scholarly journals Receptor-Ligand Interactions Studied with Homogeneous Fluorescence-Based Assays Suitable for Miniaturized Screening

2001 ◽  
Vol 6 (1) ◽  
pp. 11-18
Author(s):  
Andreas A. Scheel ◽  
Bettina Funsch ◽  
Michael Busch ◽  
Gabriele Gradl ◽  
Johannes Pschorr ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
High Throughput Screening ◽  
Free Ligand ◽  
Membrane Vesicles ◽  
Membrane Receptors ◽  
Receptor Expression ◽  
Model Systems ◽  
Fluorescence Signal ◽  
Distribution Analysis

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and β2-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1,IL without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.

Single-Molecule Detection Technologies in Miniaturized High-Throughput Screening: Fluorescence Intensity Distribution Analysis

2003 ◽  
Vol 8 (1) ◽  
pp. 19-33 ◽  
Author(s):  
Ulrich Haupts ◽  
Martin Rüdiger ◽  
Stephen Ashman ◽  
Sandra Turconi ◽  
Ryan Bingham ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Fluorescence Intensity ◽  
Intensity Distribution ◽  
High Throughput Screening ◽  
Biological Data ◽  
Single Molecule Detection ◽  
Distribution Analysis ◽  
Fluorescence Intensity Distribution Analysis ◽  
Detection Technologies

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence. ( Journal of Biomolecular Screening 2003:19-33)

A Novel High-Throughput Screening Assay for HCN Channel Blocker Using Membrane Potential-Sensitive Dye and FLIPR

2009 ◽  
Vol 14 (9) ◽  
pp. 1119-1128 ◽  
Author(s):  
Dmitry V. Vasilyev ◽  
Qin J. Shan ◽  
Yan T. Lee ◽  
Veronica Soloveva ◽  
Stanley P. Nawoschik ◽  
...  
Keyword(s):  
Membrane Potential ◽  
High Throughput ◽  
High Throughput Screening ◽  
Channel Blocker ◽  
Fluorescent Imaging ◽  
Fluorescence Signal ◽  
Hcn Channel ◽  
Plating Density ◽  
High Throughput Screening Assay ◽  
Cell Plating

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the wellto-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 ± 1500 FLIPR3 relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 µM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r2 = 0.56) between the respective IC50s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds. (Journal of Biomolecular Screening 2009:1119-1128)

scholarly journals Single-Molecule Detection Technologies in Miniaturized High Throughput Screening: Binding Assays for G Protein-Coupled Receptors Using Fluorescence Intensity Distribution Analysis and Fluorescence Anisotropy

2001 ◽  
Vol 6 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Martin Rudiger ◽  
Ulrich Haupts ◽  
Keith J. Moore ◽  
Andrew J. Pope
Keyword(s):  
Single Molecule ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
G Protein Coupled Receptors ◽  
Single Molecule Detection ◽  
Distribution Analysis ◽  
Binding Assays ◽  
Fluorescence Intensity Distribution Analysis ◽  
Fluorescent Ligands ◽  
G Protein Coupled

G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.

scholarly journals Hypothesis Testing in High-Throughput Screening for Drug Discovery

2012 ◽  
Vol 17 (4) ◽  
pp. 519-529 ◽  
Author(s):  
Michael Prummer
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Single Point ◽  
Error Rates ◽  
P Value ◽  
Distribution Analysis ◽  
Use Of Resources ◽  
Compound Screening

Following the success of small-molecule high-throughput screening (HTS) in drug discovery, other large-scale screening techniques are currently revolutionizing the biological sciences. Powerful new statistical tools have been developed to analyze the vast amounts of data in DNA chip studies, but have not yet found their way into compound screening. In HTS, characterization of single-point hit lists is often done only in retrospect after the results of confirmation experiments are available. However, for prioritization, for optimal use of resources, for quality control, and for comparison of screens it would be extremely valuable to predict the rates of false positives and false negatives directly from the primary screening results. Making full use of the available information about compounds and controls contained in HTS results and replicated pilot runs, the Z score and from it the p value can be estimated for each measurement. Based on this consideration, we have applied the concept of p-value distribution analysis (PVDA), which was originally developed for gene expression studies, to HTS data. PVDA allowed prediction of all relevant error rates as well as the rate of true inactives, and excellent agreement with confirmation experiments was found.

scholarly journals Identification of Translational Activators of Glial Glutamate Transporter EAAT2 through Cell-Based High-Throughput Screening

2010 ◽  
Vol 15 (6) ◽  
pp. 653-662 ◽  
Author(s):  
Craig K. Colton ◽  
Qiongman Kong ◽  
Liching Lai ◽  
Michael X. Zhu ◽  
Kathleen I. Seyb ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Neuronal Damage ◽  
Glutamate Uptake ◽  
Glutamate Transporter ◽  
Membrane Receptors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Screening Assay ◽  
Protein Levels ◽  
Glial Glutamate Transporter

Excitotoxicity has been implicated as the mechanism of neuronal damage resulting from acute insults such as stroke, epilepsy, and trauma, as well as during the progression of adult-onset neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. One potential approach to protect against excitotoxic neuronal damage is enhanced glutamate reuptake. The glial glutamate transporter EAAT2 is the quantitatively dominant glutamate transporter and plays a major role in clearance of glutamate. Expression of EAAT2 protein is highly regulated at the translational level. In an effort to identify compounds that can induce translation of EAAT2 transcripts, a cell-based enzyme-linked immunosorbent assay was developed using a primary astrocyte line stably transfected with a vector designed to identify modulators of EAAT2 translation. This assay was optimized for high-throughput screening, and a library of approximately 140,000 compounds was tested. In the initial screen, 293 compounds were identified as hits. These 293 hits were retested at 3 concentrations, and a total of 61 compounds showed a dose-dependent increase in EAAT2 protein levels. Selected compounds were tested in full 12-point dose-response experiments in the screening assay to assess potency as well as confirmed by Western blot, immunohistochemistry, and glutamate uptake assays to evaluate the localization and function of the elevated EAAT2 protein. These hits provide excellent starting points for developing therapeutic agents to prevent excitotoxicity.

scholarly journals A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors

2015 ◽  
Vol 20 (7) ◽  
pp. 849-857 ◽  
Author(s):  
Line Vedel ◽  
Hans Bräuner-Osborne ◽  
Jesper Mosolff Mathiesen
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Phosphodiesterase Inhibitors ◽  
Receptor Expression ◽  
High Signal ◽  
Intracellular Camp ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Pde Inhibitors ◽  
Camp Levels

Cyclic adenosine 3′,5′-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein–coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or Gi-coupled GPCRs, which leads to increased or decreased cAMP levels, respectively. Here we describe a real-time Förster resonance energy transfer (FRET)–based cAMP high-throughput screening (HTS) assay for identification and characterization of Gs-coupled GPCR ligands and phosphodiesterase (PDE) inhibitors in living cells. We used the β2-adrenergic receptor (β2AR) as a representative Gs-coupled receptor and characterized two cell lines with different expression levels. Low receptor expression allowed detection of desensitization kinetics and delineation of partial agonism, whereas high receptor expression resulted in prolonged signaling and enabled detection of weak partial agonists and/or ligands with low potency, which is highly advantageous in large HTS settings and hit identification. In addition, the assay enabled detection of β2AR inverse agonists and PDE inhibitors. High signal-to-noise ratios were also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS.

Development and Implementation of a Highly Miniaturized Confocal 2D-FIDA–Based High-Throughput Screening Assay to Search for Active Site Modulators of the Human Heat Shock Protein 90β

2004 ◽  
Vol 9 (7) ◽  
pp. 569-577 ◽  
Author(s):  
Alain Schilb ◽  
Virginie Riou ◽  
Joseph Schoepfer ◽  
Johannes Ottl ◽  
Kurt Müller ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Anticancer Drugs ◽  
High Throughput ◽  
High Throughput Screening ◽  
Liver Toxicity ◽  
Distribution Analysis ◽  
Screening Assay ◽  
Small Molecular Weight ◽  
High Throughput Screening Assay

The beta isoform of the heat shock protein 90 (Hsp90β) is a cellular chaperone required for the maturation of key proteins involved in growth response to extracellular factors as well as oncogenic transformation of various cell types. Compounds that inhibit the function of Hsp90β are thus believed to have potential as novel anticancer drugs. To date, 2 fungal metabolites are known to inhibit Hsp90β. However, insolubility and liver toxicity restrict the clinical use of these molecules. The limitation to identify novel and safe Hsp90β inhibitors is that presently no suitable high-throughput screening assay is available. Here, the authors present the development of a homogenous assay based on 2-dimensional fluorescence intensity distribution analysis of tetramethyl-rhodamine (TAMRA)-labeled radicicol bound to Hsp90β. Furthermore, the assay has been shown to be compatible with the confocal nanoscreening platform Mark II™ from Evotec-Technologies and can therefore be used for miniaturized high-throughput screening. The applied detection technology provides critical information about the nature of biomolecular interaction at the thermodynamic equilibrium, such as affinity constants and stoichiometric parameters of the binding. The assay is used to identify small molecular weight compounds displacing TAMRA-radicicol. Such compounds are believed to be important molecules in the discovery of novel anticancer drugs.

Sign in / Sign up

Export Citation Format

Share Document