Development of an automated, high-throughput assay to detect angiotensin AT 2 -receptor agonistic compounds by nitric oxide measurements in vitro.

Autor: Souza-Silva IM; Institute for Molecular Medicine, Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark., Peluso AA; Institute for Molecular Medicine, Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark; Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark., Mortensen C; Department of Public Health, University of Southern Denmark, Odense, Denmark., Nazarova AL; Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA; Center for New Technologies in Drug Discovery and Development, Bridge Institute, Michelson Center for Convergent Biosciences, University of Southern California, Los Angeles, CA, USA., Stage TB; Department of Public Health, University of Southern Denmark, Odense, Denmark., Sumners C; Department of Physiology and Aging, University of Florida, Gainesville, USA., Katritch V; Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA; Center for New Technologies in Drug Discovery and Development, Bridge Institute, Michelson Center for Convergent Biosciences, University of Southern California, Los Angeles, CA, USA; Department of Chemistry, University of Southern California, Los Angeles, CA, USA., Steckelings UM; Institute for Molecular Medicine, Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark. Electronic address: usteckelings@health.sdu.dk.
Jazyk: angličtina
Zdroj: Peptides [Peptides] 2024 Feb; Vol. 172, pp. 171137. Date of Electronic Publication: 2023 Dec 22.
DOI: 10.1016/j.peptides.2023.171137
Abstrakt: Angiotensin AT 2 -receptor (AT 2 R) agonists have shown a wide range of protective effects in many preclinical disease models. However, the availability of AT 2 R-agonists is very limited due to the lack of high-throughput assays for AT 2 R-agonist identification. Therefore, we aimed to design and validate an assay for high-throughput screening of AT 2 R-agonist candidates. The assay is based on nitric oxide (NO) release measurements in primary human aortic endothelial cells (HAEC), in AT 2 R-transfected CHO cells (AT 2 R-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 AT 2 R-agonists C21, CGP42112A and angiotensin II significantly increased NO release from AT 2 R-CHO cells, while the non-AT 2 R-agonists angiotensin-(1-7) and acetylcholine had no effect. Assay reliability was shown by high-throughput screening of a library comprised of 40 potential AT 2 R-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 AT 2 R-agonistic activity was proven to be sensitive, selective, and reliable. This method is suitable for the screening of potential AT 2 R-agonists in future drug development programs.
Competing Interests: Declaration of interest The authors report no conflict of interest.
(Copyright © 2024. Published by Elsevier Inc.)
Databáze: MEDLINE
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