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

Igor Maciel Souza-Silva, A. Augusto Peluso, Christina Mortensen, Antonina L. Nazarova, Tore Bjerregaard Stage, Colin Sumners, Vsevolod Katritch, U. Muscha Steckelings*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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.

Original languageEnglish
Article number171137
JournalPeptides
Volume172
Number of pages7
ISSN0196-9781
DOIs
Publication statusPublished - 2024

Bibliographical note

Publisher Copyright:
© 2024

Keywords

  • Angiotensin AT-receptor
  • Drug development
  • High-throughput screening
  • Nitric oxide

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