Automated BRET-based screening platform for fungal ATG4 inhibitor discovery and fungicide development
An automated high-throughput screening platform that identifies small-molecule inhibitors of fungal ATG4, a cysteine protease essential for autophagy in plant pathogens such as Fusarium oxysporum. The BRET-based assay converts ATG4-mediated ATG8 cleavage into a quantitative signal, enabling mechanism-based discovery of novel fungicide chemistries targeting an underexploited pathway.
Overview
This platform enables discovery of a new class of fungicides by targeting fungal autophagy, a cellular recycling process essential for the growth, development, and pathogenicity of plant pathogens such as Fusarium oxysporum. F. oxysporum is a globally significant soilborne pathogen responsible for vascular wilt and root diseases in economically important crops. The platform screens chemical libraries for small-molecule inhibitors of ATG4, a conserved cysteine protease that processes ATG8 proteins required for autophagosome formation. By disrupting this process, candidate compounds aim to inhibit fungal growth and reduce pathogenicity, offering a novel, mechanism-based approach for crop protection.
Technical specifications
Core technology:
- BRET-based assay: Active ATG4 cleaves an ATG8-based Bioluminescence Resonance Energy Transfer (BRET) substrate, separating donor and acceptor molecules and reducing the BRET ratio, providing a quantitative, mechanism-based readout of ATG4 activity
- Automation: Integration with automated liquid handling enables reproducible, high-throughput screening of large chemical libraries
- Hit prioritization: Compounds are prioritized based on BRET response, concentration dependence, and reproducibility
- Validation workflow: Confirmed hits undergo concentration-response assays and orthogonal biochemical approaches to eliminate nonspecific artifacts
- Antifungal testing: Validated inhibitors are evaluated against Fusarium oxysporum to establish target-dependent effects on fungal growth and pathogenicity
Lead compounds will be prioritized based on potency and antifungal efficacy, providing a foundation for developing novel fungicide candidates.
Technology readiness level
The platform is in the early research and development stage. Current efforts focus on optimizing recombinant fungal ATG4 and the ATG8-based BRET substrate, establishing assay conditions and controls, and integrating the assay with automated liquid handling into a standardized workflow. Subsequent steps include screening chemical libraries, validating hits through orthogonal approaches, and evaluating confirmed inhibitors against Fusarium oxysporum to demonstrate target-dependent antifungal activity.
About University of Wyoming
A comprehensive public land‑grant university serving Wyoming from its Laramie campus and regional sites. Industry engages through co‑located high‑bay and pilot‑scale facilities, field stations across the state, and an incubator network that provides mentorship and startup space. A partnership with a national supercomputing center in Cheyenne enables advanced modeling, visualization, and large‑dataset workflows, while the extension network connects companies to talent and end users in every county. Research is supported by competitive federal funding from agencies such as NSF, DOE, USDA, and NIH, alongside state and industry collaboration. A dedicated technology transfer office advises on IP, licensing, and commercialization.
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