scholarly journals Validation of FLIPR Membrane Potential Dye for High Throughput Screening of Potassium Channel Modulators

2001 ◽  
Vol 6 (5) ◽  
pp. 305-312 ◽  
Author(s):  
Kristi L. Whiteaker ◽  
Sujatha M. Gopalakrishnan ◽  
Duncan Groebe ◽  
Char-Chang Shieh ◽  
Usha Warrior ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Potassium Channel ◽  
High Throughput ◽  
High Throughput Screening ◽  
Time Course ◽  
Functional Characterization ◽  
K Channel ◽  
Potassium Channel Modulators ◽  
Alternative Choice ◽  
Fluorescent Compounds

A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive Ki channel; KATP) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference KATP channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.

Identification of Positive Allosteric Modulators of Glycine Receptors from a High-Throughput Screen Using a Fluorescent Membrane Potential Assay

2016 ◽  
Vol 21 (10) ◽  
pp. 1042-1053 ◽  
Author(s):  
Clara Stead ◽  
Adam Brown ◽  
Cathryn Adams ◽  
Sarah J. Nickolls ◽  
Gareth Young ◽  
...  
Keyword(s):  
Membrane Potential ◽  
High Throughput ◽  
Glycine Receptor ◽  
Functional Characterization ◽  
In Vitro Assays ◽  
Allosteric Modulators ◽  
Inhibitory Neurotransmission ◽  
Starting Point ◽  
Positive Allosteric Modulators

Glycine receptor 3 (GlyRα3) is a ligand-gated ion channel of the cys-loop family that plays a key role in mediating inhibitory neurotransmission and regulation of pain signaling in the dorsal horn. Potentiation of GlyRα3 function is therefore of interest as a putative analgesic mechanism with which to target new therapeutics. However, to date, positive allosteric modulators (PAMs) of this receptor with sufficient selectivity to enable target validation studies have not been described. To address this lack of pharmacological tools, we developed a suite of in vitro assays comprising a high-throughput fluorescent membrane potential screen and a medium-throughput electrophysiology assay using IonFlux HT together with conventional manual patch clamp. Using these assays, we conducted a primary screening campaign and report the structures of hit compounds identified as GlyR PAMs. Our functional characterization data reveal a hit compound with high efficacy relative to current known potentiators and selectivity over GABAAR, another major class of inhibitory neurotransmission receptors of importance to pain. These small-molecule GlyR PAMs have high potential both as early tool compounds to enable pharmacological studies of GlyR inhibitory neurotransmission and as a starting point for the development of potent, selective GlyRα3 PAMs as novel analgesics.

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 Development of a High Throughput Screening Assay for Mitochondrial Membrane Potential in Living Cells

2002 ◽  
Vol 7 (4) ◽  
pp. 383-389 ◽  
Author(s):  
Shu-Gui Huang
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mitochondrial Membrane ◽  
Living Cells ◽  
Screening Assay ◽  
Mitochondrial Inner Membrane ◽  
Obesity And Diabetes ◽  
High Throughput Screening Assay

The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC50 values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.

scholarly journals High-Throughput Screening Reveals a Small-Molecule Inhibitor of the Renal Outer Medullary Potassium Channel and Kir7.1

2009 ◽  
Vol 76 (5) ◽  
pp. 1094-1103 ◽  
Author(s):  
L. Michelle Lewis ◽  
Gautam Bhave ◽  
Brian A. Chauder ◽  
Sreedatta Banerjee ◽  
Katharina A. Lornsen ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitor ◽  
Molecule Inhibitor

scholarly journals A Continuous, Fluorescence-based Assay of µ-Opioid Receptor Activation in AtT-20 Cells

2012 ◽  
Vol 18 (3) ◽  
pp. 269-276 ◽  
Author(s):  
Alisa Knapman ◽  
Marina Santiago ◽  
Yan Ping Du ◽  
Philip R. Bennallack ◽  
Macdonald J. Christie ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Opioid Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Receptor Activation ◽  
Maximum Effect ◽  
Individual Response ◽  
Dependent Manner ◽  
Membrane Hyperpolarization ◽  
Opioid Ligands

Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary “no-wash” fluorescent membrane potential dye to act as a reporter of µ–opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.

A High-Throughput Screening Campaign for Detection of Ca2+–Activated K+ Channel Activators and Inhibitors Using a Fluorometric Imaging Plate Reader–Based Tl+-Influx Assay

2013 ◽  
Vol 11 (3) ◽  
pp. 163-172 ◽  
Author(s):  
Susanne Jørgensen ◽  
Tino Dyhring ◽  
David T. Brown ◽  
Dorte Strøbæk ◽  
Palle Christophersen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
K Channel ◽  
Imaging Plate ◽  
Plate Reader

scholarly journals High-Throughput Quantitative Intrinsic Thiol Reactivity Evaluation Using a Fluorescence-Based Competitive Endpoint Assay

2017 ◽  
Vol 22 (9) ◽  
pp. 1168-1174 ◽  
Author(s):  
Tomoya Sameshima ◽  
Ikuo Miyahisa ◽  
Seiji Yamasaki ◽  
Mika Gotou ◽  
Toshitake Kobayashi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Time Course ◽  
Reaction Rate ◽  
Evaluation Method ◽  
Chemical Reactivity ◽  
Competitive Reaction ◽  
Quantitative Calculation ◽  
Thiol Reactivity ◽  
Endpoint Assay

In a high-throughput screening (HTS) process, the chemical reactivity of test samples should be carefully examined because such reactive compounds may lead to false-positive results and adverse effects in vivo. Among all natural amino acids, the thiol side chain in cysteine has the highest nucleophilicity; thus, assessment of intrinsic thiol group reactivity in the HTS processes is expected to accelerate drug discovery. In general, kchem (M−1s−1), the secondary reaction rate constant of a compound to thiol, can be evaluated via time course measurements of thiol-compound adducts using liquid chromatography–mass spectroscopy; this requires time-consuming and labor-intensive procedures. To overcome this issue, we developed a fluorescence-based competitive endpoint assay that allows quantitative calculation of the reaction rate of test compounds in an HTS format. Our assay is based on the competitive reaction for a free thiol (e.g., glutathione) between the test compounds and a fluorescent probe, o-maleimide BODIPY. Our assay provides robust data with a satisfactory throughput at an affordable cost. Our kchem evaluation method has advantages over previous assays in terms of higher throughput and quantitativeness. Thus, it contributes to early elimination of reactive compounds as well as quantitative evaluation of the kchem values of covalent inhibitors.

scholarly journals High-Throughput Generation of Product Profiles for Arabinoxylan-Active Enzymes from Metagenomes

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Maria João Maurício da Fonseca ◽  
Zachary Armstrong ◽  
Stephen G. Withers ◽  
Yves Briers
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Functional Characterization ◽  
Enzymatic Reaction ◽  
Added Value ◽  
Screening Methods ◽  
Reaction Products ◽  
High Throughput Analysis ◽  
Substrate Preferences ◽  
Promising Source

ABSTRACT Metagenomics is an exciting alternative to seek carbohydrate-active enzymes from a range of sources. Typically, metagenomics reveals dozens of putative catalysts that require functional characterization for further application in industrial processes. High-throughput screening methods compatible with adequate natural substrates are crucial for an accurate functional elucidation of substrate preferences. Based on DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) analysis of enzymatic-reaction products, we generated product profiles to consequently infer substrate cleavage positions, resulting in the generation of enzymatic-degradation maps. Product profiles were produced in high throughput for arabinoxylan (AX)-active enzymes belonging to the glycoside hydrolase families GH43 (subfamilies 2 [MG432], 7 [MG437], and 28 [MG4328]) and GH8 (MG8) starting from 12 (arabino)xylo-oligosaccharides. These enzymes were discovered through functional metagenomic studies of feces from the North American beaver (Castor canadensis). This work shows how enzyme loading alters the product profiles of all enzymes studied and gives insight into AX degradation patterns, revealing sequential substrate preferences of AX-active enzymes. IMPORTANCE Arabinoxylan is mainly found in the hemicellulosic fractions of rice straw, corn cobs, and rice husk. Converting arabinoxylan into (arabino)xylo-oligosaccharides as added-value products that can be applied in food, feed, and cosmetics presents a sustainable and economic alternative for the biorefinery industries. Efficient and profitable AX degradation requires a set of enzymes with particular characteristics. Therefore, enzyme discovery and the study of substrate preferences are of utmost importance. Beavers, as consumers of woody biomass, are a promising source of a repertoire of enzymes able to deconstruct hemicelluloses into soluble oligosaccharides. High-throughput analysis of the oligosaccharide profiles produced by these enzymes will assist in the selection of the most appropriate enzymes for the biorefinery.

Overcoming Problems of Compound Storage in DMSO: Solvent and Process Alternatives

2009 ◽  
Vol 14 (6) ◽  
pp. 708-715 ◽  
Author(s):  
Timothy J. Waybright ◽  
John R. Britt ◽  
Thomas G. McCloud
Keyword(s):  
Water Absorption ◽  
High Throughput ◽  
High Throughput Screening ◽  
Time Course ◽  
Molar Concentration ◽  
Dmso Solution ◽  
Compound Library ◽  
Plate Production ◽  
The Common ◽  
Drug Solution

The common practice of preparing storage libraries of compounds in 100% DMSO solution well in advance of bioassay brings with it difficulties that affect the accuracy of the data obtained. This publication presents a series of studies done on a subset of compounds that are difficult to bioassay because they precipitate from DMSO solution. These compounds are members of a frequently used, diverse compound library of the sort commonly used in the high-throughput screening (HTS) environment. Experiments were performed to determine the concentration of drug in solution above the precipitate, observe the time course and effect of various mixtures of solvents upon precipitation, measure the viscosity of cosolvents to determine compatibility with HTS, determine water absorption rates for various solvent combinations, and investigate resolubilization techniques to ensure proper drug solution for HTS. Recommendations are made on how to best maximize the probability that problem compounds will remain in solution, be accurately transferred during assay plate production, and, as a result, be accurately bioassayed at the specified molar concentration. ( Journal of Biomolecular Screening 2009:708-715)

Sign in / Sign up

Export Citation Format

Share Document