Development of a Fluorescence Based High Throughput Assay for Antagonists of the Human Chorionic Gonadotropin Receptor Extracellular Domain: Analysis of Peptide Inhibitors

A simple method for prompt fluorescent detection of inhibitors of human chorionic gonadotropin (hCG) binding to the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLHICG) receptor was developed for high throughput screening (HTS). Construction and analysis of a recombinant phage that displays the extracellular binding domain of the hLH/CG receptor on its surface and specifically binds hCG was previously described. To facilitate the identification of molecules that disrupt the interaction of hCG with its receptor, a method for prompt fluorescent detection of these phage bound to hCG was developed. This technique is extremely sensitive and employs fluorescent labels (PBXL dyes) that are derived from red and blue-green algae. Antibodies labeled with PBXL dye were able to specifically detect phage that display the extracellular domain of the hLH/CG receptor when bound to hCG immobilized in 96-well microplates. Decreases in fluorescence correlate with the concentration of exogenous hCG or hCG antagonists in the assay. This prompt fluorescence detection assay was optimized in a 96-well format as a model system for HTS applications that target the receptors for the group of hormones known as the gonadotropins. Low-affinity molecules that disrupt binding of the phage-displayed receptor extracellular domain to hCG can be rapidly identified in this high throughput screen.

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Development of a High-Throughput Cell-Based Assay for Superoxide Production in HL-60 Cells

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109359687 ◽

2010 ◽

Vol 15 (4) ◽

pp. 388-397 ◽

Cited By ~ 8

Author(s):

Patricia M. Seitz ◽

Rona Cooper ◽

Gregory J. Gatto ◽

Fernando Ramon ◽

Thomas D. Sweitzer ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Superoxide Production ◽

Test Set ◽

Cellular Processes ◽

Detection Assay ◽

Z Value ◽

Compound Concentration ◽

Compound Test ◽

Early Drug

Superoxide affects many normal and pathogenic cellular processes, and the detection of superoxide produced by cells is therefore of interest for potential therapeutic applications. To develop a high-throughput cell-based assay for the detection of extracellular superoxide production that could be run in a 384-well or 1536-well format, 2 luminescent reagents, Lucigenin and Diogenes, and one fluorescent reagent, Oxyburst Green BSA, were tested. HL-60 cells, which had been differentiated to a neutrophil-like phenotype with DMSO and frozen in large batches, were used in assays. All 3 superoxide detection reagents performed well statistically in terms of IC50 reproducibility and met a desired Z′ value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 µM compound concentration, a higher hit rate was obtained with the 2 luminescent reagents compared with that obtained with the fluorescent Oxyburst Green BSA reagent. The Oxyburst Green BSA assay was ultimately chosen for compound profiling and high-throughput screening activities. This 1536 superoxide detection assay using cryopreserved differentiated HL-60 cells represents a shifting paradigm toward the utilization of more therapeutically relevant cells in early drug development activities.

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Multiplexing Fluorescence Polarization Assays to Increase Information Content Per Screen: Applications for Screening Steroid Hormone Receptors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104264420 ◽

2004 ◽

Vol 9 (4) ◽

pp. 294-302 ◽

Cited By ~ 13

Author(s):

Paul Blommel ◽

George T. Hanson ◽

Kurt W. Vogel

Keyword(s):

Information Content ◽

High Throughput ◽

High Throughput Screening ◽

Hormone Receptors ◽

Scale Up ◽

Cost Savings ◽

Estrogen Receptor Α ◽

Steroid Hormone Receptors ◽

Simple Method ◽

Liquid Handling

As the push to reduce cost per well in high-throughput screening reaches the practical limitations of liquid handling, future cost savings will likely arise from an increase in information content per well. One strategy to increase information content is to perform discreet assays against multiple targets in a single well. In such assays, reagent usage and liquid handling steps do not scale-up in direct proportion to the increase in information content, providing for a simple method to increase data points per screen without further reductions in assay volume. The authors have used tracers incorporating the spectrally distinct fluorophores fluorescein and TAMRA to develop a high-throughput assay to identify selective estrogen receptor α or proges-terone receptor ligands. Selectivity is assessed immediately in this assay, with no requirement for separate follow-up screening to determine selectivity. This methodology is easily adaptable to other target classes.

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Revisiting and Questioning Functional Rescue between Dimerized LH Receptor Mutants

Molecular Endocrinology ◽

10.1210/me.2011-1285 ◽

2012 ◽

Vol 26 (4) ◽

pp. 655-668 ◽

Cited By ~ 20

Author(s):

Meilin Zhang ◽

Rongbin Guan ◽

Deborah L. Segaloff

Keyword(s):

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Hormone Receptors ◽

Resonance Energy ◽

Extracellular Domain ◽

Glycoprotein Hormone ◽

Lh Receptor ◽

Defective Mutant

Abstract The glycoprotein hormone receptors are G protein-coupled receptors containing a large extracellular domain fused to a prototypical serpentine domain. cis-activation occurs when binding of hormone to the extracellular domain stabilizes the serpentine domain in an active conformation. Studies by others suggested that these receptors can also signal by trans-activation, where hormone binding to one receptor protomer activates the serpentine domain of an associated protomer, as documented by the partial rescue of hormone-dependent signaling when a binding defective mutant is coexpressed with a signaling defective mutant. However, our characterizations of several LH receptor (LHR) mutants used in previous studies differ markedly from those originally reported. Also, when examining a pair of LHR mutants previously shown to functionally rescue in vitro as well as in vivo, in addition to finding that the properties of the individual mutants differ significantly from those originally described, we determined that when this pair of mutants was coexpressed in vitro, quantitative analyses did not indicate functional rescue. Additional data are presented that provide a plausible alternate explanation for the apparent in vivo trans-activation that was reported. Finally, using LHR mutants that we have documented to be expressed at the cell surface but to lack human chorionic gonadotropin binding activity or to be severely impaired in their ability to activate Gs, we did not observe functional rescue of human chorionic gonadotropin-stimulated cAMP when the mutants were coexpressed, even though bioluminescence resonance energy transfer analyses confirmed that the coexpressed mutants formed dimers. Taken altogether, our data substantively question the concept of functional rescue between LHR mutants.

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Twelve of fourteen surface epitopes of receptor-bound human chorionic gonadotropin (hCG) being antibody-inaccessible suggest an extensive involvement of the long extracellular domain of the hCG receptor

Molecular and Cellular Endocrinology ◽

10.1016/0303-7207(91)90010-p ◽

1991 ◽

Vol 82 (1) ◽

pp. 71-79 ◽

Cited By ~ 4

Author(s):

S. Schwarz ◽

H. Krude ◽

G. Wick ◽

P. Berger

Keyword(s):

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Extracellular Domain ◽

Extensive Involvement ◽

Hcg Receptor

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Characterization of the 5-HT2b Receptor in Evaluation of Aequorin Detection of Calcium Mobilization for Miniaturized GPCR High-Throughput Screening

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108319212 ◽

2008 ◽

Vol 13 (6) ◽

pp. 486-493 ◽

Cited By ~ 17

Author(s):

Mark A. Gilchrist ◽

Angela Cacace ◽

David G. Harden

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Rank Order ◽

Calcium Mobilization ◽

Fluorescent Detection ◽

Calcium Flux ◽

High Signal ◽

Agonist And Antagonist ◽

Cell Densities ◽

Small Molecule Modulators

Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein—coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen™), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (<10 µL) 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550™) and kinetic flash luminometry (CyBi Lumax™). The enhanced high signal-to-background ratios observed relative to fluorescence were readily manipulated by altering per-well cell densities and yielded acceptable screening statistics in miniaturized format for both agonist and antagonist screening scenarios. In addition, the authors demonstrate the feasibility of using agonist concentrations less than EC50 in a miniaturized antagonist assay. These features, coupled with improved sample handling, should enhance sensitivity and provide the benefits of miniaturization including cost reduction and throughput gains. ( Journal of Biomolecular Screening 2008:486-493)

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