Isolation, Identification and Evaluation of New Anti-prion Compounds from Australian Marine Invertebrates using Novel Yeast-prion Screening

Abstract

Prion diseases are fatal neurodegenerative diseases caused by the build-up of a misfolded form of the prion protein in the brain. These misfolded isoforms of the prion protein are infectious and capable of catalysing the transformation of the native protein into the same misfolded ‘prion’ form. Prion diseases include Creutzfeldt-Jakob disease (CJD) in humans, as well as scrapie and bovine spongiform encephalopathy (mad cow disease) in animals. Other neurodegenerative diseases caused by the build-up of misfolded proteins include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Motor Neurone disease. An outbreak of CJD in the UK in the 1990s led to an extensive search for therapeutics to treat prion diseases. This resulted in the identification of a number of anti-prion compounds active in vitro, however, very few compounds active in vivo that could be used as drugs. Therefore, there is currently no curative therapeutic available for the treatment of these fatal diseases. The screening of large, diverse compound libraries has been considered an important strategy for the identification of novel anti-prion compounds. However, this has been hindered by a number of factors, including the limited of knowledge of prion mechanisms and the lack of suitable screening assays. For this reason, we aimed to develop a new assay that could be used to screen chemically complex natural extracts for anti-prion activity. This assay was then used to identify novel anti-prion compounds from the marine environment, a large source of chemical and biological diversity previously untapped in the search for anti-prion compounds. The isolated anti-prion compounds were further evaluated and compared to previously identified compounds that lack sufficient activity in vivo. Chapter One (Prion publication) describes the development of a new yeast-based anti-prion assay that can be used for screening naturally-derived extracts. This assay utilises prions of the yeast Saccharomyces cerevisiae as the basis for a simple colorimetric anti-prion screen. The yeast is white when infected with the yeast prion and red when the protein is in its non-infectious normal form. This assay was then used to screen 500 marine invertebrate derived extracts resulting in the identification of four extracts with activity. This chapter then describes the use of the anti-prion assay to target the isolation of the active compounds from the active extract from the Australian sponge, Suberea ianthelliformis. This resulted in the identification of three known bromotyrosine alkaloids with potent anti-prion activity. Chapter Two describes the bioassay-guided isolation of active compounds from the anti-prion extract from the Australian ascidian, Polycarpa procera. Extensive purification and structural elucidation resulted in the identification of four new butenolide and two new propanone metabolites, the procerolides and procerones, respectively. These compounds exhibited potent anti-prion activity. Chapter Three describes the bioassay-guided isolation of new anti-prion compounds from an active extract from the Australian ascidian, Didemnum sp. This resulted in the isolation and identification of a new set of sulfated poly-oxygenated sterol derivatives, the didemnisterols. These compounds were isolated in low yields but exhibit potent anti-prion activity. Further biological testing also showed that these compounds display binding to α-synuclein, another neurodegenerative disease causing misfolded protein, and inhibit its aggregation. Chapter Four describes the bioassay-guided isolation of active compounds from an anti-prion extract from the Australian sponge, Dysidea sp. This resulted in the identification of four known poly-oxygenated sterol derivatives. These Dysidea-sterols displayed potent anti-prion activity in the yeast-based anti-prion assay. Chapter Five describes the evaluation of the sixteen potent anti-prion natural products isolated in this study. We evaluate their anti-prion activity, physicochemical properties, neurotoxicity and ability to inhibit α-synuclein aggregation in vitro. This data was compared to that of previously identified anti-prion compounds and currently used CNS drugs for the selection of the most promising lead candidates. We suggest that the lead compounds from this study be further evaluated using structure-activity relationship studies to identify their important molecular fragments. This knowledge can then be used for the design of novel therapeutics to treat prion diseases.