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A Comparative Study of Impedance versus Optical Label-Free Systems Relative to Labelled Assays in a Predominantly Gi Coupled GPCR (C5aR) Signalling.

Halai R, Croker DE, Suen JY, Fairlie DP, Cooper MA - Biosensors (Basel) (2012)

Bottom Line: Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR.The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells.However, label-free read outs gave consistently lower potency values in both native and transfected cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. r.halai@uq.edu.au.

ABSTRACT
Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays.

No MeSH data available.


Three dose representative label-free profiles for C5a (red, blue and green) and baseline (black) in human monocyte-derived macrophages (HMDM) and CHO-C5aR cells on the EPIC® and xCELLigence. The average of triplicates with SEM is shown. For CHO-C5aR cells, red is 250 nM, green is 83 nM and blue is 28 nM. For HMDM, red is 1 µM, green is 0.3 µM and blue is 0.1 µM. Arrows indicate peaks that may be indicative of different signalling pathways. Black star indicates point of compound addition. Question mark indicates ambiguous, but dose-dependent response.
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biosensors-02-00273-f002: Three dose representative label-free profiles for C5a (red, blue and green) and baseline (black) in human monocyte-derived macrophages (HMDM) and CHO-C5aR cells on the EPIC® and xCELLigence. The average of triplicates with SEM is shown. For CHO-C5aR cells, red is 250 nM, green is 83 nM and blue is 28 nM. For HMDM, red is 1 µM, green is 0.3 µM and blue is 0.1 µM. Arrows indicate peaks that may be indicative of different signalling pathways. Black star indicates point of compound addition. Question mark indicates ambiguous, but dose-dependent response.

Mentions: HMDM were seeded, at 50,000 cells/well and CHO-C5aR and CHO-K1 at 7,500 cells/well, into fibronectin-coated cell-based EPIC® plates (Corning) and incubated overnight. Prior to ligand addition, media was exchanged for HBSS (Invitrogen Life Technologies): 20 mM HEPES (Sigma Aldrich) buffer, supplemented with 0.5% DMSO, and allowed to equilibrate at 26 °C for 1 h (HMDM) or 2 h (CHO-C5aR and CHO-K1) within the instrument. Agonists were prepared at a final DMSO concentration of 0.5% in HBSS:HEPES buffer. After ligand addition, measurements were taken continuously for 1–2 h at 26 °C. Peak amplitude response (Figure 2) was measured for data analysis using Corning EPIC® software.


A Comparative Study of Impedance versus Optical Label-Free Systems Relative to Labelled Assays in a Predominantly Gi Coupled GPCR (C5aR) Signalling.

Halai R, Croker DE, Suen JY, Fairlie DP, Cooper MA - Biosensors (Basel) (2012)

Three dose representative label-free profiles for C5a (red, blue and green) and baseline (black) in human monocyte-derived macrophages (HMDM) and CHO-C5aR cells on the EPIC® and xCELLigence. The average of triplicates with SEM is shown. For CHO-C5aR cells, red is 250 nM, green is 83 nM and blue is 28 nM. For HMDM, red is 1 µM, green is 0.3 µM and blue is 0.1 µM. Arrows indicate peaks that may be indicative of different signalling pathways. Black star indicates point of compound addition. Question mark indicates ambiguous, but dose-dependent response.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263554&req=5

biosensors-02-00273-f002: Three dose representative label-free profiles for C5a (red, blue and green) and baseline (black) in human monocyte-derived macrophages (HMDM) and CHO-C5aR cells on the EPIC® and xCELLigence. The average of triplicates with SEM is shown. For CHO-C5aR cells, red is 250 nM, green is 83 nM and blue is 28 nM. For HMDM, red is 1 µM, green is 0.3 µM and blue is 0.1 µM. Arrows indicate peaks that may be indicative of different signalling pathways. Black star indicates point of compound addition. Question mark indicates ambiguous, but dose-dependent response.
Mentions: HMDM were seeded, at 50,000 cells/well and CHO-C5aR and CHO-K1 at 7,500 cells/well, into fibronectin-coated cell-based EPIC® plates (Corning) and incubated overnight. Prior to ligand addition, media was exchanged for HBSS (Invitrogen Life Technologies): 20 mM HEPES (Sigma Aldrich) buffer, supplemented with 0.5% DMSO, and allowed to equilibrate at 26 °C for 1 h (HMDM) or 2 h (CHO-C5aR and CHO-K1) within the instrument. Agonists were prepared at a final DMSO concentration of 0.5% in HBSS:HEPES buffer. After ligand addition, measurements were taken continuously for 1–2 h at 26 °C. Peak amplitude response (Figure 2) was measured for data analysis using Corning EPIC® software.

Bottom Line: Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR.The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells.However, label-free read outs gave consistently lower potency values in both native and transfected cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. r.halai@uq.edu.au.

ABSTRACT
Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays.

No MeSH data available.