Two low complexity ultra-high throughput methods to identify diverse chemically bioactive molecules using...

Area of Science:

• Biochemistry
• Molecular Biology
• Yeast Genetics

Background:

• Budding yeast (Saccharomyces cerevisiae) is a crucial eukaryotic model for understanding how small molecules work.
• Developing efficient screening methods is vital for identifying novel bioactive compounds.

Purpose of the Study:

• To create and validate two high throughput screening (HTS) assays for assessing cell viability in S. cerevisiae.
• To screen a large library of low molecular weight compounds and identify potential hits.

Main Methods:

• Development of two HTS methods: one using alamarBlue® (resazurin) reduction and another using direct optical measurement of cell growth.
• Miniaturization of assays for 384- and 1536-well formats.
• Screening of approximately 1.1 million compounds and subsequent genome-wide haploinsufficiency profiling (HIP).

Main Results:

• The alamarBlue® assay demonstrated high performance (Z' >0.7, signal to basal ratio >6.5).
• Approximately 25,000 primary hits were identified from the compound library.
• 572 hits showed diverse mechanisms of action in HIP, impacting over 25% of yeast strains.

Conclusions:

• The developed HTS methods are robust and scalable for large-scale compound screening in yeast.
• The screening identified a significant number of compounds with potential therapeutic applications.
• These compounds represent valuable tools for further research into cellular mechanisms and drug discovery.