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The next posters and technical sheets have been showed in different forums, conferences and meetings.

 

Multicolored fluorescent assay for biased compound screening

  • Here we show a multiplexed assay using multicolored Nomad technology and Endothelin receptor 2 to screen compound libraries. The molecular structure of Nomad Biosensors comprises: a membrane localization peptide, a second messenger transduction protein binding peptide, a reticulum retention signal and a fluorescent peptide. Nomad biosensors are normally localized in the Plasma Membrane but an increase in the second messenger concentration leads to a change in the structural folding of the Biosensor that promotes its vesicularization. In this assay it is possible to analyze both signals: calcium and AMPc concentration changes due to the receptor activity, simultaneously. In this work we have used this multiplex assay to screen a library of 480 compounds. Endothelin-1 and BQ-788 were used as agonist control and antagonist control for this model, respectively. After the screening campaign, positive compounds were chosen for further testing, based on the strength of the initial response and the lack of cytotoxicity. Our results indicate that this multiplex model is a valid strategy for drug screening.
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    GPCR regulation through arrestin pathway using Nomad technology

    • Here we show a screening of compounds for Neurokinin 1 receptor regulation using Nomad technology through arrestin pathway. The Nomad biosensor localization is in the Plasma Membrane but the GPCR activation leads to a modification in its structural folding that promotes its vesicularization. In this work we have screened a library of 600 compounds. Substance P and L733-060 compounds were used as agonist and inhibitor control for this model. After the screening campaign, positive compounds were chosen for further testing, based on the strength of the initial response and the lack of cytotoxicity. Our results indicated that this model is a valid strategy for drug screening.
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      MULTIPLEX CELL LINES – Calcium and Arrestin NOMAD- NEUROTENSIN RECEPTOR 1

      • A novel NTSR1 Nomad Mpx cell line has been developed stably transfecting red fluorescent b-arrestin Nomad biosensor, green fluorescent Ca++ Nomad biosensor and label free human neurotensin receptor 1 termed NTSR1 into U2OS cell line. Innoprot NTSR1 Nomad Mpx cell line has been designed to assay compounds or analyze their capability to modulate neurotensin receptor 1. When the agonist binds to NTSR1 a G protein is activated, which in turn, triggers a cellular response mediated by Ca++ and at the same time β-arrestin-dependent mechanisms can be also activated. Using Mpx Nomad cell lines, it is possible to monitor both activation pathways in a single assay.
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        Multicolor fluorescent biosensors for multiplexed detection of GPCR receptor activation

        • We describe a new live-cell assay that uses fluorescent biosensors co-expressed with several GPCRs for measuring the second messenger concentration changes that occurs after GPCR activation. The molecular structure of the Biosensors comprises: a membrane localization peptide, a second messenger transduction protein binding peptide, a reticulum retention signal and a fluorescent peptide. The biosensors are normally localized in the Plasma Membrane but an increase in the second messenger concentration leads to a change in the structural folding of the Biosensor that promotes its vesicularization. The second messenger transduction protein binding peptide could be replaced depending on the second messenger, resulting three different versions of the biosensors; cAMP, Calcium and DAG Nomad Biosensor. In addition, we also present here the development of multiplexed assays using the different versions of the Biosensor enabling the analysis both different intracellular signaling and receptor internalization, simultaneously.
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          Development of a new biosensor for the measurement of the intracellular second messenger levels in living cells by High-Content Imaging

          • Innoprot has developed a novel and robust living cell assay for measuring human GPCR activity using a fluorescent tGFP-tagged polypeptidic domain. This polypeptide has been cloned in U2OS cells and is used as a biosensor that allows the measurement of changes in the cellular second messenger concentration. This fusion polypeptide comprises a membrane localization peptide, a second messenger transduction protein binding peptide, a reticulum retention signal and a fluorescent peptide. The biosensor shuttles between cytoplasmic membrane to the retention vesicles upon and increase of second messengers concentration within the cell cytoplasm. Second messenger activity is easily quantified by image analysis, measuring cytoplasmic granularity changes following the changes in its concentration. There are several second messenger transduction protein binding peptide options allowing the measurement of Calcium, cAMP and DAG changes. This biosensor provides a sensitive method for high content screening of drug libraries in living cells, to identify compounds that modulate receptors that induce changes in the second messenger concentration. In this work we have used a U2OS cell line co-expressing b-Adrenergic 2 receptor and cAMP-biosensor to screen a library of 1200 compounds. Isoprotenerol was used as positive control. After the screening campaign, the results validate the use of our biosensor as a reliable strategy for drug screening..
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            Please contact us for detailed information regarding Nomad Biosensors Technology at innoprot@innoprot.com