An activation-based high throughput screen identifies caspase-10 inhibitors

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Caspases are critical cysteine proteases involved in inflammation and apoptosis.
• Developing selective inhibitors for highly homologous caspases is a significant challenge.
• Targeting zymogen forms offers a strategy to overcome selectivity issues due to reduced structural homology.

Purpose of the Study:

• To establish a screening assay for discovering zymogen-directed caspase-10 selective inhibitors.
• To identify novel small molecule inhibitors of procaspase-10.
• To characterize the inhibitory activity and mechanism of identified compounds.

Main Methods:

• Engineered a tobacco etch virus (TEV)-activated caspase-10 protein for a high-activity, low-background screening assay.
• Conducted a high-throughput screen of approximately 100,000 compounds.
• Employed counter screening against TEV protease and confirmatory studies to validate inhibitors.

Main Results:

• The screening platform achieved an average Z' value of 0.58.
• Identified a class of thiadiazine-containing compounds as cysteine-reactive procaspase-10 inhibitors.
• Pifithrin-μ (PFTμ) was identified as a promiscuous caspase inhibitor, also showing preferential zymogen inhibition.

Conclusions:

• The developed screening platform is effective for identifying state-specific protease inhibitors.
• The identified thiadiazine compounds represent a promising class for procaspase-10 inhibition.
• The study highlights the potential of targeting zymogen forms for selective caspase inhibition.