scholarly journals Mapping Out Receptors: High Throughput Screening of Endothelial Monocyte Activating Polypeptide II (EMAPII) Using PRESTO-TANGO

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Nathan Lam ◽  
Bernhard Maier ◽  
Sarvesh Chelvanambi ◽  
Takashi Hato ◽  
Matthias Clauss
Keyword(s):  
Colorectal Cancer ◽  
Cardiovascular Disease ◽  
Receptor Binding ◽  
High Throughput ◽  
High Throughput Screening ◽  
Neutralizing Antibodies ◽  
Signal To Noise Ratio ◽  
Receptor System ◽  
Gpcr Activation ◽  
Cxcr3 Ligand

Background: Secreted endothelial monocyte activating polypeptide II (EMAPII/AIMP1) is a pro-apoptotic, pro-inflammatory ligand implicated in diseases such as colorectal cancer, cardiovascular disease, and emphysema. Thus, EMAPII has been shown to induce apoptosis through CXCR3 receptor binding. However, not all EMAPII functions have been attributed to CXCR3. Discovery of new receptors interacting with EMAPII could lead to development of new therapies blocking cognate ligand-receptor binding. We hypothesize the existence of at least one unknown secondary receptor for EMAPII.  Methods: The PRESTO-TANGO assay, a construct which converts G-protein coupled receptor (GPCR) ligand binding into luciferase activity measurable by luminometer, was validated using transfection with TANGO-modified CXCR3 and S1P1R as test receptors in HTLA cells. Protocols for cell transfection, adherence, and cultivation were optimized with IP10, EMAPII, and S1P as test ligands.  Results: The assay was successfully validated using several GPCR activation readouts. Binding curves were generated for S1P/S1P1 (EC50= 569nM), IP10/CXCR3 (EC50= 47.1 nM), and EMAPII/CXCR3 (EC50= 628 nM). Conditions for the PRESTO-TANGO assay were further refined for maximal signal-to-noise ratio and robust inter-assay reproducibility in preparation for high-throughput screening. We are currently testing 314 TANGO-modified GPCRs for EMAPII affinity.  Conclusion: We have validated the Tango assay for the known EMAPII-CXCR3 ligand-receptor system, a valuable tool for evaluation of anti-EMAPII therapeutics. Discovery of a novel EMAPII receptor would allow for the development of therapies including neutralizing antibodies (analogous to the PD1 receptor antibody Pembrolizumab for solid tumors) in diseases such as colorectal cancer, cardiovascular disease, and emphysema.

scholarly journals A Homogeneous Enzyme Fragment Complementation-Based β-Arrestin Translocation Assay for High-Throughput Screening of G-Protein-Coupled Receptors

2008 ◽  
Vol 13 (8) ◽  
pp. 737-747 ◽  
Author(s):  
Xiaoning Zhao ◽  
Adrie Jones ◽  
Keith R. Olson ◽  
Kun Peng ◽  
Tom Wehrman ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Somatostatin Receptor ◽  
Gene Families ◽  
G Protein Coupled Receptors ◽  
Functional Assay ◽  
Gpcr Activation ◽  
Fragment Complementation ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized α fragment peptide (termed ProLink™) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Gαi-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. ( Journal of Biomolecular Screening 2008:737-747)

Development of a High Throughput Screening Assay for Inhibitors of Fibroblast Growth Factor-Receptor-Heparin Interactions

2001 ◽  
Vol 6 (3) ◽  
pp. 171-177 ◽  
Author(s):  
David Aviezer ◽  
Andrew P. Seddon ◽  
Mary Jo Wildey ◽  
Peter Böhlen ◽  
Avner Yayon
Keyword(s):  
Alkaline Phosphatase ◽  
Growth Factors ◽  
Growth Factor ◽  
Tumor Progression ◽  
Receptor Binding ◽  
High Throughput ◽  
High Throughput Screening ◽  
Growth Factor Receptor ◽  
Fibroblast Growth ◽  
Fgf Receptor

High throughput screening (HTS) of large compound libraries for inhibitors of growth factors raises the requirement for simple yet reliable assays. Fibroblast growth factors (FGFs) play a pivotal role in the multistep pathway of malignant transformation, tumor progression, metastasis, and angiogenesis. FGF-2 (basic FGF) requires a cooperative interaction with heparin or heparan sulfate proteoglycans in order to form functional growth factor-receptor complexes that are essential for receptor binding and activation. We have developed a simple screening system, devised to identify molecules that modulate heparin-FGF-receptor interactions. The system is composed of a heparin matrix, FGF-2, and a FGF receptor-1 protein engineered by genetically fusing the extracellular domain of FGF receptor-1 to alkaline phosphatase (FRAP). The screen is conducted using 96-well plates to which heparin has been covalently attached. FGF-2 is then bound to the plates through heparin-FGF interactions, followed by the addition of FRAP and compounds to be screened for modulation of heparin-FGF, receptor-heparin, and receptor-FGF interactions. The endpoint of the assay is measured enzymatically using the alkaline phosphatase (AP)-catalyzed formation of a chromogenic product, which is directly proportional to the amount of FRAP present on the plates as a heparin-FGF-FRAP ternary complex. Reduced AP values relative to control, as measured by spectrophotometry, indicate inhibition of the formation of an active FGF-receptor-heparin complex. The simple and versatile nature of the assay makes it an attractive HTS system. The screen has identified several potent inhibitors of FGF-2 receptor binding and activation. Furthermore, secondary screening of the HTS-recognized compounds identified several compounds that have the capacity to block growth factor-mediated tumor progression and angiogenesis in vivo.

scholarly journals Searching for Chemokine Receptor Binding Antagonists by High Throughput Screening

1997 ◽  
Vol 2 (3) ◽  
pp. 153-157 ◽  
Author(s):  
Geoffrey W. Mellor ◽  
Simon J. Fogarty ◽  
M. Shane O'Brien ◽  
Miles Congreve ◽  
Martyn N. Banks ◽  
...  
Keyword(s):  
Receptor Binding ◽  
High Throughput ◽  
Chemokine Receptor ◽  
Combinatorial Chemistry ◽  
High Throughput Screening ◽  
Biological Targets ◽  
Drug Candidates ◽  
Lead Compounds ◽  
Selective Antagonists ◽  
Biological Sources

Identification of putative drug candidates by high throughput screening is assuming enormous importance within the pharmaceutical industry, driven by increasing numbers of valid therapeutic targets from both classical and molecular biological sources. Screening is an applied discipline that requires equipment and, more importantly, thinking that is fundamentally different from more traditional, lower throughput assay methodology. This article describes the process as applied to the discovery of selective antagonists of three chemokine receptor binding systems, from the original biological targets to chemically prosecutable lead compounds, which are currently being investigated using traditional medicinal and combinatorial chemistry methods.

scholarly journals Improved Green Fluorescent Protein Reporter Gene-Based Microplate Screening for Antituberculosis Compounds by Utilizing an Acetamidase Promoter

2003 ◽  
Vol 47 (12) ◽  
pp. 3682-3687 ◽  
Author(s):  
Chartchai Changsen ◽  
Scott G. Franzblau ◽  
Prasit Palittapongarnpim
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antimicrobial Agents ◽  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Enhanced Fluorescence ◽  
Signal To Noise ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

ABSTRACT The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc2155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.

scholarly journals High-throughput screening in colorectal cancer tissue-originated spheroids

2018 ◽  
Vol 110 (1) ◽  
pp. 345-355 ◽  
Author(s):  
Jumpei Kondo ◽  
Tomoya Ekawa ◽  
Hiroko Endo ◽  
Kanami Yamazaki ◽  
Norio Tanaka ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cancer Tissue ◽  
Colorectal Cancer Tissue

scholarly journals Monitoring Parallel Robotic Cultivations with Online Multivariate Analysis

Processes ◽  
2020 ◽  
Vol 8 (5) ◽  
pp. 582 ◽  
Author(s):  
Sebastian Hans ◽  
Christian Ulmer ◽  
Harini Narayanan ◽  
Trygve Brautaset ◽  
Niels Krausch ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Signal To Noise Ratio ◽  
Model Organism ◽  
Principal Component ◽  
Optimal Operation ◽  
Aeration System ◽  
Microbial Screening ◽  
Good Signal

In conditional microbial screening, a limited number of candidate strains are tested at different conditions searching for the optimal operation strategy in production (e.g., temperature and pH shifts, media composition as well as feeding and induction strategies). To achieve this, cultivation volumes of >10 mL and advanced control schemes are required to allow appropriate sampling and analyses. Operations become even more complex when the analytical methods are integrated into the robot facility. Among other multivariate data analysis methods, principal component analysis (PCA) techniques have especially gained popularity in high throughput screening. However, an important issue specific to high throughput bioprocess development is the lack of so-called golden batches that could be used as a basis for multivariate analysis. In this study, we establish and present a program to monitor dynamic parallel cultivations in a high throughput facility. PCA was used for process monitoring and automated fault detection of 24 parallel running experiments using recombinant E. coli cells expressing three different fluorescence proteins as the model organism. This approach allowed for capturing events like stirrer failures and blockage of the aeration system and provided a good signal to noise ratio. The developed application can be easily integrated in existing data- and device-infrastructures, allowing automated and remote monitoring of parallel bioreactor systems.

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