Genetic optimization of combinatorial libraries

Most agrochemical and pharmaceutical companies have set up high-throughput screening programs which require large numbers of compounds to screen. Combinatorial libraries provide an attractive way to deliver these compounds. A single combinatorial library with four variable positions can yield more than 10(12) potential compounds, if one assumes that about 1000 reagents are available for each position. This is far more than any high-throughput screening facility can afford to screen. We have proposed a method for iterative compound selection from large databases, which identifies the most active compounds by examining only a small fraction of the database. In this article, we describe the extension of this method to the problem of selecting compounds from large combinatorial libraries. Copyright 1998 John Wiley & Sons, Inc.     

Development and validation of an automated high-throughput system for zebrafish in vivo screenings

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism.     

Visual and computational analysis of structure--activity relationships in high-throughput screening data

Novel analytic methods are required to assimilate the large volumes of structural and bioassay data generated by combinatorial chemistry and high-throughput screening programmes in the pharmaceutical and agrochemical industries. Recent work in visualisation and data mining has been used to develop structure--activity relationships from such chemical-biological datasets.     

Identification of metabotropic glutamate receptor antagonists using an automated high-throughput screening system

Antagonists to the human metabotropic glutamate receptor subtype 5a(mGluR(5a)) have been implicated as potential therapeutics for the treatment of a variety of nervous system disorders, including pain, anxiety, and Parkinson's disease. To discover novel antagonists to the mGluR(5a), a functional assay measuring agonist-induced intracellular calcium release was developed. The assay was used for the high-throughput screening of a large collection of compounds in single wells using a fully automated robotic platform. Primary high-throughput screening hits were subjected to a combination of data analysis and counterscreening assays to identify several compounds with both efficacy and selectivity for the metabotropic glutamate receptor target.     

Identification of gap junction blockers using automated fluorescence microscopy imaging

Gap junctions coordinate electrical signals and facilitate metabolic synchronization between cells. In this study, the authors have developed a novel assay for the identification of gap junction blockers using fluorescence microscopy imaging-based high-content screening technology. In the assay, the communication between neighboring cells through gap junctions was measured by following the redistribution of a fluorescent marker. The movement of calcein dye from dye-loaded donor cells to dye-free acceptor cells through gap junctions overexpressed on cell surface membranes was monitored using automated fluorescence microscopy imaging in a high-throughput compatible format. The fluorescence imaging technology consisted of automated focusing, image acquisition, image processing, and data mining. The authors have successfully performed a high-throughput screening of a 486,000- compound program with this assay, and they were able to identify false positives without additional experiments. Selective and pharmacologically interesting compounds were identified for further optimization.     

High-throughput receptor-binding methods for somatostatin receptor 2

Three high-throughput screening methods for quantitating 125I-SS14 binding to human somatostatin receptor 2 (hSST2) have been developed. Microplate-based separation assays were performed in Packard Unifilter and Millipore Multiscreen plates. A homogeneous ligand-binding assay was developed by employing wheat germ agglutinin (WGA)-coated Flashplates. Apparent dissociation constants for 125I-SS14 binding to hSST2 were obtained with each method. IC(50) values were determined for 12 compounds using each of the methods. Similar IC(50) values were obtained for each compound with all of the methods. The WGA-Flashplate is suitable for fully automated high-throughput screening whereas the Unifilter and Multiscreen methods are more suitable for semiautomated and manual screening applications.     

SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands

A high-throughput screening methodology tailored to the discovery of ligands for known and orphan proteins is presented. With this method, labeling of neither target protein nor screened compounds is required, as the ligands are affinity selected by incubation of the protein with mixtures of compounds in aqueous binding buffer. Unbound small-molecular-weight compounds are removed from the target protein:ligand complex by rapid size-exclusion chromatography in the 96-well format. The protein fraction is analyzed subsequently by liquid chromatography-mass spectrometry for detection and identification of the bound ligand. This screening method was validated with known protein:ligand model systems and optimized for selection of high-affinity binders in an industrial screening environment. All sample handling steps and the analytics are rapid, robust, and largely automated, adopting this technology to the needs of present high-throughput screening processes. This affinity-selection technology, termed SpeedScreen, is currently an integral part of our lead discovery process.     

Single-cell microliter-droplet screening system (MISS Cell): An integrated platform for automated high-throughput microbial monoclonal cultivation and picking

Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor- and resource-intensive for high-throughput requirements. Currently, several instruments have been integrated for automated and high-throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single-cell microliter-droplet screening system (MISS Cell), for automated, high-throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high-throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate-producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high-throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.     

Discovery of pyridine-based agrochemicals by using Intermediate Derivatization Methods

Pyridine-based compounds have been playing a crucial role as agrochemicals or pesticides including fungicides, insecticides/acaricides and herbicides, etc. Since most of the agrochemicals listed in the Pesticide Manual were discovered through screening programs that relied on trial-and-error testing and new agrochemical discovery is not benefiting as much from the in silico new chemical compound identification/discovery techniques used in pharmaceutical research, it has become more important to find new methods to enhance the efficiency of discovering novel lead compounds in the agrochemical field to shorten the time of research phases in order to meet changing market requirements. In this review, we selected 18 representative known agrochemicals containing a pyridine moiety and extrapolate their discovery from the perspective of Intermediate Derivatization Methods in the hope that this approach will have greater appeal to researchers engaged in the discovery of agrochemicals and/or pharmaceuticals.     

Development of a novel,high-throughput screening tool for efficient perfusion-based cell culture process development

Perfusion technology has been successfully used for the commercial production of biotherapeutics, in particular unstable recombinant proteins, for more than a decade. However, there has been a general lack of high-throughput cell culture tools specifically for perfusion-based cell culture processes. Here, we have developed a high-throughput cell retention operation for use with the ambr® 15 bioreactor system. Experiments were run in both 24 and 48 reactor configurations for comparing perfusion mimic models, media development, and clone screening. Employing offline centrifugation for cell retention and a variable volume model developed with MATLAB computational software, the established screening model has demonstrated cell culture performance, productivity, and product quality were comparable to bench scale bioreactors. The automated, single use, high-throughput perfusion mimic is a powerful tool that enables us to have rapid and efficient process development of perfusion-based cell culture processes.     

A High-Throughput Assay for Small Molecule Destabilizers of the KRAS Oncoprotein

Mutations in the Ras family of small GTPases, particularly KRAS, occur at high frequencies in cancer and represent a major unmet therapeutic need due to the lack of effective targeted therapies. Past efforts directed at inhibiting the activity of the Ras oncoprotein have proved difficult. We propose an alternative approach to target Ras by eliminating Ras protein from cells with pharmacological means. In this study, we developed a cell-based, high-content screening platform to identify small molecules that could promote the degradation of the KRAS oncoprotein. We generated an EGFP-KRAS^G12V fluorescence reporter system and implemented it for automated screening in 1536-well plates using high-throughput cellular imaging. We screened a library of clinically relevant compounds at wide dose range and identified Ponatinib and AMG-47a as two candidate compounds that selectively reduced the levels of EGFP-KRAS^G12V protein but did not affect EGFP protein in cells. This proof-of-principle study demonstrates that it is feasible to use a high-throughput screen to identify compounds that promote the degradation of the Ras oncoprotein as a new approach to target Ras.     

Quantum dots go with the flow

RNA interference (RNAi) and automated high-throughput screening is a promising combination. But the first systematic large-scale mapping of genetic interactions in an animal shows that manual methods still have advantages over sophisticated automated screens.     

Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes

Mitochondrial dysfunction has been associated with a variety of currently marketed therapeutics and has also been implicated in many disease states. Alterations in the rate of oxygen consumption are an informative indicator of mitochondrial dysfunction, but the use of such assays has been limited by the constraints of traditional measurement approaches. Here, we present a high-throughput, fluorescence-based methodology for the analysis of mitochondrial oxygen consumption using a phosphorescent oxygen-sensitive probe, standard microtitre plates and plate reader detection. The protocol describes the isolation of mitochondria from animal tissue, initial establishment and optimization of the oxygen consumption assay, subsequent screening of compounds for mitochondrial toxicity (uncoupling and inhibition), data analysis and generation of dose-response curves. It allows dozens of compounds (or hundreds of assay points) to be analyzed in a single day, and can be further up-scaled, automated and adapted for other enzyme- and cell-based screening applications.     

Inclusion counter: a public domain image analysis system for measurement of Chlamydiaceae growth in vitro

The traditional method of measuring chlamydial growth in vitro, counting Chlamydiaceae inclusions by eye, is time-consuming and error prone. This paper describes a novel automated image analysis system suitable for high-throughput screening of novel anti-Chlamydiaceae compounds. The software, Inclusion Counter v3.0, is freely available in the public domain (http://www.image-analysis.co.uk).     

Industrial biocatalysis

The number of industrial processes for the synthesis of fine and commodity chemicals, pharmaceutical and agrochemical intermediates and drug substances utilizing biological catalysts continues to grow. The combination of new molecular biology techniques, such as directed evolution and pathway engineering, with new and efficient high-throughput screening methods is poised to bolster this field and further advance the contribution of biocatalysis to the chemical and the pharmaceutical industries.     

Development of a high-throughput robotic fluorescence-based assay for HsEg5 inhibitor screening

HsEg5 has microtubule-activated ATPase activity and plays essential roles in bipolar spindle formation. Because HsEg5 is validated as an attractive cancer target, in vitro biochemical assays have been developed for identifying compounds with high inhibitory activity. Several compounds, including quinazoline ring-containing compounds, have been identified and are currently in clinical trials. Although considerable progress has been made during recent years, limitations of HsEg5 in vitro screening assays still reside in two main aspects. First, colorimetric-based assays exhibit relatively low sensitivity and limited dynamic range that are unable to accurately measure compounds with nanomolar potencies. Second, current fluorescence assays are relatively low throughput without "mix and read" homogeneous features. In this study, we describe a sensitive fluorescence-based assay for HsEg5-specific inhibitors. By coupling several enzymes' activities, the release of ADP was measured quantitatively through red fluorescent resorufin. The Km for ATP hydrolysis in this assay was calculated as 23 microM. The known HsEg5 inhibitors CK0106023 and CK0238273 gave IC50 values of 9.8 and 30.6 nM, respectively. Our fluorescence assay has a 20-fold increase in sensitivity with broader dynamic range when compared with a colorimetric assay. We further automated this assay for high-throughput screening with a Z' factor of 0.8.     

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Futhermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.     

Development of a high-throughput screening system for the compounds that inhibit collagen–protein interactions

Collagen-binding proteins (CBPs) play important roles in various physiological events. Some CBPs are regarded as targets for drug development; for example, platelet glycoprotein VI (GPVI) and heat shock protein 47 (HSP47) are promising targets for the development of novel antiplatelet and antifibrotic drugs, respectively. However, no systematic screening method to search compounds that inhibit collagen–CBP interactions have been developed, and only a few CBP inhibitors have been reported to date. In this study, a facile turbidimetric multiwell plate assay was developed to evaluate inhibitors of CBPs. The assay is based on the finding that CBPs retard spontaneous collagen fibril formation in vitro and that fibril formation is restored in the presence of compounds that interfere with the collagen–CBP interactions. Using the same platform, the assay was performed in various combinations of fibril-forming collagen types and CBPs. This homogeneous assay is simple, convenient, and suitable as an automated high-throughput screening system.     

High-throughput screening by mass spectrometry: comparison with the scintillation proximity assay with a focused-file screen of AKT1/PKB alpha

Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire) with the scintillation proximity assay (SPA). The cancer therapy target AKT1/PKBalpha was screened against a focused library of kinase inhibitors and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds that exhibited 相似文献