High Throughput Screening for Small-molecules Facilitating Prion Study

Project: Research project

Description

Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of unusual infectious mammalian neurodegenerative disorders. The pathogen of TSEs is believed to be a misfolded form (PrPSc) of a normal functional host cellular protein (PrPC). Partly due to the complication of protein-based pathology, TSEs remain incurable and currently there is no treatment that can halt their rapid progressions. Intriguingly, the budding yeast, Saccharomyces cerevisiae, contains several non-Mendelian genetic elements that are transmitted as altered protein conformations and are termed as yeast prions. Its simplicity and genetic trackability has made yeast a powerful model organism for prion research. In this proposal, we plan to use the yeast prion [SWI+] system as a platform to identify small molecules that can inhibit prion propagation through a high-throughput screen approach. [SWI+] was discovered in our laboratory, whose protein determinant is Swi1, a subunit of an evolutionarily conserved chromatin-remodeling complex – SWI/SNF. We found that the expression of FLO1, a SWI/SNF target gene encoding a cell wall protein required for yeast filamentous growth is severely suppressed by [SWI+]. By replacing the FLO1-ORF with the URA3-coding region, we created a faithful FLO1promoter-URA3-based chromosomal reporter. While [SWI+] cannot grow in media lacking uracil, the isogenic non-prion cells can. Thus, chemical compounds that can eliminate [SWI+] can be positively selected in a simple, one-step growth assay in media lacking uracil. We demonstrate that this cost-effective assay is suitable for high-throughput screens in a 384-well format. Our pilot screens have already yielded a number of hits (chemical compounds that can effectively eliminate [SWI+]). We anticipate that some anti-prion compounds obtained from this study will likely become valuable molecular probes for prion research and further investigation of their prion-curing mechanism will lead to identification of novel components important for prion formation and propagation and development of promising anti-prion therapeutic drugs. Due to the amyloid nature of the [SWI+] prion, we also expect that some identified anti-prion compounds are also effective in suppressing non-prion amyloidogenic diseases resulted from protein misfolding, such as Alzheimer’s disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS).
StatusFinished
Effective start/end date7/1/146/30/18

Funding

  • National Institute of General Medical Sciences (5R01GM110045-03)

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Prions
Prion Diseases
Uracil
Yeasts
PrPC Proteins
Proteostasis Deficiencies
Molecular Probes
Protein Conformation
Saccharomycetales
Chromatin Assembly and Disassembly
Fungal Proteins
Amyotrophic Lateral Sclerosis
Growth
Research
Amyloid
Neurodegenerative Diseases
Cell Wall
Open Reading Frames
Parkinson Disease
Saccharomyces cerevisiae