UCLA researchers in the Department of Biological Chemistry have developed a high throughput screening platform to identify state-specific protease inhibitors, enabling identification of two inhibitor classes that preferentially show zymogen-directed caspase-10 inhibition.
BACKGROUND: Caspases are a conserved family of cysteine proteases that play important roles in apoptosis and inflammation. Because this family of enzymes is highly homologous, current small-molecule inhibitors often lack isoform specificity, limiting their utility as research tools. Emerging studies suggest that precursor (zymogen) caspases exhibit greater structural divergence, offering an underexploited opportunity for selective inhibitor design. Thus, there is a need for scalable assays optimized for discovering inhibitors that preferentially target caspase zymogens rather than active proteases.
INNOVATION: UCLA researchers have engineered a low-background, high-activity caspase-10 protein that can be selectively activated by the tobacco etch virus (TEV) protease. This enables sensitive detection of compounds that inhibit the zymogen form of caspase-10. Researchers performed a 100,000-compound high throughput screen with robust performance. They additionally screened against TEV protease to differentiate true procaspase-10 inhibitors. This screen yielded two inhibitor classes with preferential zymogen binding, including a known small molecule p53 inhibitor that was not previously known to function as a caspase inhibitor. This screening platform represents a valuable technology to identify state-specific protease inhibitors in widespread applications.
POTENTIAL APPLICATIONS:
- Development of selective inhibitors for zymogen form caspases
- Tool discovery for studying apoptosis, inflammation, etc.
- Extension to other zymogen-directed protease targets
ADVANTAGES:
- Platform is engineered specifically to select for procaspase-selective inhibitors
- Robust performance for high throughput screens
- Revealed previously unknown caspase inhibitory properties of existing molecules
- Generalizable approach for other caspases or proteases
DEVELOPMENT-TO-DATE: UCLA researchers have conducted an in vitro high throughput screen using the engineered TEV-activatable procaspase-10 construct. They conducted further biochemical assays to confirm procaspase-10-specific inhibition and identify a class of low-PH activatable caspase-10 inhibitors.
Related Papers (from the inventors only): Castellón JO, Yuen C, Han B, Andrews KH, Ofori S, Julio AR, Boatner LM, Palafox MF, Perumal N, Damoiseaux R, Backus KM. An activation-based high throughput screen identifies caspase-10 inhibitors. RSC Chem Biol. 2025 Feb 4;6(4):604-617. doi: 10.1039/d5cb00017c. PMID: 40013156; PMCID: PMC11854450.
Keywords: High throughput screen, caspase, zymogen, tobacco etch virus, small molecule inhibitors, procaspase inhibition, selective inhibition, homology, screening platform, drug discovery