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PubMed2006

Iterative ACORN as a high throughput tool in structural genomics.

High throughput macromolecular structure determination is very essential in structural genomics as the available number of sequence information far exceeds the number of available 3D structures. ACORN, a freely available resource in the CCP4 suite of programs is a comprehensive and efficient program for phasing in the determination of protein structures, when atomic resolution data are available. ACORN with the automatic model-building program ARP/wARP and refinement program REFMAC is a suitable combination for the high throughput structural genomics. ACORN can also be run with secondary structural elements like helices and sheets as inputs with high resolution data. In situations, where ACORN phasing is not sufficient for building the protein model, the fragments (incomplete model/dummy atoms) can again be used as a starting input. Iterative ACORN is proved to work efficiently in the subsequent model building stages in congerin (PDB-ID: lis3) and catalase (PDB-ID: 1gwe) for which models are available.

Animals↗
PubMedDate not supplied

High-throughput screening approaches for investigating drug metabolism and pharmacokinetics.

1. High-throughput screening approaches have been adopted throughout the pharmaceutical industry to aid in the rapid discovery of new chemical entities. Because it is now well recognized that the selection of a robust candidate requires a balance of potency, safety and pharmacokinetics, the role of drug metabolism departments has widened from their traditional one of supporting drug development to include the screening of compounds during the discovery process. To put drug metabolism and pharmacokinetic (DMPK) studies in context, the evolving role of DMPK screening in the drug discovery strategy of pharmaceutical companies will be discussed and a generalized approach will be presented. 2. With the increasing numbers of compounds requiring screening, DMPK optimization methods have had to be adapted for high throughput. There have been many developments in this field over the past decade and this review will focus on the high-throughput DMPK screening methodologies used today and in the recent past. 3. In vitro and in silico (computer-based) methods have proven most amenable to high-throughput approaches and these will firm the bulk of the review, but some advances with in vivo methods will also be discussed. As there has been a vast increase in published material on the topic of high-throughput DMPK methodologies in the past 10 years, it would be impossible to cover every method in detail, so this review will concentrate on the key areas and refer the reader to other, more detailed reviews wherever possible. 4. Most high-throughput methods would not be possible without the enabling technologies of computing, automation, new sample preparation technologies, and highly sensitive and selective detection systems, and these will also be reviewed. 5. The advantages and disadvantages of the screening methods will be presented, in particular the issue of handling the false-positives and -negatives that arise. 6. In concluding the review, future developments in this field will be discussed along with key issues that will need to be addressed.

Absorption↗
PubMed2001

Experimental strategies for combinatorial and high-throughput materials development.

As high-throughput experimental techniques have become common in the area of materials research, entirely new types of experimental strategies have appeared. The kinds of problems, the desired outcomes, and the appropriate patterns are significantly different from those associated with conventional experimentation. Classical experimental design (design of experiments, DOE) strategies grew up in a period of slow, laborious, error-prone experimentation; a modern high-throughput laboratory can test more materials in a week than was previously done in a year. The goal of this Account is to identify and critically discuss some of the strategies that are being developed and used in this new, exciting area of research.

Combinatorial Chemistry Techniques↗
PubMed2005

Cholesterol modulated antibody binding in supported lipid membranes as determined by total internal reflectance microscopy on a microfabricated high-throughput glass chip.

A high-throughput microfabricated all-glass microchip, lipid biochip, was created and used to measure fluorescently tagged antibody binding to dinitrophenol (DNP) haptens in planar supported phospholipid/cholesterol lipid bilayers as a function of cholesterol-to-lipid molar ratio (X(CHOL)). Multiple parallel microchannels etched in the lipid biochip allowed simultaneous measurement of antibody binding to hapten-containing and hapten-free lipid bilayers, for a range of aqueous antibody concentrations. Specific and nonspecific antibody binding to the supported lipid bilayers was determined from the internally calibrated intensity of the surface fluorescence using total internal reflectance fluorescence (TIRF) microscopy. The TIRF intensity data of the specific antibody binding were fitted to the Langmuir isotherm and Hill equation models to determine the apparent dissociation constant K(d), the maximum fluorescence parameter F(infinity), and binding cooperativity n. As X(CHOL) increased from 0 to 0.50, K(d) exhibited a minimum of approximately 4 microM and n reached a maximum of approximately 2.2 at X(CHOL) approximately 0.20. However, F(infinity) appeared to be insensitive to the cholesterol content. The nonspecific binding fraction (NS), defined as the ratio of the TIRF intensity for hapten-free bilayers to that with hapten, showed a minimum of approximately 0.08 also at X(CHOL) approximately 0.20. The results suggest that cholesterol regulates the specific binding affinity and cooperativity, as well as suppresses nonspecific binding of aqueous antibody to a planar supported lipid bilayer surface at an optimal cholesterol content of X(CHOL) approximately 0.20. Interestingly, for X(CHOL) approximately 0.40, NS reached a maximum of approximately 0.57, suggesting significant packing defects in the lipid bilayer surface, possibly as a result of lipid domain formation as predicted by the lipid superlattice model. We conclude that cholesterol plays a significant role in regulating both specific and nonspecific antibody/antigen binding events on the lipid bilayer surface and that our lipid biochip represents a new and useful high-resolution microfluidic device for measuring lipid/protein surface binding activities in a parallel and high-throughput fashion.

Binding Sites, Antibody↗
PubMed2001

Advances in high-throughput mass spectrometry.

The evolution of high-throughput drug discovery is readily apparent as the pharmaceutical industry continues to stress the rapid progression of new chemical entities and biological agents through drug discovery and development pipelines. Mass spectrometry and high performance liquid chromatography-mass spectrometry have played an instrumental role in the support and advancement of all facets of high-throughput drug discovery. The introduction of new instrumentation has extended the breadth of mass spectrometric-based capabilities from the characterization of high-throughput organic synthesis products to early adsorption, distribution, metabolism and excretion profiling. Additionally, advances in the capacity and throughput of mass spectrometry systems have concurrently led to the introduction of data management tools to address automated data reduction, archival and mining, as well as analytical data integration to chemical and biological databases.

Chromatography, High Pressure Liquid↗
PubMed2006

High-throughput techniques in breast cancer: a clinical perspective.

High-throughput technologies such as DNA-microarrays, RT-PCR and proteomics can improve the prognostic and predictive information acquired from classical parameters. Unlike information gathered by classical methods, high-throughput technologies can accurately inform clinicians on patient response to adjuvant therapy or those who will resist the effect of that therapy. Studies performed in breast cancer with high-throughput techniques have focused on tumour biology, prognosis, prediction of response to a few agents and, more recently, early diagnosis. However, further refinement is needed before these techniques become part of clinical routine. In the meantime, they will be used in clinical investigation, particularly in the areas of hormonal therapy and adjuvant chemotherapy, where modest improvements in the capacity of prediction can benefit many women. Close cooperation among clinicians, pathologists and basic investigators is essential to take high-throughput techniques to daily practice. New diagnostic tools will be complex but they will provide valuable patient information.

Breast Neoplasms↗
PubMed2006

A collaborative hit-to-lead investigation leveraging medicinal chemistry expertise with high throughput library design, synthesis and purification capabilities.

High throughput screening (HTS) campaigns, where laboratory automation is used to expose biological targets to large numbers of materials from corporate compound collections, have become commonplace within the lead generation phase of pharmaceutical discovery. Advances in genomics and related fields have afforded a wealth of targets such that screening facilities at larger organizations routinely execute over 100 hit-finding campaigns per year. Often, 10(5) or 10(6) molecules will be tested within a campaign/cycle to locate a large number of actives requiring follow-up investigation. Due to resource constraints at every organization, traditional chemistry methods for validating hits and developing structure activity relationships (SAR) become untenable when challenged with hundreds of hits in multiple chemical families per target. To compound the issue, comparison and prioritization of hits versus multiple screens, or physical chemical property criteria, is made more complex by the informatics issues associated with handling large data sets. This article describes a collaborative research project designed to simultaneously leverage the medicinal chemistry and drug development expertise of the Novartis Institutes for Biomedical Research Inc. (NIBRI) and ArQule Inc.'s high throughput library design, synthesis and purification capabilities. The work processes developed by the team to efficiently design, prepare, purify, assess and prioritize multiple chemical classes that were identified during high throughput screening, cheminformatics and molecular modeling activities will be detailed.

Chemistry, Pharmaceutical↗
PubMed2005

Techniques: high-throughput measurement of intracellular Ca(2+) -- back to basics.

High-throughput screening techniques continue to provide important tools to the pharmaceutical industry for the efficient identification of drug leads. However, high-throughput techniques are now being exploited to address a variety of pharmacological and cellular signaling research questions, including the regulation and role of intracellular Ca(2+) in a plethora of biological systems. Although an awareness of specific assay conditions is crucial for reliable and reproducible measurements of intracellular free Ca(2+) whichever system of study is used, the complex temporal nature of Ca(2+) signals has posed some unique limitations for its measurement in high-throughput mode. Progress in high-throughput design has overcome many of these limitations and will complement other technical approaches to understanding the underlying regulation and role of intracellular Ca(2+).

Animals↗
PubMed1994

Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins.

The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.

Animals↗
PubMed1996

From hybridization image to numerical values: a practical, high throughput quantification system for high density filter hybridizations.

Hybridization to sets of bacterial colonies or PCR products arrayed on high density filters is used in a number of experimental schemes. In many cases it is desirable to collect quantitative information ('hybridization signatures') rather than indications on 'positive' and 'negative' colonies. We present a practical system, based on an imaging plate analyser and a customized version of commercial software, that makes such quantification feasible, and define its performance in terms of reproducibility and linearity. The system is far superior to methods based on autoradiography and should be useful in many projects that involve the increasingly popular high density filter format.

Animals↗
PubMed1999

High-throughput polymorphism screening and genotyping with high-density oligonucleotide arrays.

A highly reliable and efficient technology has been developed for high-throughput DNA polymorphism screening and large-scale genotyping. Photolithographic synthesis has been used to generate miniaturized, high-density oligonucleotide arrays. Dedicated instrumentation and software have been developed for array hybridization, fluorescent detection, and data acquisition and analysis. Specific oligonucleotide probe arrays have been designed to rapidly screen human STSs, known genes and full-length cDNAs. This has led to the identification of several thousand biallelic single-nucleotide polymorphisms (SNPs). Meanwhile, a rapid and robust method has been developed for genotyping these SNPs using oligonucleotide arrays. Each allele of an SNP marker is represented on the array by a set of perfect match and mismatch probes. Prototype genotyping chips have been produced to detect 400, 600 and 3000 of these SNPs. Based on the preliminary results, using oligonucleotide arrays to genotype several thousand polymorphic loci simultaneously appears feasible.

Genetic Markers↗
PubMed2003

Emerging trends in high-throughput screening.

Novel technologies are emerging for high-throughput screening, driven by the needs and fine-tuning of established drug discovery activities, as well as by the emergence of novel target classes resulting from the deciphering of the human genome. Disciplines other than biology have now entered the screening scene, as bioinformatics, micro-technology and analytics provide powerful methodologies and applications that were not previously suitable for high-throughput screening. Many of these will move high-throughput screening from a numbers game to a content- and information-based approach to identify leads for novel disease targets.

Combinatorial Chemistry Techniques↗
PubMed2006

High-throughput protein purification using an automated set-up for high-yield affinity chromatography.

One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity.

Chromatography, Affinity↗
PubMedDate not supplied

Photodegradation catalyst discovery by high-throughput experiment.

A high-throughput experimental methodology was developed for photocatalysis reaction. In this work, a CCD imaging analysis system and photocatalytic reactor for UV light was designed and tested. By making use of the technologies, we have screened several catalyst libraries. From the SiO2-supported single component catalyst library, we found that TiO2, ZrO2, Nb2O5, and WO3 were good candidates for highly active catalyst formulation. We designed and screened several triangle catalyst libraries and found that the WO3- and Nb2O5-codoped TiO2 catalyst showed much higher photodegradation activities for the degradation of 1,6-hexamethylenediamine than did the pure TiO2 catalyst. The doping of ZrO2 into TiO2 did not generate apparent positive effects on catalytic activity.

Catalysis↗
PubMed2003

Application of a neural network in high-throughput protein crystallography.

High-throughput protein crystallography requires the automation of multiple steps used in the protein structure determination. One crucial step is to find and monitor the crystal quality on the basis of its diffraction pattern. It is often time-consuming to scan protein crystals when selecting a good candidate for exposure. The use of neural networks for this purpose is explored. A dynamic neural network algorithm to achieve a fast convergence and high-speed image recognition has been developed. On the test set a 96% success rate in identifying properly the quality of the crystal has been achieved.

Algorithms↗
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↗