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Results for “High Throughput Screening”

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PubMed2026

From complexity to clarity: Building dashboards for hit selection in high throughput screens.

High throughput screening produces large, complex datasets that are difficult to interrogate without programming expertise, making hit selection time-consuming and inflexible. While instrument software and commercial tools offer partial solutions, they often lack adaptability or require costly infrastructure. Interactive dashboards provide an effective alternative by enabling dynamic filtering and integrated visualization within a single interface. Here, we present simple R Markdown-based templates for creating customizable, modular dashboards for screen data analysis. Built using the flexdashboard and crosstalk R packages, and HTML widgets, these lightweight, easy-to-build HTML dashboards require no complex installation process or installation of licensed software. They support linked visualizations, threshold-based filtering (e.g., Z-score, p-value, fold change), and interactive data exploration and are shared as a standalone HTML file. This framework enables rapid, flexible hit selection across diverse high throughput screening applications and is designed for users with basic R experience.

High-Throughput Screening Assays↗

PubMed2005

Discovery and optimization of new chromium catalysts for ethylene oligomerization and polymerization aided by high-throughput screening.

High throughput screening (HTS) of a 205 member Schiff base salicylaldimine ligand library derived from salicylaldehydes bearing bulky ortho-substituents, i.e., 9-anthracenyl, 1,4,5,8-tetramethylanthracenyl or triptycenyl, reacted in-situ with (p-tolyl)CrCl2(thf)3, identified two new classes of highly active chromium based systems for the oligomerization and polymerization of ethylene, respectively. The polymerization system comprises bidentate ortho-substituted anthracenyl Schiff bases bearing small primary or secondary alkyl imine substituents. The oligomerization catalysts are based upon tridentate ortho-triptycenyl-substituted Schiff bases with pyridylmethyl or quinolyl substituents. Validation tests confirmed polymerization productivities of up to 3000 g x mmol(-1)h(-1)bar(-1) for the polymerization catalyst systems while the oligomerization catalysts gave productivities up to 10 000 g x mmol(-1)h(-1)bar(-1). Key catalyst precursors have been characterized by X-ray crystallography.

Journal Article↗

PubMed2006

A set of UV-inducible autolytic vectors for high throughput screening.

A high throughput screening scheme is often a prerequisite for directed evolution of enzymes or metagenomic analysis of DNA samples. For assaying intracellular enzymes of interest (e.g. when Escherichia coli is used), it requires cell lysis in many cases, chemical or enzymatic, which can be tedious and cost-consuming. In this study, a set of UV-inducible autolytic vectors was constructed to offer a simpler means of cell lysis that is free of additional liquid handling. The SRRz lysis gene cassette from bacteriophage Lambda was cloned downstream of a UV-inducible promoter, the recA promoter or the umuDC promoter, and further inserted into the backbone of pUC18, and transformed into E. coli BL21 cells. The SRRz expression and cell lysis was induced by UV irradiation. For both the recA and umuDC promoters, at 30 degrees C the lysis efficiency was found to be consistent and above 60% as measured using beta-galactosidase as the reporter. However, at 37 degrees C the lysis profiles were found to be erratic. UV lysis in 96-well plates also produced consistent lysis results that were comparable to those obtained by lysozyme treatment, demonstrating the utility of these autolytic vectors in high throughput screening. This set of artificial SRRz autolysis units should be transferable to other vectors. Surprisingly, it was found that the E. coli BL21(DE3) was also partially disrupted under UV irradiation, with a lysis efficiency of 44.5% at 30 degrees C, and 22.5% at 37 degrees C.

Bacteriolysis↗

PubMed2002

Novel miniaturized systems in high-throughput screening.

High-throughput screening (HTS) using high-density microplates is the primary method for the discovery of novel lead candidate molecules. However, new strategies that eschew 2D microplate technology, including technologies that enable mass screening of targets against large combinatorial libraries, have the potential to greatly increase throughput and decrease unit cost. This review presents an overview of state-of-the-art microplate-based HTS technology and includes a discussion of emerging miniaturized systems for HTS. We focus on new methods of encoding combinatorial libraries that promise throughputs of as many as 100,000 compounds per second.

Combinatorial Chemistry Techniques↗

PubMed2004

Technological advances in high-throughput screening.

High-throughput screening (HTS) is the process of testing a large number of diverse chemical structures against disease targets to identify 'hits'. Compared to traditional drug screening methods, HTS is characterized by its simplicity, rapidness, low cost, and high efficiency, taking the ligand-target interactions as the principle, as well as leading to a higher information harvest. As a multidisciplinary field, HTS involves an automated operation-platform, highly sensitive testing system, specific screening model (in vitro), an abundant components library, and a data acquisition and processing system. Various technologies, especially the novel technologies such as fluorescence, nuclear-magnetic resonance, affinity chromatography, surface plasmon resonance, and DNA microarray, are now available, and the screening of more than 100,000 samples per day is already possible. Fluorescence-based assays include the scintillation proximity assay, time-resolved energy transfer, fluorescence anisotropy, fluorescence correlation spectroscopy, and fluorescence fluctuation spectroscopy. Fluorescence-based techniques are likely to be among the most important detection approaches used for HTS due to their high sensitivity and amenability to automation, giving the industry-wide drive to simplify, miniaturize, and speed up assays. The application of NMR technology to HTS is another recent trend in drug research. One advantage afforded by NMR technology is that it can provide direct information on the affinity of the screening compounds and the binding location of protein. The structure-activity relationship acquired from NMR analysis can sharpen the library design, which will be very important in furnishing HTS with well-defined drug candidates. Affinity chromatography used for library screening will provide the information on the fundamental processes of drug action, such as absorption, distribution, excretion, and receptor activation; also the eluting curve can give directly the possibility of candidate drug. SPR can measure the quantity of a complex formed between two molecules in real-time without the need for fluorescent or radioisotopic labels. SPR is capable of characterizing unmodified biopharmaceuticals, studying the interaction of drug candidates with macromolecular targets, and identifying binding partners during ligand fishing experiments. DNA microarrays can be used in HTS be used to further investigate the expression of biological targets associated with human disease, which then opens new and exciting opportunities for drug discovery. Without doubt, the addition of new technologies will further increase the application of HTS in drug screening and its related fields.

Animals↗

PubMed2001

[Assay in high throughput screening].

HTS (High Throughput Screening) has been put into practice in recent years. HTS is aiming to discover lead compounds for medicinal drugs. High efficiency must be achieved in all the processes including sample preparation, assay procedure, automation and data management. This review will focus on the aspects concerned with the assay technology and the efficiency in HTS. One of the major trends in HTS is assay miniaturization using high-density microplates with 384 and 1536 wells. This allows us to increase the throughput and to decrease the cost. The so-called "mix and measure" or "homogeneous" assay system, which has no separation steps such as washing or filtration, is effective for this purpose. The homogeneous assays, such as scintillation proximity assay (SPA), fluorescence energy transfer (HTRF, LANCE) and fluorescence polarization (FP), are frequently used. The reporter gene assay or the cell proliferation assay can be adapted for the homogeneous assay using high-density plates. In addition, HTS measuring the intracellular Ca2+ influx is also possible using a CCD Imager. The assay quality as well as the efficiency is also important especially in HTS. The Z'-factor provides a good tool for evaluating the quality of assays.

Biological Assay↗

PubMed2003

Improved statistical methods for hit selection in high-throughput screening.

High-throughput screening (HTS) plays a central role in modern drug discovery, allowing the rapid screening of large compound collections against a variety of putative drug targets. HTS is an industrial-scale process, relying on sophisticated automation, control, and state-of-the art detection technologies to organize, test, and measure hundreds of thousands to millions of compounds in nano- to microliter volumes. Despite this high technology, hit selection for HTS is still typically done using simple data analysis and basic statistical methods. The authors discuss in this article some shortcomings of these methods and present alternatives based on modern methods of statistical data analysis. Most important, they describe and show numerous real examples from the biologist-friendly Stat Server HTS application (SHS), a custom-developed software tool built on the commercially available S-PLUS and StatServer statistical analysis and server software. This system remotely processes HTS data using powerful and sophisticated statistical methodology but insulates users from the technical details by outputting results in a variety of readily interpretable graphs and tables.

Algorithms↗

PubMed2004

Complex phenotypic assays in high-throughput screening.

High-throughput screening (HTS), systematically testing thousands of small molecules to find candidates for lead optimization, primarily involves exposure of purified proteins to arrayed collections of small molecules. More complex phenotypic assays, such as cell-based or whole-organism assays, traditionally have flanked HTS, preceding it to validate new therapeutic targets, and following it to characterize new lead compounds in cellular contexts. Recently, however, cell- and organism-based phenotypic assays have increasingly been adopted as a primary screening platform for annotating small molecules.

Combinatorial Chemistry Techniques↗

PubMed2003

Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening.

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-beta-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml(-1). The assay is linear for sugar concentrations from 0 to 86 microg ml(-1) and can also be used to assay protein concentrations (0 to 143 microg ml(-1)) on the same plate. A variety of temperatures and pH conditions can be used and, after incubation, the assay requires only one detection reagent and one heating step.

Carbohydrates↗

PubMed2003

Multiplexing nuclear receptors for agonist identification in a cell-based reporter gene high-throughput screen.

High-throughput screening (HTS) has become an essential part of the drug discovery process. Due to the rising requirements for both data quality and quantity, along with increased screening cost and the demand to shorten the time for lead identification, increasing throughput and cost-effectiveness has become a necessity in the hit identification process. The authors present a multiplexed HTS for 2 nuclear receptors, the farnesoid X-activated receptor and the peroxisome proliferator-activated receptor delta in a viable cell-based reporter gene assay. The 2 nuclear receptors were individually transfected into human hepatoma cells, and the transient transfected cell lines were pooled for the multiplexed screen. Hits identified by the multiplexed screen are similar to those identified by the individual receptor screens. Furthermore, the multiplexed screen provides selectivity information if ligands selective for one and not the other receptor are one of the hit criteria. The data demonstrate that multiplexing nuclear receptors can be a simple, efficient, cost-effective, and reliable alternative to traditional HTS of individual targets without compromising data quality.

Cell Line, Tumor↗

PubMed2005

Statistical analysis of systematic errors in high-throughput screening.

High-throughput screening (HTS) is an efficient technology for drug discovery. It allows for screening of more than 100,000 compounds a day per screen and requires effective procedures for quality control. The authors have developed a method for evaluating a background surface of an HTS assay; it can be used to correct raw HTS data. This correction is necessary to take into account systematic errors that may affect the procedure of hit selection. The described method allows one to analyze experimental HTS data and determine trends and local fluctuations of the corresponding background surfaces. For an assay with a large number of plates, the deviations of the background surface from a plane are caused by systematic errors. Their influence can be minimized by the subtraction of the systematic background from the raw data. Two experimental HTS assays from the ChemBank database are examined in this article. The systematic error present in these data was estimated and removed from them. It enabled the authors to correct the hit selection procedure for both assays.

Algorithms↗

PubMed2006

Assay concordance between SPA and TR-FRET in high-throughput screening.

High-throughput screening (HTS) of large chemical libraries has become the main source of new lead compounds for drug development. Several specialized detection technologies have been developed to facilitate the cost- and time-efficient screening of millions of compounds. However, concerns have been raised, claiming that different HTS technologies may produce different hits, thus limiting trust in the reliability of HTS data. This study was aimed to investigate the reliability of the authors most frequently used assay techniques: scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET). To investigate the data concordance between these 2 detection technologies, the authors screened a large subset of the Schering compound library consisting of 300,000 compounds for inhibitors of a nonreceptor tyrosine kinase. They chose to set up this study in realistic HTS scale to ensure statistical significance of the results. The findings clearly demonstrate that the choice of detection technology has no significant impact on hit finding, provided that assays are biochemically equivalent. Data concordance is up to 90%. The little differences in hit findings are caused by threshold setting but not by systematic differences between the technologies. The most significant difference between the compared techniques is that in the SPA format, more false-positive primary hits were obtained.

Biological Assay↗

PubMed2002

A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening.

High-throughput and virtual screening are widely used to discover novel leads for drug design. On examination, many screening hits appear non-drug-like: they act noncompetitively, show little relationship between structure and activity, and have poor selectivity. Attempts to develop these peculiar molecules into viable leads are often futile, and much time can be wasted on the characterization of these "phony" hits. Despite their common occurrence, the mechanism of action of these promiscuous molecules remains unknown. To investigate this problem, 45 diverse screening hits were studied. Fifteen of these were previously reported as inhibitors of various receptors, including beta-lactamase, malarial protease, dihydrofolate reductase, HIV Tar RNA, thymidylate synthase, kinesin, insulin receptor, tyrosine kinases, farnesyltransferase, gyrase, prions, triosephosphate isomerase, nitric oxide synthase, phosphoinositide 3-kinase, and integrase; 30 were from an in-house screening library of a major pharmaceutical company. In addition to their original targets, 35 of these 45 compounds were shown to inhibit several unrelated model enzymes. These 35 screening hits included compounds, such as fullerenes, dyes, and quercetin, that have repeatedly shown activity against diverse targets. When tested against the model enzymes, the compounds showed time-dependent but reversible inhibition that was dramatically attenuated by albumin, guanidinium, or urea. Surprisingly, increasing the concentration of the model enzymes 10-fold largely eliminated inhibition, despite a 1000-fold excess of inhibitor; a well-behaved competitive inhibitor did not show this behavior. One model to explain these observations was that the active form of the promiscuous inhibitors was an aggregate of many individual molecules. To test this hypothesis, light scattering and electron microscopy experiments were performed. The nonspecific inhibitors were observed to form particles of 30-400 nm diameter by both techniques. In control experiments, a well-behaved competitive inhibitor and an inactive dye-like molecule were not observed to form aggregates. Consistent with the hypothesis that the aggregates are the inhibitory species, the particle size and IC(50) values of the promiscuous inhibitors varied monotonically with ionic strength; a competitive inhibitor was unaffected by changes in ionic strength. Unexpectedly, aggregate formation appears to explain the activity of many nonspecific inhibitors and may account for the activity of many promiscuous screening hits. Molecules acting via this mechanism may be widespread in drug discovery screening databases. Recognition of these compounds may improve screening results in many areas of pharmaceutical interest.

Dialysis↗

PubMed2002

Integration of virtual and high-throughput screening.

High-throughput and virtual screening are important components of modern drug discovery research. Typically, these screening technologies are considered distinct approaches, as one is experimental and the other is theoretical in nature. However, given their similar tasks and goals, these approaches are much more complementary to each other than often thought. Various statistical, informatics and filtering methods have recently been introduced to foster the integration of experimental and in silico screening and maximize their output in drug discovery. Although many of these ideas and efforts have not yet proceeded much beyond the conceptual level, there are several success stories and good indications that early-stage drug discovery will benefit greatly from a more unified and knowledge-based approach to biological screening, despite the many technical advances towards even higher throughput that are made in the screening arena.

Drug Design↗

PubMed1996

Inhibition of self-splicing group I intron RNA: high-throughput screening assays.

High-throughput screening assays have been developed to rapidly identify small molecule inhibitors targeting catalytic group I introns. Biochemical reactions catalyzed by a self-splicing group I intron derived from Pneumocystis carinii or from bacteriophage T4 have been investigated. In vitro biochemical assays amenable to high-throughput screening have been established. Small molecules that inhibit the functions of group I introns have been identified. These inhibitors should be useful in better understanding ribozyme catalysis or in therapeutic intervention of group I intron-containing microorganisms.

Bacteriophage T4↗

PubMed2001

A virtual high throughput screen for high affinity cytochrome P450cam substrates. Implications for in silico prediction of drug metabolism.

Structure-based virtual screening techniques require reliable scoring functions to discriminate potential substrates effectively. In this study we compared the performance of GOLD, PMF, DOCK and FlexX scoring functions in FlexX flexible docking to cytochrome P450cam binding site. Crystal structures of protein-substrate complexes were most effectively reproduced by the FlexX/PMF method. On the other hand, the FlexX/GOLD approach provided the best correlation between experimental binding constants and predicted scores. Binding modes selected by the FlexX/PMF approach were rescored by GOLD to obtain a reliable measure of binding energetics. The effectiveness of the FlexX/PMF/GOLD method was demonstrated by the correct classification of 32 out of the 33 experimentally studied compounds and also in a virtual HTS test on a library of 10,000 compounds. Although almost all the available functions were developed to be general, our study on cytochrome P450cam substrates suggests that careful selection or even tailoring the scoring function might increase the prediction power of virtual screens significantly. The FlexX/PMF/GOLD methodology was tested on cytochrome P450 3A4 substrates and inhibitors. This preliminary study revealed that the combined function was able to recognise 334 out of the 345 compounds bound to 3A4.

Binding Sites↗

PubMed1999

Stacker Modules Used in a High-Capacity Robotics System for High Throughput Screening Compound Replication.

High throughput screening is now established as a key component of the pharmaceutical lead identification process in many pharmaceutical companies. Over recent years, thanks to advances in assay technology, process automation, and logistics control, the throughput capacity of HTS groups has increased significantly. It is now entirely possible to screen corporate compound collections against an individual pharmacological target within a timescale of several weeks. Despite these improvements, many HTS groups find that their capacity is limited by the rate at which they can provide test compounds in a "screen-ready" format. This limitation is usually imposed by the capacity and productivity of the single-armed robotic systems utilized. We have recently constructed a robotic system aimed at overcoming this particular problem. This system uses purpose-built microplate stacker units that provide high-capacity microplate storage and, importantly, provide an easy and fast interface between the robotic system and the human operators. This paper describes this automation project and the benefits that have resulted from its deployment.

Journal Article↗

PubMed2005

Calculating the probability of detection for inhibitors in enzymatic or binding reactions in high-throughput screening.

In high-throughput screening (HTS) for drug candidates from a library containing tens of thousands to millions of chemical compounds, one problem is assessing the sensitivity of an assay for detecting compounds with a particular potency. For example, when looking for inhibitors of an enzyme, what is the potency of an inhibitor that will be readily detected by an enzyme inhibition assay? Similarly, when assessing compounds that inhibit binding between receptors and ligands or similar molecule-to-molecule interactions, what potency of an inhibitor will be readily detected? In this article, the well-established concepts of Michaelis-Menten kinetics and Langmuir binding isotherms are combined with fundamental statistical principles to yield a measure of assay sensitivity. The approach is general and can be modified to accommodate situations where the reaction kinetics is known to be more complicated than situations described by the Michaelis-Menten and Langmuir equations. The calculations presented take into account the concentration of inhibitor used, the variability of the assay, the relationship between the K(m) or K(d) of the reaction and the substrate or ligand concentration used, the threshold or cutoff value used for determining "hits," and the number of replicates used in screening.

Drug Evaluation, Preclinical↗