Screen for phosphatidylserine synthase inhibitors: antifungals & lipid probes

描述（由申请人提供）：这是一项开发高通量筛选的提案，用于鉴定白色念珠菌（人类最常见的真菌病原体）中抑制真菌磷脂酰丝氨酸（PS）合成酶的小分子。这些化合物既可以作为抗真菌药物开发的前治疗药物，也可以作为真菌模式生物中磷脂酰丝氨酸（PS）生物学研究的探针。Cho1p PS合成酶抑制剂将通过选择性筛选来鉴定，其中白色念珠菌只有在PS合成被抑制时才会生长，否则真菌将被PS结合毒素papuamide a （PapA）杀死。这一筛选是基于这样的观察，即PapA杀死具有PS野生型水平的白色念珠菌细胞，但PS合成缺陷的突变体对毒素具有抗性。鉴定PS合成酶抑制剂有两个理由：(A)治疗前抗真菌分子：需要新的抗真菌药物，因为只有3类药物用于治疗侵袭性真菌病，而且毒性、耐药性和口服可得性差的综合限制了它们的有效性。Cho1p PS合成酶抑制剂将成为抗真菌开发的优秀先导化合物，因为1)白色念珠菌cho1 /突变体在动物模型中不能引起疾病。2)在哺乳动物中没有发现Cho1p，所以抑制剂应该对人类没有毒性。3) Cho1p在整个真菌中都是保守的，所以抑制剂应该影响其他真菌。(B) PS合成探针：Cho1p抑制剂将是获得更全面了解的有力工具

DESCRIPTION (provided by applicant): 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.