High-throughput yeast-based reporter assay to identify compounds with anti-inflammatory potential

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Abstract

The association between altered proteostasis and inflammatory responses has been increasingly recognized, therefore the identification and characterization of novel compounds with anti-inflammatory potential will certainly have a great impact in the therapeutics of protein-misfolding diseases such as degenerative disorders. Although cell-based screens are powerful approaches to identify potential therapeutic compounds, establishing robust inflammation models amenable to high-throughput screening remains a challenge. To bridge this gap, we have exploited the use of yeasts as a platform to identify lead compounds with anti-inflammatory properties. The yeast cell model described here relies on the high-degree homology between mammalian and yeast Ca2+/calcineurin pathways converging into the activation of NFAT and Crz1 orthologous proteins, respectively. It consists of a recombinant yeast strain encoding the lacZ gene under the control of Crz1-recongition elements to facilitate the identification of compounds interfering with Crz1 activation through the easy monitoring of β-galactosidase activity. Here, we describe in detail a protocol optimized for high-throughput screening of compounds with potential anti-inflammatory activity as well as a protocol to validate the positive hits using an alternative β-galactosidase substrate.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages441-452
Number of pages12
Volume1449
DOIs
Publication statusPublished - 1 Sep 2016

Publication series

NameMethods in Molecular Biology
Volume1449
ISSN (Print)1064-3745

Keywords

  • Calcineurin
  • Cell-based assays
  • Crz1
  • Drug screening
  • Inflammation
  • NFAT
  • Yeast

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  • Cite this

    Garcia, G., do Santos, C. N., & Menezes, R. (2016). High-throughput yeast-based reporter assay to identify compounds with anti-inflammatory potential. In Methods in Molecular Biology (Vol. 1449, pp. 441-452). (Methods in Molecular Biology; Vol. 1449). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-3756-1_29