High-Throughput Yeast-Based Reporter Assay to Identify Compounds with Anti-inflammatory Potential.
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 Ca(2+)/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.
Molecular Nutrition and Health, Instituto de Biologia Experimental Tecnológica, Av. da República, Estação Agronómica Nacional, Oeiras, 2780-157, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Oeiras, Portugal.
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Garcia
Molecular Nutrition and Health, Instituto de Biologia Experimental Tecnológica, Av. da República, Estação Agronómica Nacional, Oeiras, 2780-157, Portugal.|Instituto de Tecnologia Química e Biológica António Xavier, Oeiras, Portugal.
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Santos
Molecular Nutrition and Health, Instituto de Biologia Experimental Tecnológica, Av. da República, Estação Agronómica Nacional, Oeiras, 2780-157, Portugal. rmenezes@ibet.pt.|Instituto de Tecnologia Química e Biológica António Xavier, Oeiras, Portugal. rmenezes@ibet.pt.
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Menezes
D000893:Anti-Inflammatory Agents; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D015966:Gene Expression Regulation, Fungal; D057166:High-Throughput Screening Assays; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D029701:Saccharomyces cerevisiae Proteins; D019703:Calcineurin
Calcineurin; Cell-based assays; Crz1; Drug screening; Inflammation; NFAT; Yeast