Anti-prion drug screening system in Saccharomyces cerevisiae based on an artificial [LEU2+] prion

Abstract

Proteinaceous infectious particles causing mammalian transmissible spongiform encephalopathies or prions are being extensively studied. However due to their hazardous nature, the initial screening of potential anti-prion drugs is often made in a yeast-based screening system utilizing a well-characterized [PSI⁺] prion (amyloid formed by the translation termination factor Sup35p). In the [PSI⁺] prion screening system (white/red colony assay), the prion phenotype yields white colonies while addition of an anti-prion drug will yield red colonies. However, this system has some limitations. It is difficult to quantify the effectiveness of the anti-prion compound, the diffusion of the studied compound may affect the result, and the deficiency of glutathione in cells may prevent the formation of red pigment in cured cells. Therefore, alternative yeast prion screening systems are still needed. This article aims to present an alternative yeast-based system to evaluate anti-prion activity of chemical compounds. The method that was used is based on an artificial [LEU2⁺] prion created by fusing Leu2p with the prion-forming domain of Sup35p in Saccharomyces cerevisiae. Phenotypic analysis and semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) confirmed the presence of the artificial [LEU2⁺] prion in yeast cells. This screening system verified the anti-prion activity of 3 drugs that were found to have been active in the white/red colony assay, while one compound (6-chlorotacrine) that was active in the white/red colony assay was found to be inactive in the [LEU2⁺] system. This new system also appears to be more sensitive than the white/red colony assay.

Keywords: Artificial prion; Drug screening; Genetic engineering; Neurodegenerative diseases; Organic synthesis; Yeast-based assay.