Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro. / Souza-Silva, Igor Maciel; Peluso, A. Augusto; Mortensen, Christina; Nazarova, Antonina L.; Stage, Tore Bjerregaard; Sumners, Colin; Katritch, Vsevolod; Steckelings, U. Muscha.

In: Peptides, Vol. 172, 171137, 2024.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Souza-Silva, IM, Peluso, AA, Mortensen, C, Nazarova, AL, Stage, TB, Sumners, C, Katritch, V & Steckelings, UM 2024, 'Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro', Peptides, vol. 172, 171137. https://doi.org/10.1016/j.peptides.2023.171137

APA

Souza-Silva, I. M., Peluso, A. A., Mortensen, C., Nazarova, A. L., Stage, T. B., Sumners, C., Katritch, V., & Steckelings, U. M. (2024). Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro. Peptides, 172, [171137]. https://doi.org/10.1016/j.peptides.2023.171137

Vancouver

Souza-Silva IM, Peluso AA, Mortensen C, Nazarova AL, Stage TB, Sumners C et al. Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro. Peptides. 2024;172. 171137. https://doi.org/10.1016/j.peptides.2023.171137

Author

Souza-Silva, Igor Maciel ; Peluso, A. Augusto ; Mortensen, Christina ; Nazarova, Antonina L. ; Stage, Tore Bjerregaard ; Sumners, Colin ; Katritch, Vsevolod ; Steckelings, U. Muscha. / Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro. In: Peptides. 2024 ; Vol. 172.

Bibtex

@article{7e6e6ef813f3491ab53c9ee6c836fbe2,
title = "Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro",
abstract = "Angiotensin AT2-receptor (AT2R) agonists have shown a wide range of protective effects in many preclinical disease models. However, the availability of AT2R-agonists is very limited due to the lack of high-throughput assays for AT2R-agonist identification. Therefore, we aimed to design and validate an assay for high-throughput screening of AT2R-agonist candidates. The assay is based on nitric oxide (NO) release measurements in primary human aortic endothelial cells (HAEC), in AT2R-transfected CHO cells (AT2R-CHO) or in non-transfected CHO cells (Flp-CHO) using the fluorescent probe DAF-FM diacetate. It is run in 96-well plates and fluorescence signals are semi-automatically quantified. The assay was tested for sensitivity (recognition of true positive results), selectivity (recognition of true negative results), and reliability (by calculating the repeatability coefficient (RC)). The high-throughput, semi-automated method was proven suitable, as the NO-releasing agents C21, CGP42112A, angiotensin-(1−7) and acetylcholine significantly increased NO release from HAEC. The assay is sensitive and selective, since the established AT2R-agonists C21, CGP42112A and angiotensin II significantly increased NO release from AT2R-CHO cells, while the non-AT2R-agonists angiotensin-(1−7) and acetylcholine had no effect. Assay reliability was shown by high-throughput screening of a library comprised of 40 potential AT2R-agonists, of which 39 met our requirements for reliability (RC ≤ 20% different from RC for C21). Our newly developed high-throughput method for detection of AT2R-agonistic activity was proven to be sensitive, selective, and reliable. This method is suitable for the screening of potential AT2R-agonists in future drug development programs.",
keywords = "Angiotensin AT-receptor, Drug development, High-throughput screening, Nitric oxide",
author = "Souza-Silva, {Igor Maciel} and Peluso, {A. Augusto} and Christina Mortensen and Nazarova, {Antonina L.} and Stage, {Tore Bjerregaard} and Colin Sumners and Vsevolod Katritch and Steckelings, {U. Muscha}",
note = "Publisher Copyright: {\textcopyright} 2024",
year = "2024",
doi = "10.1016/j.peptides.2023.171137",
language = "English",
volume = "172",
journal = "Peptides",
issn = "0196-9781",
publisher = "Elsevier",

}

RIS

TY - JOUR

T1 - Development of an automated, high-throughput assay to detect angiotensin AT2-receptor agonistic compounds by nitric oxide measurements in vitro

AU - Souza-Silva, Igor Maciel

AU - Peluso, A. Augusto

AU - Mortensen, Christina

AU - Nazarova, Antonina L.

AU - Stage, Tore Bjerregaard

AU - Sumners, Colin

AU - Katritch, Vsevolod

AU - Steckelings, U. Muscha

N1 - Publisher Copyright: © 2024

PY - 2024

Y1 - 2024

N2 - Angiotensin AT2-receptor (AT2R) agonists have shown a wide range of protective effects in many preclinical disease models. However, the availability of AT2R-agonists is very limited due to the lack of high-throughput assays for AT2R-agonist identification. Therefore, we aimed to design and validate an assay for high-throughput screening of AT2R-agonist candidates. The assay is based on nitric oxide (NO) release measurements in primary human aortic endothelial cells (HAEC), in AT2R-transfected CHO cells (AT2R-CHO) or in non-transfected CHO cells (Flp-CHO) using the fluorescent probe DAF-FM diacetate. It is run in 96-well plates and fluorescence signals are semi-automatically quantified. The assay was tested for sensitivity (recognition of true positive results), selectivity (recognition of true negative results), and reliability (by calculating the repeatability coefficient (RC)). The high-throughput, semi-automated method was proven suitable, as the NO-releasing agents C21, CGP42112A, angiotensin-(1−7) and acetylcholine significantly increased NO release from HAEC. The assay is sensitive and selective, since the established AT2R-agonists C21, CGP42112A and angiotensin II significantly increased NO release from AT2R-CHO cells, while the non-AT2R-agonists angiotensin-(1−7) and acetylcholine had no effect. Assay reliability was shown by high-throughput screening of a library comprised of 40 potential AT2R-agonists, of which 39 met our requirements for reliability (RC ≤ 20% different from RC for C21). Our newly developed high-throughput method for detection of AT2R-agonistic activity was proven to be sensitive, selective, and reliable. This method is suitable for the screening of potential AT2R-agonists in future drug development programs.

AB - Angiotensin AT2-receptor (AT2R) agonists have shown a wide range of protective effects in many preclinical disease models. However, the availability of AT2R-agonists is very limited due to the lack of high-throughput assays for AT2R-agonist identification. Therefore, we aimed to design and validate an assay for high-throughput screening of AT2R-agonist candidates. The assay is based on nitric oxide (NO) release measurements in primary human aortic endothelial cells (HAEC), in AT2R-transfected CHO cells (AT2R-CHO) or in non-transfected CHO cells (Flp-CHO) using the fluorescent probe DAF-FM diacetate. It is run in 96-well plates and fluorescence signals are semi-automatically quantified. The assay was tested for sensitivity (recognition of true positive results), selectivity (recognition of true negative results), and reliability (by calculating the repeatability coefficient (RC)). The high-throughput, semi-automated method was proven suitable, as the NO-releasing agents C21, CGP42112A, angiotensin-(1−7) and acetylcholine significantly increased NO release from HAEC. The assay is sensitive and selective, since the established AT2R-agonists C21, CGP42112A and angiotensin II significantly increased NO release from AT2R-CHO cells, while the non-AT2R-agonists angiotensin-(1−7) and acetylcholine had no effect. Assay reliability was shown by high-throughput screening of a library comprised of 40 potential AT2R-agonists, of which 39 met our requirements for reliability (RC ≤ 20% different from RC for C21). Our newly developed high-throughput method for detection of AT2R-agonistic activity was proven to be sensitive, selective, and reliable. This method is suitable for the screening of potential AT2R-agonists in future drug development programs.

KW - Angiotensin AT-receptor

KW - Drug development

KW - High-throughput screening

KW - Nitric oxide

U2 - 10.1016/j.peptides.2023.171137

DO - 10.1016/j.peptides.2023.171137

M3 - Journal article

C2 - 38142816

AN - SCOPUS:85181837972

VL - 172

JO - Peptides

JF - Peptides

SN - 0196-9781

M1 - 171137

ER -

ID: 380695704