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Results for “High Throughput Screening”

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PubMed2003

Potential high-throughput assay for screening inhibitors of West Nile virus replication.

Prevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encoding Renilla luciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses.

Animals↗
PubMed2001

High-density miniaturized thermal shift assays as a general strategy for drug discovery.

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

Estrogen Receptor alpha↗
PubMed1999

Use of recursive partitioning in the sequential screening of G-protein-coupled receptors.

High-throughput screening (HTS) is changing as more compounds and better assay techniques become available. HTS is also generating a large amount of data. There is a need to rationalize the HTS process, because, in some cases, the screening of all available compounds is not economically feasible. In addition to the selection of promising compounds, there is a need to learn from the data that we collect. In this paper, we use a data-mining method, recursive partitioning, to help uncover and understand structure-activity relations and to help biology and chemistry experts make better decisions on which compounds to screen next and better characterize. The sequential-screening process is presented and the results of applying that process to 14 G-protein-coupled receptor assays are reported.

Animals↗
PubMed2005

High throughput P450 inhibition screens in early drug discovery.

This review of high throughput (HT) P450 inhibition technologies and their impact on early drug discovery finds the field at a mature stage. The relationship between P450 inhibition and drug-drug interactions is well understood. A wide variety of P450 inhibition detection technologies are readily available off-the-shelf, but what seems still to be missing is a general agreement on how much weight one should give to the various types of early discovery HT P450 inhibition data. Method-dependent potency differences are a cause of concern, and to resolve this issue the authors advocate calibration of the HT methods with a large set of marketed drugs.

Calibration↗
PubMed2002

A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists.

In the new high-throughput screening (HTS) campaign, receptor functional assays, 3',5'-cyclic adenosine monophosphate (cAMP), intracellular [Ca(2)+](i), phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter trade mark enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Galpha(s)-coupled receptor. In the EFC-cAMP assay, the beta-galactosidase (beta-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the beta-gal enzyme acceptor (EA) fragment to form an active beta-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Galpha(s) to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC(50) of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC(50)~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 microM in suspension cells. The assay is very robust, with a Z' value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.

Animals↗
PubMed2005

NMR fragment screening: tackling protein-protein interaction targets.

High-throughput screening of libraries containing compounds of 'drug-like' molecular weight has frequently resulted in no or poor drug candidates, especially when screening against demanding drug targets such as protein-protein interactions. Fragment-based lead discovery and optimization has evolved as a promising solution to this problem by combining the universal adaptability of low-molecular-weight fragments with immediate structural information on fragment binding modes. This review focuses on nuclear magnetic resonance (NMR) fragment screening techniques, which provide a unique combination of medium-throughput, direct binding site information and broad applicability. The utility and exemplary data of chemical shift-detected NMR fragment screening applied to the challenging protein-protein interaction target PDZ domains are summarized.

Animals↗
PubMed2004

Affinity ultrafiltration of DNA topoisomerases-targeted compounds determined with HPLC/ESI-MS for drug candidate screening.

A method of screening assay is demonstrated. The approach is based on the affinity of antitumor candidates for topoisomerases. In this method, antitumor candidates are fished out using topoisomerases as targets. Traditional analysis of complex compounds typically encounters signal suppression due to the relatively low concentrations, but enzyme-affinity screening for the active compounds can effectively concentrate the desired analysts into a small volume of high concentration. Active compounds are separated from non-affinity compounds by ultrafiltration. The molecules-enzymes complexes that are retained on the filter are subsequently separated by acidification to obtain the topoisomerases-affinity compounds for analysis on High Performance Liquid Chromatography coupled with electrospray ionization mass spectrometric detection (ESI-MS). This enzyme-affinity based screening assay provides a highly specific and efficient method that can directly screen, identify, and acquire drug candidates thus improving the accuracy and speed of high-throughput screening activities.

Chromatography, High Pressure Liquid↗
PubMed1996

Development of a high volume screen to identify inhibitors of endothelial cell activation.

We have developed a high throughput screen to identify inhibitors of endothelial cell activation using E-selection cell-surface expression as a marker. Endothelial cell activation is an important component of both acute and chronic inflammatory disease. Inhibitors of this process represent potential therapeutic agents. A cell culture system for primary human umbilical vein endothelial cells was generated, including an analysis of donor variability, passage number, seeding density, and cost. The effects of IL-1 beta, TNF alpha, LPS, and an LPS-conditioned plasma product on E-selectin expression were characterized. E-selectin expression on the surface of IL-1-stimulated endothelial cells was quantified with a direct ELISA on fixed cell monolayers. Automation of the ELISA necessitated identification of methods for cell fixation, liquid handling, and compound addition which would maintain the integrity of the cell monolayer. The ELISA is inexpensive, reproducible, and suitable for high throughput primary cell assays, supporting a screening rate of 10,000 compounds/ week. The method is compatible with a broad chemical diversity, and the cellular format provides early information on the cellular uptake and cytotoxicity of compounds. We describe a screening paradigm which allowed us to identify inhibitors of endothelial cell activation and simultaneously discriminate their activity from their cytotoxic effects.

Cell Survival↗
PubMed2005

Electroacupuncture up-regulates natural killer cell activity Identification of genes altering their expressions in electroacupuncture induced up-regulation of natural killer cell activity.

As an important cellular component of the innate immune system, NK cells constitute a first line of defense against various infections and malignancies. Previous studies have reported electroacupuncture (EA) modulation of natural killer cell (NK cell) activities. Our study confirmed that EA treatment increases NK cell activity using (51)Cr release assay. Furthermore, in order to better understand the activation mechanism of NK cell by EA, we employed a cDNA microarray technique to elucidate how EA alters gene expressions in the spleen of rats. We screened EA responsive genes using a high-throughput screening and identified 154 genes. Among those genes we selected 4 genes that are known to play a crucial role in NK cell activation and examined their mRNA expressions after EA treatment using RT-PCR. Our data shows that EA treatment increased CD94, PTK and VCAM-1 expressions while decreased PTP and SHP-1. These results imply that EA treatment increase PTK expression, which increases NK cell activity, through induction of CD94 while decreases SHP-1, which inhibits NK cell activity, simultaneously so that it activates NK cell with high efficacy. It seems that increased VCAM-1 expression is due to INF-gamma produced by activated NK cell. Increased production of VCAM-1 is expected to play an important role in binding of NK cell to the target cell. The result of our study may provide key insights in understanding the mechanisms of activation of NK cell induced by EA.

Acupuncture Points↗
PubMed2004

A strategy for discovery of novel broad-spectrum antibacterials using a high-throughput Streptococcus pneumoniae transcription/translation screen.

The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact. After filtering luciferase inhibitors and several known classes of antibiotics, approximately 50 hits remained. These compounds were examined for their ability to inhibit Escherichia coli TT, uncoupled S. pneumoniae translation or transcription, rabbit reticulocyte translation, and in vitro toxicity in human and bacterial cells. One of these compounds had the desired profile of broad-spectrum biochemical activity in bacteria and selectivity versus mammalian biochemical and whole-cell assays.

Anti-Bacterial Agents↗
PubMed2006

Structure-based virtual screening of chemical libraries for drug discovery.

One of the main goals in drug discovery is to identify new chemical entities that have a high likelihood of binding to the target protein to elicit the desired biological response. To this end, virtual screening is being increasingly used as a complement to high-throughput screening to improve the speed and efficiency of the drug discovery and development process. The availability of inexpensive high-performance computing platforms in recent years has transformed this field into one that is highly diverse and rapidly evolving, where large chemical databases have been successfully screened to identify hits for a wide range of targets such as Bcl-2 family proteins, G protein-coupled receptors, kinases, metalloproteins, nuclear hormone receptors, proteases and many more.

Acetylcholinesterase↗
PubMed2006

High-throughput Plasmodium falciparum growth assay for malaria drug discovery.

New therapeutic agents for the treatment of malaria, the world's most deadly parasitic disease, are urgently needed. Malaria afflicts 300 to 500 million people and results in 1 to 2 million deaths annually, and more than 85% of all malaria-related mortality involves young children and pregnant women in sub-Saharan Africa. The emergence of multidrug-resistant parasites, especially in Plasmodium falciparum, has eroded the efficacy of almost all currently available therapeutic agents. The discovery of new drugs, including drugs with novel cellular targets, could be accelerated with a whole-organism high-throughput screen (HTS) of structurally diverse small-molecule libraries. The standard whole-organism screen is based on incorporation of [3H]hypoxanthine and has liabilities, such as limited throughput, high cost, multiple labor-intensive steps, and disposal of radioactive waste. Recently, screens have been reported that do not use radioactive incorporation, but their reporter signal is not robust enough for HTS. We report a P. falciparum growth assay that is technically simple, robust, and compatible with the automation necessary for HTS. The assay monitors DNA content by addition of the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) as a reporter of blood-stage parasite growth. This DAPI P. falciparum growth assay was used to measure the 50% inhibitory concentrations (IC50s) of a diverse set of known antimalarials. The resultant IC50s compared favorably with those obtained in the [3H]hypoxanthine incorporation assay. Over 79,000 small molecules have been tested for antiplasmodial activity using the DAPI P. falciparum growth assay, and 181 small molecules were identified as highly active against multidrug-resistant parasites.

Animals↗
PubMed2003

Use of SAM2 biotin capture membrane in microarrayed compound screening (muARCS) format for nucleic acid polymerization assays.

Microarrayed compound screening format (muARCS) is a novel high-throughput screening technology that uses agarose matrices to integrate various biochemical or biological reagents in the assay. To evaluate the feasibility of using the muARCS technology for nucleic acid polymerization assays, the authors developed HIV reverse transcription (RT) and E1-dependent human papillomavirus (HPV) replication assays in this format. HIV RT is an RNA-dependent DNA polymerase, whereas HPV E1 is a DNA helicase. To ensure the efficient capture of the nucleic acid polymerization reaction and to minimize the nonspecific binding, the authors used a SAM(2) biotin capture membrane in the assay. In both studies, the nucleic acid substrate was biotinylated on one end and was bound to the SAM(2) membrane. A low melting-point agarose gel containing the rest of the reaction components was first placed on a polystyrene sheet spotted with compounds to allow passive diffusion of the compounds into the gel. The gel was removed from the compound sheet and applied to the SAM(2) membrane with the immobilized nucleic acid template to initiate the polymerization. After the incubation, the membrane was washed with a high-salt buffer and exposed for imaging. Potential inhibitors can be seen as white spots on a dark background. The sensitivity for the known inhibitors appears to be comparable in muARCS as in a traditional 96-well plate assay. The methodology described in this paper further expands the applications of muARCS technology.

Alkynes↗
PubMed2025

Construction and In Vitro and In Vivo Analysis of Coxsackievirus B4 Reporter Viruses: Attenuated Virulence but Highly Efficient for Antiviral Drug Screening and Evaluation.

Coxsackievirus B4 (CVB4) is an enterovirus with one of the highest mortality rates following infection, yet research on it remains limited. To enhance the efficiency of CVB4 research, we developed the rCVB4-EGFP and rCVB4-NanoLuc reporter viruses. The replication kinetics of these reporter viruses in SH-SY5Y and HeLa cells were essentially consistent with those of the wild-type CVB4. A strong correlation was observed between the fluorescence and bioluminescence signals of rCVB4-EGFP and rCVB4-NanoLuc and viral titers at specific times postinfection. When evaluating the anti-CVB4 drug fluoxetine using these reporter viruses, the half-maximal effective concentrations derived from fluorescence signals, bioluminescence signal intensities, and viral genome copies were consistent. In In Vivo drug evaluations, because CVB4 can infect various tissues and organs, the bioluminescence signal of rCVB4-NanoLuc effectively demonstrated the antiviral effects of drugs, offering significant advantages over traditional tissue viral titer analysis. The reporter viruses exhibited reduced virulence compared with wild-type CVB4 both In Vitro, in SH-SY5Y and HeLa cells, and In Vivo, in ICR suckling mice. Although this reduced virulence may limit their application for studying pathogenic mechanisms, these reporter viruses can serve as highly efficient tools for high-throughput screening and evaluation of anti-CVB4 drugs, vaccines, and neutralizing antibodies.

Humans↗
PubMed2026

The Use of Deep Learning in RNA Therapeutic Development.

Ribonucleic acid (RNA)-based therapeutics have emerged as promising methods of disease treatment due to their ability to target the human genome and influence protein production, their versatility, and their relative lack of toxicity compared to other gene therapies. However, the RNA therapeutic design space is extremely large, encompassing multiple variables, including codon identities, secondary structure, and design of specific regions. RNA therapeutic optimization is difficult due to the impracticality of exploring such a vast design space experimentally. To address this limitation, deep learning methods have been employed to optimize RNA therapeutic development. In this review, we examine the application of deep learning models across three key aspects of RNA therapeutic development (RNA structure prediction, CRISPR activity, and RNA delivery), highlighting major contributions in these fields and analyzing how deep learning model architectures could affect model performance. We then discuss challenges associated with using deep learning for RNA therapeutics, such as computational and data limitations. Finally, we offer perspectives on areas for future exploration, such as emerging model architectures and methods of integration with more advanced high-throughput screening techniques. Ultimately, this review provides an overview of how deep learning is used in RNA therapeutic development and how it can evolve in the future.

Deep Learning↗
PubMed2000

Development of a fluorescent ligand-binding assay using the AcroWell filter plate.

One of the most powerful tools for receptor research and drug discovery is the use of receptor-ligand affinity screening of combinatorial libraries. Early work involved the use of radioactive ligands to identify a binding event; however, there are numerous limitations involved in the use of radioactivity for high throughput screening. These limitations have led to the creation of highly sensitive, nonradioactive alternatives to investigate receptor-ligand interactions. Pall Gelman Laboratory has introduced the AcroWell, a patented low-fluorescent-background membrane and sealing process together with a filter plate design that is compatible with robotic systems. Taken together, these allow the AcroWell 96-well filter plate to detect trace quantities of lanthanide-labeled ligands for cell-, bead-, or membrane-based assays using time-resolved fluorescence. Using europium-labeled galanin, we have demonstrated that saturation binding experiments can be performed with low-background fluorescence and signal-to-noise ratios that rival traditional radioisotopic techniques while maintaining biological integrity of the receptor-ligand interaction. In addition, the ability to discriminate between active and inactive compounds in a mock galanin screen is demonstrated with low well-to-well variability, allowing reliable determination of positive hits even for low-affinity interactions.

Cell Line↗
PubMed2000

Implementation of a continuous, enzyme-coupled fluorescence assay for high-throughput analysis of glutamate-producing enzymes.

Enzymatic formation of glutamate is critical to numerous biological pathways. However, current methods for assaying the activities of glutamate-forming enzymes are not particularly suitable for high-throughput screening in drug discovery. We present a continuous-read, fluorometric assay for high-throughput analysis of glutaminases. This assay is adapted to a microplate format and employs glutamate oxidase and horseradish peroxidase to couple glutamate formation to production of the fluorescent reporter molecule, resorufin, for enhancement of sensitivity (M. Zhou, Z. Diwu, N. Panchuk-Voloshina, and R. P. Haughland, 1997, Anal. Biochem. 253, 162-168). Described herein is the selection of suitable levels of coupling enzymes for optimal kinetic response and lag time of the reporter system, based on the kinetic characteristics of the individual coupling enzymes. Finally, implementation of the assay in a format for high-throughput kinetic analysis of glutaminases is demonstrated for Escherichia coli carbamoyl phosphate synthase. Derived kinetic constants are comparable to literature values determined using a variety of assay techniques.

Glutamic Acid↗
PubMed2005

Discovery of novel inhibitor of human leukocyte common antigen-related phosphatase.

Human leukocyte common antigen-related phosphatase (LAR) may play a role in type 2 diabetes and cancer, and in the development of the nervous system, and it may be an attractive target for the treatment of diabetes and cancer. We identified eight hits from the random screening of LAR D1 with a high-throughput screening assay. Further validation of the eight hits showed that the meD insertion was associated with inhibition of LAR D1D2 and LAR D1Q. These data suggest that the inserted meD peptide influences the interaction of the enzyme and inhibitor, which is consistent with the kinetic catalysis constants of the substrate pNPP. Our data showed that Hit 1, the first published novel inhibitor of LAR, is a competitive inhibitor with a K(i) of 330 nM that displays obvious selectivity for LAR and mouse PTPsigma, but not for other protein tyrosine phosphatases.

Animals↗