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PubMed2004

A powerful antiradiation compound revealed by a new high-throughput screening method.

We present a new high-throughput screening method for the selection of powerful water-soluble antiradiation compounds. This method, which uses conventional immunoassay techniques, allowed the capacity of a given compound to protect thymidine from irradiation to be evaluated. By applying this assay to an antioxidant library, we showed for the first time that norbadione A, a well-known mushroom pigment, has pronounced atypical antiradiation properties.

4-Butyrolactone↗
PubMed2006

Use of mixture distributions to deconvolute the behavior of "hits" and controls in high-throughput screening data.

The stochastic nature of high-throughput screening (HTS) data indicates that information may be gleaned by applying statistical methods to HTS data. A foundation of parametric statistics is the study and elucidation of population distributions, which can be modeled using modern spreadsheet software. The methods and results described here use fundamental concepts of statistical population distributions analyzed using a spreadsheet to provide tools in a developing armamentarium for extracting information from HTS data. Specific examples using two HTS kinase assays are analyzed. The analyses use normal and gamma distributions, which combine to form mixture distributions. HTS data were found to be described well using such mixture distributions, and deconvolution of the mixtures to the constituent gamma and normal parts provided insight into how the assays performed. In particular, the proportion of hits confirmed was predicted from the original HTS data and used to assess screening assay performance. The analyses also provide a method for determining how hit thresholds--values used to separate active from inactive compounds--affect the proportion of compounds verified as active and how the threshold can be chosen to optimize the selection process.

Chemistry, Pharmaceutical↗
PubMed2005

Multidimensional high-throughput screening of displacers.

A multidimensional, batch high-throughput screening (MD-HTS) protocol was developed to investigate the effects of various parameters on the selectivity of ion-exchange protein displacement systems. A variety of molecules were screened, and the results were employed to provided insights into the influence of displacer chemistry and concentration, resin chemistry, and mobile-phase salt counterion on the efficacy and selectivity of these nonlinear chromatographic systems. These results open up the possibility of tailoring the selectivity of displacement separations by choosing appropriate combinations of operating conditions using the MD-HTS technique. The screens were also employed for the identification of displacers and conditions for the separation of a challenging protein mixture by selective displacement chromatography. Column displacements were carried out with potential lead compounds identified from the MD-HTS screens, and the results confirmed that selective displacement could indeed be achieved for this model mixture. Furthermore, the results indicated that this approach is particularly useful when the order of elution is not changed, but the inherent selectivity is increased in the presence of the displacer. The results presented in this paper demonstrate the utility of the MD-HTS technique for rapid method development in protein ion-exchange displacement chromatography.

Calcium↗
PubMed2006

In praise of manual high-throughput screening.

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.

Animals↗
PubMed2005

Statistical evaluation of a self-deconvoluting matrix strategy for high-throughput screening of the CXCR3 receptor.

In high-throughput screening (HTS), compounds can be tested in self-deconvoluting matrices (SDMs) of 10 compounds per well. The SDM setup is based upon a systematic mixing of compound samples such that each compound appears twice in the screening assay, in two independent mixtures. In order to test the quality of the SDM approach, we compared it with a standard single-compound screening approach. In a CXCR3 scintillation proximity assay, we performed five multiple screening trials of 26,400 compounds at a 10 microM screening concentration to estimate false positive and false negative rates in the compound population. No potent hits (<6.2 microM IC50) were missed in any screening method. Forty-eight percent of all actives were found in every screening trial independent of compound handling method. The SDM strategy had an average of 25 false positives and 15 false negatives as compared with an average of 34 false positives and 15 false negatives with a more conventional single-compound screening approach. Most of the variability resulted from day-to-day variation around the hit cutoff criterion, rather than from any particular screening technique. In the two most extreme examples, a compound with a 7.5 microM IC50 was missed in one out of two mixture trials, and a compound with a 6.2 microM IC50 was missed in one out of three single-compound trials. In the CXCR3 assay presented herein, the SDM screening method had better predictive value than the single-compound screening approach.

Animals↗
PubMed2000

Development of a 5-hydroxytryptamine(2A) receptor binding assay for high throughput screening using 96-well microfilter plates.

A high throughput screening method for the analysis of 5-hydroxytryptamine(2A) (5-HT(2A)) receptor binding parameters has been developed, using 96-well filter plates of the Millipore MultiScreen system in combination with a MicroBeta PLUS microplate scintillation counter. MAFB filter plates (GF/B filter over a Durapore membrane) were used because of the lower nonspecific binding of the radioligand to GF/B filter material than to GF/C filters. Comparing different scintillation cocktails, highest counting efficiency and shortest equilibration time were detected with Betaplatescint, after drying the plates at 50 degrees C for 2 h. Measuring the plates without the plastic underdrain increased the counting efficiency by about 39% as compared with counting the plate with the underdrain intact. Presoaking the wells with 0.5% polyethyleneimine for 2 h reduced the nonspecific binding to the filter material by about 50%. A linear relationship of protein concentration and radioligand binding was established up to a protein concentration of 165 microg of protein/well. In the assays, 70 microg of protein/well was generally used, which has turned out to be favorable with respect to the number of counts obtained. When a higher concentration of protein was used, the period of time needed to aspirate the plate was too long because of obstruction of the filter material. Receptor-radioligand equilibration was reached after about 20 min at concentrations less than 0.05 nM [(3)H]ketanserin-HCl; at higher concentrations it was reached after about 10 min. Saturation analysis of [(3)H]ketanserin-HCl resulted in a mean B(max) of 393 fmol/mg protein and a K(D) of 2.0 nM using rat frontal cortex as a receptor source. Competition experiments with known 5-HT(2A) receptor ligands-DOB-HCl (K(i) = 59 nM), DOET-HCl (K(i) = 137 nM), DOM-HCl (K(i) = 533 nM), DMT (K(i) = 1,985 nM), and TMA-HCl (K(i) = 22,340 nM)-were in accordance with literature values.

Animals↗
PubMed2006

A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening.

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.

Aldehyde-Lyases↗
PubMed2006

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening.

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.

Animals↗
PubMed2004

Application of chemoinformatics to high-throughput screening: practical considerations.

The objective of this chapter is to summarize and evaluate some of the most common chemoinformatic methods that are applied to the analysis of high-throughput-screening data. The chapter will briefly describe current high-throughput-screening practices and will stress how the major constraint on the application of chemoinformatics is often the quality of high-throughput-screening data. Discussion of the NCI dataset and how it differs from most high-throughput-screening datasets will be made to highlight this point.

Chemical Phenomena↗
PubMedDate not supplied

Micro-X-ray fluorescence as a general high-throughput screening method for catalyst discovery and small molecule recognition.

A powerful high-throughput screening technique is described for the rapid screening of bead-based libraries for catalyst discovery and molecular recognition. Micro-X-ray fluorescence (MXRF) screens materials for elemental composition with mesoscale analysis. This method is nondestructive and requires minimal sample preparation and no special tags for analysis, and the screening time is dependent on the desired sensitivity. The speed, sensitivity, and simplicity of MXRF as a high-throughput screening technique were applied to screen bead-based libraries of oligopeptides for phosphate hydrolysis catalysts and molecular recognition of selective receptors for the degradation products and analogues of chemical warfare agents. This paper demonstrates the analytical or HTS capability of MXRF for combinatorial screening. It is meant only to show the capabilities of MXRF and is not meant as an exhaustive study of the catalyst and molecular recognition systems presented.

Catalysis↗
PubMed2002

Integration of NMR and high-throughput screening.

NMR-based screening has become a powerful method for the identification and analysis of low-molecular weight organic compounds that bind to protein targets and can be utilized in drug discovery programs. In particular, heteronuclear NMR-based screening can yield information about both the affinity and binding location of potential lead compounds. In addition, heteronuclear NMR-based screening has wide applications in complementing and facilitating conventional high-throughout screening programs. This article will describe several strategies for the integration of NMR-based screening and high-throughput screening. The marriage of these two techniques promises to be of tremendous benefit in the triage of hits that come from HTS, and can aid the medicinal chemist in the identification of quality leads that have high potential for further optimization.

Combinatorial Chemistry Techniques↗
PubMed2002

Virtual screening: a real screening complement to high-throughput screening.

Virtual screening is being routinely used as an integral part of today's hit-identification strategies for, on one hand, prioritizing large corporate screening collections and, on the other hand, to extend the scope of screening to external databases. A brief description of the essential elements required for virtual screening and an application example to the identification of agonist hits for the oestrogen receptor subtype ER alpha are presented.

Drug Evaluation, Preclinical↗
PubMed2005

A high-throughput screening for phosphatases using specific substrates.

A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.

Alkaline Phosphatase↗
PubMed2006

A high-throughput screening assay for hydroxynitrile lyase activity.

A high-throughput screening assay for hydroxynitrile lyase activity accepting a wide range of HNL-substrates is presented, which is useful either for enzyme fingerprinting or screening of huge variant libraries generated in metagenome or directed evolution approaches.

Aldehyde-Lyases↗
PubMed2000

Detection of oligonucleotide-ligand complexes by ESI-MS (DOLCE-MS) as a component of high throughput screening.

With the advent of combinatorial chemistry and high throughput screening, a major bottleneck in the pharmaceutical industry has changed from quickly finding active compounds to limiting them to a manageable number for proper follow-up. With hundreds to thousands of active compounds identified by a multitude of biological screens, there need to be rapid and unambiguous methods for eliminating false positive, toxic, or otherwise difficult compounds from further scrutiny. We have used electrospray ionization mass spectrometry as a rapid screening method to identify compounds from viral screens that yield a positive assay response by interaction with DNA rather than inhibiting the target enzyme. Both the sample preparation and data acquisition have been automated, allowing the screening of all hits from relevant biological screens (up to 1,000/week). The assay was validated using several known DNA intercalators and minor groove binders. These "standards" and many but not all of our "active compounds" were shown to form noncovalent complexes with a variety of different DNA:DNA and DNA:RNA duplexes.

Base Sequence↗
PubMed2006

Molecular descriptors and methods for ligand based virtual high throughput screening in drug discovery.

The aim of virtual high throughput screening is the identification of biologically relevant molecules amongst either tangible or virtual (large) collections of compounds. Amongst the various virtual screening approaches, those that are ligand based are becoming very popular due to the possibility to screen millions of molecules in a timely way. Descriptors and methods are briefly introduced and reviewed with more emphasis for those approaches that are based on fingerprint descriptors and that seems to be more utilized during the drug discovery process.

Artificial Intelligence↗
PubMed2004

[The role of high throughput screening in the early stage of drug research].

High throughput screening (HTS) became an integral part of the early phase drug discovery process. Although the methodology itself is widely accepted as a powerful tool of lead discovery there are a number of positive and also negative opinions associated to its application. This paper gives a short introduction to HTS technology, summarizes primary experiences and points out latest changes in screening strategy and technology. Development and application of the new screening facility of Gedeon Richter is also reported.

Animals↗
PubMed1999

Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology.

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-µmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.

Journal Article↗