-4,-pentylbiphenyl (5CB), when doped with 4,-pentyl-biphenyl-4-carboxylic acid (PBA), shows a bright-to-dark optical response to a very small change in pH (from 6.9 to 7.0). The pH-driven optical response can be explained by using orientational transitions of 5CB induced by the protonation and deprotonation of PBA at the aqueous/LC interface. Because of its high pH sensitivity, the LC-based sensor is further exploited for monitoring local pH changes resulting from enzymatic reactions. As a proof of concept, the hydrolysis of penicillin G by surface-immobilized penicillinase is monitored using the system, even when the concentration of penicillin G is as low as 1,nM. This type of LC-based sensor may find potential utilities in high-throughput screening of enzyme substrates and enzyme inhibitors. [source] Small-Molecule-Directed Assembly: A Gold Nanoparticle-Based Strategy for Screening of Homo-Adenine DNA Duplex Binders,ADVANCED MATERIALS, Issue 4 2008G. Song By using AuNP-modified homo-adenine DNA conjugate as a model system, simple colorimetric and resonance Rayleigh scattering assays have been developed for screening small molecules that trigger the formation of the non-Watson,Crick homo-adenine duplexes. The assay presented here is more simplified in format as it involves only one type of ssDNA modified Au-NP, and can be easily adapted to high-throughput screening. [source] Novel Inhibitors of Alkaline Phosphatase Suppress Vascular Smooth Muscle Cell Calcification,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2007Sonoko Narisawa Abstract We report three novel inhibitors of the physiological pyrophosphatase activity of alkaline phosphatase and show that these compounds are capable of reducing calcification in two models of vascular calcification (i.e., they suppress in vitro calcification by cultured Enpp1,/, VSMCs and they inhibit the increased pyrophosphatase activity in a rat aortic model). Introduction: Genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) leads to accumulation of the calcification inhibitor inorganic pyrophosphate (PPi). TNALP deficiency ameliorates the hypermineralization phenotype in Enpp1,/, and ank/ank mice, two models of osteoarthritis and soft tissue calcification. We surmised that the pharmacological inhibition of TNALP pyrophosphatase activity could be used to prevent/suppress vascular calcification. Materials and Methods: Comprehensive chemical libraries were screened to identify novel drug-like compounds that could inhibit TNALP pyrophosphatase function at physiological pH. We used these novel compounds to block calcification by cultured vascular smooth muscle cells (VSMCs) and to inhibit the upregulated pyrophosphatase activity in a rat aortic calcification model. Results: Using VSMC cultures, we determined that Enpp1,/, and ank/ank VSMCs express higher TNALP levels and enhanced in vitro calcification compared with wildtype cells. By high-throughput screening, three novel compounds, 5361418, 5923412, and 5804079, were identified that inhibit TNALP pyrophosphatase function through an uncompetitive mechanism, with high affinity and specificity when measured at both pH 9.8 and 7.5. These compounds were shown to reduce the calcification by Enpp1,/, VSMCs. Furthermore, using an ex vivo rat whole aorta PPi hydrolysis assay, we showed that pyrophosphatase activity was inhibited by all three lead compounds, with compound 5804079 being the most potent at pH 7.5. Conclusions: We conclude that TNALP is a druggable target for the treatment and/or prevention of ectopic calcification. The lead compounds identified in this study will serve as scaffolds for medicinal chemistry efforts to develop drugs for the treatment of soft tissue calcification. [source] A personal account of the role of peptide research in drug discovery: the case of hepatitis C,JOURNAL OF PEPTIDE SCIENCE, Issue 1 2001Antonello Pessi Abstract Although peptides themselves are not usually the end products of a drug discovery effort, peptide research often plays a key role in many aspects of this process. This will be illustrated by reviewing the experience of peptide research carried out at IRBM in the course of our study of hepatitis C virus (HCV). The target of our work is the NS3/4A protease, which is essential for maturation of the viral polyprotein. After a thorough examination of its substrate specificity we fine-tuned several substrate-derived peptides for enzymology studies, high-throughput screening and as fluorescent probes for secondary binding assays. In the course of these studies we made the key observation: that the protease is inhibited by its own cleavage products. Single analog and combinatorial optimization then derived potent peptide inhibitors. The crucial role of the NS4A cofactor was also addressed. NS4A is a small transmembrane protein, whose central domain is the minimal region sufficient for enzyme activation. Structural studies were performed with a peptide corresponding to the minimal activation domain, with a series of product inhibitors and with both. We found that NS3/4A is an induced fit enzyme, requiring both the cofactor and the substrate to acquire its bioactive conformation; this explained some puzzling results of ,serine-trap' type inhibitors. A more complete study on NS3 activation, however, requires the availability of the full-length NS4A protein. This was prepared by native chemical ligation, after sequence engineering to enhance its solubility; structural studies are in progress. Current work is focused on the P, region of the substrate, which, at variance with the P region, is not used for ground state binding to the enzyme and might give rise to inhibitors showing novel interactions with the enzyme. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd. [source] Characterization of drug,protein interactions in blood using high-performance affinity chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 5-6 2009David S. Hage Abstract The binding of drugs with proteins in blood, serum, or plasma is an important process in determining the activity, distribution, rate of excretion, and toxicity of drugs in the body. High-performance affinity chromatography (HPAC) has received a great deal of interest as a means for studying these interactions. This review examines the various techniques that have been used in HPAC to examine drug,protein binding and discusses the types of information that can be obtained through this approach. A comparison of these techniques with traditional methods for binding studies (e.g., equilibrium dialysis and ultrafiltration) will also be presented. The use of HPAC with specific serum proteins and binding agents will then be discussed, including HSA and ,1 -acid glycoprotein (AGP). Several examples from the literature are provided to illustrate the applications of such research. Recent developments in this field are also described, such as the use of improved immobilization techniques, new data analysis methods, techniques for working directly with complex biological samples, and work with immobilized lipoproteins. The relative advantages and limitations of the methods that are described will be considered and the possible use of these techniques in the high-throughput screening or characterization of drug,protein binding will be discussed. [source] Combinatorial Ink-Jet Printer for Ceramics: CalibrationJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 10 2003Mohammad Masoud Mohebi This article describes an ink-jet printer for the construction of combinatorial libraries and functionally graded ceramics. It can mix and print all possible compositions for high-throughput screening. The number of components is set by the number of mixing valves that deliver ceramic ink from pressurized reservoirs into a circulation chamber. Compositional control is by either complete or incremental change. Organic liquids and ceramic inks are used in a systematic three-stage calibration. The calibration protocol accounts for the effects of ink viscosity, reservoir pressure, valve-opening time, and temperature, but reveals unexpected segregation effects that occur in the ink after deposition. [source] Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screeningLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2009F. Alberto Abstract Aims:, The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants. Methods and Results:, The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1·2 g l,1 of FaeB was selected (12-fold more than the A. niger overproducing strain). Conclusions:, This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, time-saving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days. Significance and Impact of the Study:, This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening. [source] Measurement of porto-systemic shunting in mice by novel three-dimensional micro-single photon emission computed tomography imaging enabling longitudinal follow-upLIVER INTERNATIONAL, Issue 8 2010Christophe Van Steenkiste Abstract Background and aims: The reference method for diagnosing porto-systemic shunting (PSS) in experimental portal hypertension involves measuring 51Chrome (51Cr)-labelled microspheres. Unfortunately, this technique necessitates the sacrifice of animals. Alternatively, 99mtechnetium-macroaggregated albumin (99mTc-MAA) has been used; however, planar scintigraphy imaging techniques are not quantitatively accurate and adequate spatial information is not attained. Here, we describe a reliable, minimally invasive and rapid in vivo imaging technique, using three-dimensional single photon emission computed tomography (3D SPECT) modus, that allows more accurate quantification, serial measurements and spatial discrimination. Methodology: Partial portal vein ligation, common bile duct ligation and sham were induced in male mice. A mixture of 51Cr microspheres and 99mTc-macroaggregated albumin particles was injected into the splenic pulpa. All mice were scanned in vivo with ,SPECT (1 mm spatial resolution) and, when mandatory for localisation, a ,SPECT-CT was acquired. A relative quantitative analysis was performed based on the 3D reconstructed datasets. Additionally, 51Cr was measured in the same animals to calculate the correlation coefficient between the 99mTc detection and the gold standard 51Cr. In each measuring modality, the PSS fraction was calculated using the formula: [(lung counts)/(lung counts+liver counts)] × 100. Results: A significant correlation between the 99mTc detection and 51Cr was demonstrated in partial portal vein ligation, common bile duct ligation and sham mice and there was a good agreement between the two modalities. ,SPECT scanning delivers high spatial resolution and 3D image reconstructions. Conclusion: We have demonstrated that quantitative high-resolution ,SPECT imaging with 99mTc-MAA is useful for detecting the extent of PSS in a non-sacrificing set-up. This technology permits serial measurements and high-throughput screening to detect baseline PSS, which is especially important in pharmacological studies. [source] Biochemical applications of mass spectrometry in pharmaceutical drug discoveryMASS SPECTROMETRY REVIEWS, Issue 3 2005Kieran F. Geoghegan Abstract Biochemical applications of mass spectrometry (MS) are important in the pharmaceutical industry. They comprise compositional analyses of biomolecules, especially proteins, and methods that measure molecular functions such as ligand binding. In early drug discovery, MS is used to characterize essential reagents and in structural biology. A number of MS-based methods have been proposed for use in high-throughput screening (HTS), but are unlikely to supplant established radiometric and fluorometric methods for this purpose. These methods, which include pulsed-ultrafiltration MS, frontal affinity chromatography-MS, and size-exclusion chromatography-MS, may ultimately be most successful in the post-screening lead development phase. In full development, MS is used heavily in the search for biomarkers that can be used to gauge disease progression and drug action. This review gives equal attention to the technical aspects of MS-based methods and to selective pressures present in the industrial environment that influence their chances of gaining wide application. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:347,366, 2005 [source] | |||||||||||||||||||||||||||||||