Virtual high-throughput in silico screening

In silico methods may benefit drug discovery and development significantly by saving an average of $130 million and 0.8 years per drug. Virtual high-throughput screening (vHTS) applies in silico approaches, such as docking and alignment, to large virtual molecular databases to enrich biologically active compounds in order to yield lead structures. In an industrial environment, the commonly used ligand-based and receptor-based methods outlined here need to be computationally faster to return the utmost benefit. Intelligent database searching using new fast feedback-driven screening methods appears to be particularly rewarding in terms of both cost and time benefits.     

Virtual screening to enrich hit lists from high-throughput screening: a case study on small-molecule inhibitors of angiogenin

"Hit lists" generated by high-throughput screening (HTS) typically contain a large percentage of false positives, making follow-up assays necessary to distinguish active from inactive substances. Here we present a method for improving the accuracy of HTS hit lists by computationally based virtual screening (VS) of the corresponding chemical libraries and selecting hits by HTS/VS consensus. This approach was applied in a case study on the target-enzyme angiogenin, a potent inducer of angiogenesis. In conjunction with HTS of the National Cancer Institute Diversity Set and ChemBridge DIVERSet E (approximately 18,000 compounds total), VS was performed with two flexible library docking/scoring methods, DockVision/Ludi and GOLD. Analysis of the results reveals that dramatic enrichment of the HTS hit rate can be achieved by selecting compounds in consensus with one or both of the VS functions. For example, HTS hits ranked in the top 2% by GOLD included 42% of the true hits, but only 8% of the false positives; this represents a sixfold enrichment over the HTS hit rate. Notably, the HTS/VS method was effective in selecting out inhibitors with midmicromolar dissociation constants typical of leads commonly obtained in primary screens.     

Agrosuppression: a bioassay for the hypersensitive response suited to high-throughput screening

We describe a novel method, agrosuppression, that addresses the need for an assay of the hypersensitive response (HR) in intact plants that is rapid and adapted to high-throughput functional screening of plant and pathogen genes. The agrosuppression assay is based on inoculation of intact plants with a mixture of Agrobacterium tumefaciens strains carrying (i) a binary plasmid with one or more candidate HR-inducing genes and (ii) a tumor-inducing (oncogenic) T-DNA. In the absence of HR induction, tumor formation is initiated, resulting in a typical crown gall phenotype. However, upon induction of the HR, tumor formation by the oncogenic T-DNA is suppressed, resulting in a phenotype that can be readily scored. We tested and optimized agrosuppression in Nicotiana benthamiana using the inf1 elicitin gene from the oomycete pathogen Phytophthora infestans, which specifically induces the HR in Nicotiana spp., and the gene-for-gene pair Avr9/Cf-9 from the fungal pathogen Cladosporium fulvum and Lycopersicon pimpinellifolium (currant tomato), respectively. Agrosuppression protocols that can be rapidly performed using simple mechanical wounding of petioles of intact N. benthamiana plants were developed and appeared particularly adapted to intensive high-throughput screening. This assay promises to greatly facilitate the cloning of novel plant R genes and pathogen Avr genes and to accelerate functional analyses and structure-function studies of these genes.     

高通量筛选工具的设计与应用

定向进化方法作为新兴的高效蛋白质工程手段,其内容包括蛋白质突变体文库的构建和有效突变体的快速筛选。高通量筛选方法是定向进化方法的重要组成部分,是成功获得有效突变体的关键。筛选的突变体数量越多,获得有效突变体的几率越大。以下介绍了目前已经成功应用于或有潜力应用于定向进化改造蛋白质的几种高通量筛选工具。高通量筛选工具的不断设计与开发将推动蛋白质工程领域的技术革新。     

基于酵母水平的高通量药物筛选

随着后基因组时代的到来,越来越多的药物靶标蛋白将会被发现,基于靶标蛋白设计出的化合物也将大量涌现,高通量药物筛选日趋重要。酵母基因组的易操作性及其简单稳定的培养条件,使得该真核微生物成为一种理想的药物筛选工程细胞。本文讨论了选择酵母系统进行细胞水平筛选的优缺点,并从基于靶点和表型两种筛选模式对酵母水平的高通量药物筛选做一总结。     

Force in numbers: high-throughput screening approaches to unlock microbial transport

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New options to engineer biofuel microbes: Development and application of a high-throughput screening system

The number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium acetobutylicum are quite limited, demonstrating the physiological complexity of solventogenic clostridia. Since multiple largely unknown parameters determine a particular phenotype, an inverse strategy to select a phenotype of interest can be useful. However, the major constraint for explorative or combinatorial metabolic engineering approaches is the availability of a feasible screening method to select the desired phenotype from a large population in a high-throughput manner. Therefore, a semi-quantitative assay was developed to monitor alcohol production in microtiter cultures of C. acetobutylicum. The applicability of the screening system was evaluated by two examples. First, C. acetobutylicum ATCC 824 was chemically mutagenized and subjected to high butanol concentrations as a pre-selection step. Screening of the butanol-tolerant population resulted in the identification of mutants with >20% increased butanol production as compared to the wildtype. The second application example was based on a pre-engineered C. acetobutylicum strain with low acetone biosynthetic activity, but concomitantly reduced butanol titer. After chemical mutagenesis, a total of 4390 clones was analyzed and mutants with significantly increased butanol concentrations and similarly low acetone levels as the parental strain were selected. Thus, the suitability of the semi-quantitative screening system was validated, opening up new perspectives for combinatorial strategies to improve solventogenic clostridia and other biofuel microbes.     

Evaluating real-life high-throughput screening data

High-throughput screening (HTS) is the result of a concerted effort of chemistry, biology, information technology, and engineering. Many factors beyond the biology of the assay influence the quality and outcome of the screening process, yet data analysis and quality control are often focused on the analysis of a limited set of control wells and the calculated values derived from these wells. Taking into account the large number of variables and the amount of data generated, multiple views of the screening data are necessary to guarantee quality and validity of HTS results. This article does not aim to give an exhaustive outlook on HTS data analysis but tries to illustrate the shortfalls of a reductionist approach focused on control wells and give examples for further analysis.     

The future of high-throughput screening

High-throughput screening (HTS) is a well-established process in lead discovery for pharma and biotech companies and is now also being set up for basic and applied research in academia and some research hospitals. Since its first advent in the early to mid-1990s, the field of HTS has seen not only a continuous change in technology and processes but also an adaptation to various needs in lead discovery. HTS has now evolved into a quite mature discipline of modern drug discovery. Whereas in previous years, much emphasis has been put toward a steady increase in capacity ("quantitative increase") via various strategies in the fields of automation and miniaturization, the past years have seen a steady shift toward higher content and quality ("quality increase") for these biological test systems. Today, many experts in the field see HTS at the crossroads with the need to decide either toward further increase in throughput or more focus toward relevance of biological data. In this article, the authors describe the development of HTS over the past decade and point out their own ideas for future directions of HTS in biomedical research. They predict that the trend toward further miniaturization will slow down with the implementation of 384-well, 1536-well, and 384 low-volume-well plates. The authors predict that, ultimately, each hit-finding strategy will be much more project related, tailor-made, and better integrated into the broader drug discovery efforts.     

Enzyme assays for high-throughput screening

Assaying enzyme-catalyzed transformations in high-throughput is crucial to enzyme discovery, enzyme engineering and the drug discovery process. In enzyme assays, catalytic activity is detected using labelled substrates or indirect sensor systems that produce a detectable spectroscopic signal upon reaction. Recent advances in the development of high-throughput enzyme assays have identified new labels and chromophores to detect a wide range of enzymes activities. Enzyme activity profiling and fingerprinting have also been used as tools for identification and classification, while microarray formats have been devised to increase throughput.     

Evaluating the high-throughput screening computations

The judges evaluated the submissions for the McMaster University High-Throughput Data-Mining and Docking Competition based on 3 criteria: identification of active compounds, percent enrichment, and overview of the competition. Using these metrics, 4 of the participating groups found meaningful enrichment, and 3 groups made perceptive comments about the general nature of the competition.     

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

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

A high-throughput screening approach to anthrax lethal factor inhibition

A high-throughput screening approach was used to identify new inhibitors of the metallo-protease lethal factor from Bacillus anthracis. A library of approximately 14,000 compounds was screened using a fluorescence-based in vitro assay and hits were further characterized enzymatically via measurements of IC50 and Ki values against a small panel of metallo-proteases. This study led to the identification of new scaffolds that inhibit LF and the Botulinum Neurotoxin Type A in the low micromolar range, while sparing the human metallo-proteases MMP-2 and MMP-9. Therefore, these scaffolds could be further exploited for the development of potent and selective anti-toxin agents.     

A nonradioactive DNA methyltransferase assay adaptable to high-throughput screening

We have developed a nonradioactive assay method for DNA methyltransferases based on the ability to protect substrate DNA from restriction. DNA immobilized to a microplate well was treated sequentially with methyltransferase and an appropriate endonuclease. The amount of methylated DNA product is reflected by a proportional decrease in endonuclease cleavage, which is in turn reflected by increased retention of the end-labeled affinity probe. A single universal substrate was designed to assay multiple methyltransferases including those that do not have a cognate endonuclease. The methodology developed is suited to screen a large number of compounds for inhibitors of various methyltransferases.     

Anchoring high-throughput screening methods to scale-up bioproduction of siderophores

The use of high throughput strategies is of acknowledged relevance since the rational use of small-scale reactors, coupled with suitable analytic tools, is contributing to the acceleration of process development in several areas of biotechnology. These small-scale reactors are available in different working volumes and configurations, being useful in a wide array of applications, from cell screening to process optimization.The present work was focused on the development of a high-throughput strategy, combining microtiter plates and analytic methodologies, to screen an in-house library of environmental bacteria in order to identify good siderophore producers. From a library of roughly 500 marine microorganisms, it was possible to ultimately obtain 11 bacterial strains with high production capabilities. Two of them had not been previously identified as siderophore producers. The bioprocess was scaled-up from microtiter plates to a 5 L stirred tank reactor, while maintaining the overall volumetric productivity, using the k_La similarity as scale-up criterion.This novel approach is a suitable alternative to traditional screening tools.     

Toxoplasma gondii: a simple high-throughput assay for drug screening in vitro

Toxoplasma gondii is the etiologic agent of toxoplasmosis. Although the combination of sulfadiazine and pyrimethamine is used as therapy for this disease, these drugs can have serious side effects and its use is limited in pregnancy. Therefore there is a need for new anti-T. gondii drugs in the clinic. Some systems for T. gondii drug screening have been described, but these have limitations and can be difficult. In order to solve these problems, we established a system to screen drugs in vitro that involved using cell viability methods to calculate drug selectivities, which are Trypan blue, [3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuzolium, inner salt] (MTS) method and lactate dehydrogenase (LDH) assay. These assays were simple to establish and perform. The IC₅₀ values calculated from the morphological assay were not significantly different from the EC₅₀ values calculated using the other three methods. In particular, the results of the morphological assay showed a distinct association with the MTS assay (R = 0.9841). These assays could be used for a wide range of applications in the screening of new drugs and may provide an alternative to the techniques currently used to screen for candidate anti-T. gondii compounds in vitro. In this study, we also tested many compounds and identified some that had a good anti-T. gondii effect in vitro based on the MTS assay. This simple and fast system allowed us to determine which compounds to investigate further using in vivo experiments.