This is a proposal to develop a high throughput screen to identify small molecules that inhibit the fungal phosphatidylserine (PS) synthase enzyme in Candida albicans (the most common fungal pathogen of humans). These compounds can serve as both pre-therapeutics for antifungal drug development and/or probes to study the biology of phosphatidylserine (PS) in fungal model organisms. Cho1p PS synthase inhibitors will be identified using a selective screen where C. albicans will grow only if PS synthesis is inhibited, because otherwise the fungus will be killed by a PS-binding toxin, papuamide A (PapA). This screen is based on the observation that PapA kills C. albicans cells with wild-type levels of PS, but mutants with defects in PS synthesis are resistant to the toxin. There are 2 rationales for identifying PS synthase inhibitors: (A) Pre-therapeutic antifungal molecules: There is a need for new antifungals, as there are only 3 classes of these drugs used to treat invasive mycoses, and a combination of toxicity, drug resistance, and poor oral availability limit their effectiveness. Cho1p PS synthase inhibitors will be excellent lead compounds for antifungal development because 1) A C. albicans cho1 / mutant cannot cause disease in an animal model. 2) Cho1p is not found in mammals, so inhibitors should not be toxic to humans. 3) Cho1p is conserved throughout fungi, so inhibitors should affect other fungi. (B) PS synthesis probe: Cho1p inhibitors will be powerful tools for attaining a fuller understanding of the roles of PS in model fungi. A Cho1p PS synthase inhibitor will introduce a method to temporally titrate PS levels in vivo in order to measure its impact in real time. PS is crucial for endosomal trafficking and signal transduction in mammals and yeast, as well as maintenance of the fungal cell wall, a major antifungal drug target. However, the mechanisms by which PS affects these processes are not well understood. The screen will be carried out in three aims:
Specific Aim 1 : Develop primary screen for compounds that inhibit the Cho1p PS synthase in C. albicans. Pilot screening has already begun, and this data will be used to optimize the primary screen to select for compounds that inhibit PS synthase and make cells resistant to PapA in the 384 well plate format. Once optimized, it will be scaled up to screen ~500,000 compounds available at St. Jude Children's Research Hospital.
Specific Aim 2 : """"""""Hit"""""""" compounds identified in the primary screen will be further tested by a series of secondary, tertiary, and chemoinformatic screens to identify those molecules that are actual PS synthase inhibitors.
Specific Aim 3 : Counter screens will be used to assess the potential of PS synthase inhibitors as pre-therapeutics. Hit compounds identified in aims 1 and 2 will be further assessed for potential as antifungal lead compounds by measuring direct antifungal effects and toxicity against human cell lines and the Galleria mellonella (waxmoth larval) host model of fungal virulence. The Galleria model will also serve to test if the compounds can reduce fungal virulence in a live animal.
Fungal infections by the pathogenic yeast Candida albicans are a growing threat to seriously ill, hospitalized patients, but there are only a limited number o drugs for treating them. This proposal aims to discover compounds that inhibit the fungal enzyme Cho1p, which makes the lipid phosphatidylserine (PS), and is absolutely required for C. albicans to cause disease. PS synthesis inhibitors will be useful as potential lead compounds for antifungal drug development, and will also be useful for studying PS biology in fungi that serve as models of human cell biology, like the bakers'yeast Saccharomyces cerevisiae.
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