Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines

Hillger, Julia M.; Schoop, J.; Boomsma, Dorret I.; Slagboom, P. Eline; IJzerman, Adriaan P.; Heitman, Laura H.

doi:10.1016/j.bios.2015.06.031

Cited by 40 publications

(38 citation statements)

References 48 publications

(100 reference statements)

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“…The xCELLigence Real-Time Cell Analyzer (RTCA) system (ACEA Biosciences, San Diego, CA, USA) was employed to measure changes in cellular impedance correlating with cell spreading and tightness, thus being widely accepted as a morphological and functional biosensor of cell status [28][29][30]. Thus, 16-well E-plates (ACEA Biosciences) were coated with 50 µL fibronectin (10 µg/mL) at 37 • C for 1 h before being washed three times with 100 µL MilliQ-water before use.…”

Section: Label-free Cellular Impedance Assaymentioning

confidence: 99%

Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects

Lanznaster

Massari

Marková

et al. 2019

Cells

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Guanosine, a guanine-based purine nucleoside, has been described as a neuromodulator that exerts neuroprotective effects in animal and cellular ischemia models. However, guanosine's exact mechanism of action and molecular targets have not yet been identified. Here, we aimed to elucidate a role of adenosine receptors (ARs) in mediating guanosine effects. We investigated the neuroprotective effects of guanosine in hippocampal slices from A 2A R-deficient mice (A 2A R −/− ) subjected to oxygen/glucose deprivation (OGD). Next, we assessed guanosine binding at ARs taking advantage of a fluorescent-selective A 2A R antagonist (MRS7396) which could engage in a bioluminescence resonance energy transfer (BRET) process with NanoLuc-tagged A 2A R. Next, we evaluated functional AR activation by determining cAMP and calcium accumulation. Finally, we assessed the impact of A 1 R and A 2A R co-expression in guanosine-mediated impedance responses in living cells. Guanosine prevented the reduction of cellular viability and increased reactive oxygen species generation induced by OGD in hippocampal slices from wild-type, but not from A 2A R −/− mice. Notably, while guanosine was not able to modify MRS7396 binding to A 2A R-expressing cells, a partial blockade was observed in cells co-expressing A 1 R and A 2A R. The relevance of the A 1 R and A 2A R interaction in guanosine effects was further substantiated by means of functional assays (i.e., cAMP and calcium determinations), since guanosine only blocked A 2A R agonist-mediated effects in doubly expressing A 1 R and A 2A R cells. Interestingly, while guanosine did not affect A 1 R/A 2A R heteromer formation, it reduced A 2A R agonist-mediated cell impedance responses. Our results indicate that guanosine-induced effects may require both A 1 R and A 2A R co-expression, thus identifying a molecular substrate that may allow fine tuning of guanosine-mediated responses.

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Section: Label-free Cellular Impedance Assaymentioning

confidence: 99%

Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects

Lanznaster

Massari

Marková

et al. 2019

Cells

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“…Although they have largely been discussed in the context of sensing environmental pollutants (Aleksic et al, 2007;Belkin, 2003;Checa et al, 2012;Prindle et al, 2012), in principle, whole-cell biosensors can be designed to detect any compound of interest. Whole-cell biosensors are not limited to bacteria, but can include any type of cell such as mammalian (Auslander et al, 2014;Hillger et al, 2015), plant (Wong et al, 2015), algae (Podola and Melkonian, 2003), fungi (Weitz et al, 2002), etc.…”

Section: Introductionmentioning

confidence: 99%

Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

Goers

Ainsworth

Goey

et al. 2017

Biotech & Bioengineering

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Many high‐value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimize the productivity of these cultures it is important to monitor cellular metabolism, for example the utilization of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole‐cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity, and robustness. Importantly, our whole‐cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole‐cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalize on the wealth of genetic operons for metabolite sensing available in nature for the development of other whole‐cell biosensors. Biotechnol. Bioeng. 2017;114: 1290–1300. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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“…This technology has been used to monitor cellular signaling processes mediated by a number of GPCRs. [19][20][21][22][23] In some cases, impedance assays also facilitate the detection of unexpected characteristics/features of test compounds. 24 Here, we identified novel Gq-biased LPA1 negative allosteric modulators (NAMs) that distinctly modulate LPAinduced cellular impedance from nonbiased antagonists.…”

Section: Introductionmentioning

confidence: 99%

Discovery of Novel Gq-Biased LPA1 Negative Allosteric Modulators

Shimizu

Nakayama

2017

SLAS Discovery

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Lysophosphatidic acid (LPA) activates the G-protein-coupled receptor LPA1, which regulates various cellular processes, including cell proliferation and migration. Although LPA1 transduces cellular responses via Gq, Gi, and G12/13, associations between these signaling molecules and cellular phenotypes remain poorly characterized due to the lack of signal-specific pharmacological tools. Here, we characterized novel signal-biased modulators using multiple assays, including label-free impedance assays. LPA caused dramatic changes in cellular impedance in LPA1-expressing recombinant cells, which were susceptible to G-protein and protein kinase inhibitors. Subsequently, Gq-biased LPA1 negative allosteric modulators (NAMs) were identified using high-throughput screening, and a nonbiased antagonist differently affected the LPA-induced cellular impedance. These NAMs provide pharmacological tools for further investigations of the biology of LPA1.

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Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines

Cited by 40 publications

References 48 publications

Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects

Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects

Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production

Discovery of Novel Gq-Biased LPA1 Negative Allosteric Modulators

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