Abstract

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 mess-enger transduction protein binding peptide, a reticulum retention signal and a fluorescent pepti-de. 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 promo-tes its vesicularization. The second messenger transduction protein binding peptide could be re-placed 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 diffe-rent intracellular signaling and receptor internalization, simultaneously.

Figure 2. cAMP Nomad biosensor subcellular localization and dose-response curve of the positive control Isoproterenol using A2R-cAMP

Nomad cell line. To determine the EC50 value for Isoproterenol, cells were treated with a range of concentrations from 10 M to 0.1 nM during 24h. Data from measurements were fitted to a sigmoid 4-parameter fit logistic model (sigmoidal) with Sigma Plot 9 software. Error bars represent the standard deviation among 3 replicate wells. EC50 for Isoproterenol was 2,3.10-7 M and Z’ factor for this experiment was 0.70 +/-0.01.

Multicolor fluorescent biosensors for multiplexed detection of GPCR receptor activationC. Salado, R Mella, D. Kortazar, M. Roura-Ferrer and P. Villacé

Innoprot , Parque tecnológico de Bizkaia, Edf. 502-1º, Bilbao, Spain www.innoprot.com

Figure 1. Schematic representation of the Nomad biosensor functioning in living cells. Nomad biosensor is a fluorescent fusion polypeptide capable of changing its localization within the cell from the cell cytoplasmic membrane to the retention vesicles, upon an increase in the concentration of second messengers within the cell cytoplasm. A second messenger concentration increase leads to the Nomad biosensor spatial folding conversion that promotes the cellular localization change. High-content screening (HCS) in Nomad systems uses living cells as a tool in biological research to discover and optimize new drug candidates.

Methods and MaterialsMethods and MaterialsCultured cells: Cultured cells:

U2OS based model cells were cultured into 96 wells Imaging Plates BD at 15.10U2OS based model cells were cultured into 96 wells Imaging Plates BD at 15.103 cells/well in 200 cells/well in 200 l of DMEM F12 10% FBS and incubated at 37ºC and 5% CO2. After 16 l of DMEM F12 10% FBS and incubated at 37ºC and 5% CO2. After 16 hours, the cells were treated in OptiMEM and incubated with the compounds 24 hours.hours, the cells were treated in OptiMEM and incubated with the compounds 24 hours.Image acquisition:Image acquisition:

After the treatment, the nucleus was stained with DAPI and image adquisition was performed with a BD Pathway 855. Redistribution of the fluorescence was detected After the treatment, the nucleus was stained with DAPI and image adquisition was performed with a BD Pathway 855. Redistribution of the fluorescence was detected using image analysis algorithms. using image analysis algorithms.

ConclusionsConclusions

*Nomad is a fluorescent biosensor platform that covers the main GPCR signaling pathways, works in *Nomad is a fluorescent biosensor platform that covers the main GPCR signaling pathways, works in

living cells and provide accurate quantitative results.living cells and provide accurate quantitative results.

*Nomad biosensor provides a robust and homogeneous assay that is amenable to High Contect *Nomad biosensor provides a robust and homogeneous assay that is amenable to High Contect

Screening with high Z’ values.Screening with high Z’ values.

*Nomad cell-based assays require no additional reagents. The fluorescence signal can be detected on *Nomad cell-based assays require no additional reagents. The fluorescence signal can be detected on

any standard imaging system and the results can be obtained easily using images analysis algorithms.any standard imaging system and the results can be obtained easily using images analysis algorithms.

*Nomad technology can be multiplexed for simultaneous measurement of second messengers of *Nomad technology can be multiplexed for simultaneous measurement of second messengers of

different signaling pathways and tagged receptors.different signaling pathways and tagged receptors.

Figure 5. Screening of 890

compounds library using the

A2R-AMPcNomad cell line.

Representative data of vesicle number per cell normalized to control. Negative control (DMSO) is represented in white and positive control (Isoproterenol 10 μM) in green. In yellow is represented the compounds selected as hits. The selection of hits was done using a cutoff of “Mean + 3*SD”. 19 compounds were considered as hit giving a hit rate of 2.14 %.

Figure 4. DAG Nomad biosensor subcellular localization and dose-response curve for Oxotremorine in M5-DAG Nomad cell line. Cells were treated with 9 log dilution series (n=5). The Ec50 for Oxotremorine was 5.01x10-8M after a treatment of 24 h with the agonist. Cells were fixed and the nuclei were stained with DAPI. % Activity was calculated relative to positive (100μM). The internalization assay was validated with an average of Z´=0.72+/- 0.02 for High Content Screening.

Figure 3. Calcium Nomad biosensor subcellular localization and dose-response curve of the positive control Neurotesin using NTSR1-Calcium

Nomad cell line. Cells were treated with 9 log dilution series (n=5). The EC50 for the Neurotensin was ˜ 2.15x10-12M after a treatment of 3 h with the agonist. Cells were fixed and the nuclei were stained with DAPI. % Activity was calculated relative to positive (10nM). The internalization assay was validated with an average of Z´=0.93+/- 0.02 for High Content Screening.

Fig6. Multiplex cellular distribution of TACR3 combinated with Calcium Nomad biosensor and dose-response curve for Substance P . Cells were treated with 12 log dilution series (n=6). After a treatment of 24 h with the agonis, cells were fixed and the nuclei were stained with DAPI. % Activity was calculated relative to positive (10μM). Thereceptor internalization assay was validated with an average of Z´=0.8 +/- 0.01 for High Content Screening. The calcium Nomad biosensor assay was validated with an average of Z´of 0.7 +/- 0.01 for High Content Screening.