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.

scholarly journals Use of FLIPR Membrane Potential Dyes for Validation of High-Throughput Screening with the FLIPR and µARCS Technologies: Identification of Ion Channel Modulators Acting on the GABAA Receptor

2008 ◽  
Vol 13 (3) ◽  
pp. 218-228 ◽  
Author(s):  
Christoph Joesch ◽  
Emelie Guevarra ◽  
Serge P. Parel ◽  
Andreas Bergner ◽  
Peter Zbinden ◽  
...  
Keyword(s):  
Membrane Potential ◽  
High Throughput ◽  
High Throughput Screening ◽  
Constitutive Expression ◽  
Initial Assessment ◽  
Blue Dye ◽  
Imaging Plate ◽  
Allosteric Modulators ◽  
Positive Allosteric Modulators ◽  
Red Dye

Fluorometric imaging plate reader (FLIPR) membrane potential dyes (FMP-Red-Dye and FMP-Blue-Dye) were evaluated for the detection of compounds acting either as positive allosteric modulators or agonists on the GABAA receptor (GABAAR). A stable HEK293 cell line with constitutive expression of the rat GABA AR α1, β2, and γ2 genes was used to establish a functional high-throughput screening (HTS) assay based on measurement of the membrane potential change in living cells. The assay was validated with the FLIPR technology for identification of agonists and positive allosteric modulators using GABA and diazepam as model compounds. The FMP-Red-Dye showed better performance than the FMP-Blue-Dye, and the effects induced by GABA and diazepam were comparable to electrophysiology data. Subsequently, the assay was also validated with an ultra-HTS approach known as microarrayed compound screening (µARCS). The LOPAC library was used in a test screen for an initial assessment of the technology. Finally, the FLIPR and µARCS technologies were tested with a larger screening campaign. A focused library of 3520 putative positive modulators was tested with the FLIPR assay, and a diverse subset of 84,480 compounds was selected for screening with the µARCS technology. All hits were subjected to verification using the FLIPR technology, and confirmed hits were subsequently evaluated by EC50 determination. Finally, selected hits were further confirmed with electrophysiology testing. ( Journal of Biomolecular Screening 2008:218-228)

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

2021 ◽  
Vol 18 (7) ◽  
pp. 3332
Author(s):  
Olga V. Naidenko ◽  
David Q. Andrews ◽  
Alexis M. Temkin ◽  
Tasha Stoiber ◽  
Uloma Igara Uche ◽  
...  
Keyword(s):  
Immune System ◽  
High Throughput ◽  
Environmental Protection Agency ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Specific Activity ◽  
Laboratory Animals ◽  
In Vitro Assays ◽  
Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

scholarly journals Determining the Relative Efficacy of Positive Allosteric Modulators of the GABAA Receptor

2013 ◽  
Vol 19 (3) ◽  
pp. 462-467 ◽  
Author(s):  
Philippe Ghisdal ◽  
Nadine Noel ◽  
Nathalie Pacico ◽  
Murielle Martini ◽  
Patrik Foerch ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Patch Clamp ◽  
Multiple Drug ◽  
Allosteric Modulators ◽  
Relative Efficacy ◽  
Subtype Selectivity ◽  
Automated Patch Clamp ◽  
Positive Allosteric Modulators ◽  
New Generation ◽  
Different Levels

Gamma amino butyric acid receptors (GABA) are major therapeutic targets for the development of drugs in neurological and psychiatric disorders. The new generation of GABAA modulators is targeting subtype selectivity and low/partial efficacy on the receptor to potentially overcome the adverse effects described for drugs with full agonist profile. We evaluated a screening approach to measure the relative efficacy of GABAA positive allosteric modulators (PAM) using automated patch clamp and fluorescence membrane potential assays. We determined that the use of an internal comparator (zolpidem), tested on each cell in parallel to the test compound, provides a reliable approach to measure and compare the relative efficacy of PAM ligands. Patch clamp recordings on recombinant GABAA receptors, using a multiple drug addition protocol, allows us to rank PAM ligands with different levels of efficacies. We observed that fluorescence membrane potential assays are not predictive of the relative efficacies of GABAA PAM ligands.

scholarly journals Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

2020 ◽  
Vol 21 (18) ◽  
pp. 6672
Author(s):  
Zora Novakova ◽  
Nikola Belousova ◽  
Catherine A. Foss ◽  
Barbora Havlinova ◽  
Marketa Gresova ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Xenograft Model ◽  
In Vitro Assays ◽  
Experimental Therapy ◽  
Single Chain ◽  
Single Chain Fv ◽  
Murine Xenograft Model ◽  
Single Target

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

scholarly journals Formulation, High Throughput In Vitro Screening and In Vivo Functional Characterization of Nanoemulsion-Based Intranasal Vaccine Adjuvants

PLoS ONE ◽  
2015 ◽  
Vol 10 (5) ◽  
pp. e0126120 ◽  
Author(s):  
Pamela T. Wong ◽  
Pascale R. Leroueil ◽  
Douglas M. Smith ◽  
Susan Ciotti ◽  
Anna U. Bielinska ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Characterization ◽  
In Vitro Screening ◽  
Vaccine Adjuvants ◽  
Intranasal Vaccine

In Vitro and In Vivo Identification of Novel Positive Allosteric Modulators of the Human Dopamine D2 and D3 Receptor

2015 ◽  
Vol 89 (2) ◽  
pp. 303-312 ◽  
Author(s):  
Martyn Wood ◽  
Ali Ates ◽  
Veronique Marie Andre ◽  
Anne Michel ◽  
Robert Barnaby ◽  
...  
Keyword(s):  
Allosteric Modulators ◽  
D3 Receptor ◽  
Dopamine D2 ◽  
Positive Allosteric Modulators

scholarly journals Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with anIn VivoCompetition Assay

2015 ◽  
Vol 82 (4) ◽  
pp. 1050-1059 ◽  
Author(s):  
Zhao Jin ◽  
Sara C. Di Rienzi ◽  
Anders Janzon ◽  
Jeff J. Werner ◽  
Largus T. Angenent ◽  
...  
Keyword(s):  
Protein Function ◽  
Gene Diversity ◽  
Functional Characterization ◽  
Acc Deaminase ◽  
Active Site Residues ◽  
Protein Coding ◽  
Content Type ◽  
Soil Microbial ◽  
Starting Point

ABSTRACTMetagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen toEscherichia coliin vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate.

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