Glutamate decarboxylase of the parasitic arthropods Ctenocephalides felis and Rhipicephalus microplus: Gene identification, cloning, expression, assay development, identification of inhibitors by high throughput screening and comparison with the orthologs from Drosophila melanogaster and mouse

2013 ◽  
Vol 43 (2) ◽  
pp. 162-177 ◽  
Author(s):  
Thomas Ilg ◽  
Michael Berger ◽  
Sandra Noack ◽  
Andreas Rohwer ◽  
Michael Gaßel
Keyword(s):  
Drosophila Melanogaster ◽  
High Throughput ◽  
Glutamate Decarboxylase ◽  
High Throughput Screening ◽  
Rhipicephalus Microplus ◽  
Ctenocephalides Felis ◽  
Assay Development ◽  
Gene Identification

scholarly journals Cross-Platform Bayesian Optimization System for Autonomous Biological Assay Development

2021 ◽  
Vol 26 (6) ◽  
pp. 579-590
Author(s):  
Sam Elder ◽  
Carleen Klumpp-Thomas ◽  
Adam Yasgar ◽  
Jameson Travers ◽  
Shayne Frebert ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Assay Development ◽  
Bayesian Optimization ◽  
Biological Assay ◽  
Proof Of Concept ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Cross Platform ◽  
Assay Conditions

Current high-throughput screening assay optimization is often a manual and time-consuming process, even when utilizing design-of-experiment approaches. A cross-platform, Cloud-based Bayesian optimization-based algorithm was developed as part of the National Center for Advancing Translational Sciences (NCATS) ASPIRE (A Specialized Platform for Innovative Research Exploration) Initiative to accelerate preclinical drug discovery. A cell-free assay for papain enzymatic activity was used as proof of concept for biological assay development and system operationalization. Compared with a brute-force approach that sequentially tested all 294 assay conditions to find the global optimum, the Bayesian optimization algorithm could find suitable conditions for optimal assay performance by testing 21 assay conditions on average, with up to 20 conditions being tested simultaneously, as confirmed by repeated simulation. The algorithm could achieve a sevenfold reduction in costs for lab supplies and high-throughput experimentation runtime, all while being controlled from a remote site through a secure connection. Based on this proof of concept, this technology is expected to be applied to more complex biological assays and automated chemistry reaction screening at NCATS, and should be transferable to other institutions. Graphical Abstract

scholarly journals Miniaturization of Whole Live Cell-Based GPCR Assays Using Microdispensing and Detection Systems

2004 ◽  
Vol 9 (3) ◽  
pp. 186-195 ◽  
Author(s):  
Oleg Kornienko ◽  
Raul Lacson ◽  
Priya Kunapuli ◽  
Jonathan Schneeweis ◽  
Ira Hoffman ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
G Protein Coupled Receptors ◽  
Assay Development ◽  
Functional Responses ◽  
Detection Systems ◽  
G Protein Coupled ◽  
Receptor Agonists And Antagonists ◽  
Assay Volume

Cell-based β-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 μL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC50/IC50 precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed.

scholarly journals Assay Development and Screening of Human DGAT1 Inhibitors with an LC/MS-Based Assay

2010 ◽  
Vol 15 (6) ◽  
pp. 695-702 ◽  
Author(s):  
Ji-Hu Zhang ◽  
Thomas P. Roddy ◽  
Pei-I Ho ◽  
Christopher R. Horvath ◽  
Chad Vickers ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Biological Response ◽  
Fluorescent Labeling ◽  
Assay Development ◽  
Label Free ◽  
Chemical Library ◽  
Label Free Detection

Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.

Phage Display of Functional Human TNF-α Converting Enzyme Catalytic Domain: A Rapid Method for the Production of Stabilized Proteolytic Proteins for Assay Development and High-Throughput Screening

2002 ◽  
Vol 7 (5) ◽  
pp. 433-440 ◽  
Author(s):  
Yangde Chen ◽  
Katrina Diener ◽  
Indravadan R. Patel ◽  
John K. Kawooya ◽  
Gary A. Martin ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Screening ◽  
Catalytic Domain ◽  
Assay Development ◽  
Scale Production ◽  
Converting Enzyme ◽  
Display System ◽  
Tnf Α

The catalytic domain of human tumor necrosis factor-α (TNF-α) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF-α to generate the mature TNF-α in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF-α-specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative efficacy of PDT. Both PDT and BET showed a similar specific cleavage profile against the defined substrates. Activity of the BET, however, was stable at 4 °C for less than 24 h. In contrast, the PDT exhibited remarkable stability, losing very little activity even after 2 years at 4 °C. On the basis of these results, the authors concluded that the phage display system might be a useful tool for expressing proteins that have stability issues related to auto-proteolytic activity. Furthermore, the ease and low cost of large-scale production of phage should make it suitable for assay development and HTS.

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