High-Throughput Yeast-Based Reporter Assay to Identify Compounds with Anti-inflammatory Potential.

Area of Science:

• Biochemistry and Molecular Biology
• Pharmacology and Drug Discovery
• Yeast Genetics and Cell Biology

Background:

• Altered proteostasis and inflammation are linked, highlighting the need for anti-inflammatory therapeutics for protein-misfolding and degenerative diseases.
• Current cell-based screens for anti-inflammatory compounds face challenges in establishing robust, high-throughput inflammation models.
• Yeast offers a promising platform for identifying lead compounds due to conserved biological pathways.

Purpose of the Study:

• To develop and optimize a yeast-based high-throughput screening (HTS) protocol for identifying compounds with anti-inflammatory properties.
• To leverage the homology between mammalian and yeast signaling pathways (Ca(2+)/calcineurin and NFAT/Crz1) for drug discovery.
• To establish a reliable method for validating potential anti-inflammatory hit compounds.

Main Methods:

• Utilized a recombinant yeast strain engineered with the lacZ gene under the control of Crz1-recognition elements.
• Implemented a β-galactosidase activity assay for monitoring Crz1 pathway activation, indicative of anti-inflammatory compound activity.
• Developed and optimized protocols for both HTS and validation of positive hits using alternative substrates.

Main Results:

• Successfully established a detailed protocol for HTS of compounds targeting the yeast Crz1 pathway.
• Demonstrated the utility of the yeast model for identifying compounds that modulate Crz1 activation.
• Provided a validated method for assessing anti-inflammatory potential in a high-throughput format.

Conclusions:

• The developed yeast-based screening platform is effective for identifying novel anti-inflammatory compounds.
• This HTS approach provides a scalable and robust method to address the challenge of inflammation modeling in drug discovery.
• The findings contribute to the therapeutic strategies for protein-misfolding diseases by enabling efficient identification of potential drug candidates.