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Screen for phosphatidylserine synthase inhibitors: antifungals & lipid probes

筛选磷脂酰丝氨酸合酶抑制剂：抗真菌药

基本信息

批准号：
8789352
负责人：
Todd B Reynolds
金额：
$ 39.34万
依托单位：
UNIVERSITY OF TENNESSEE KNOXVILLE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-02-01 至 2018-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8789352
关键词：
Affect Animal Model Animals Antifungal Agents Binding Biological Biological Process Biology Candida Candida albicans Cell Wall Cells Cellular biology Data Defect Development Disease Dose Drug Targeting Drug resistance Drug usage Effectiveness Ensure Enzymes Ethanolamines Fungi Model Growth Health HepG2 Human Human Cell Line Lead Lecithin Libraries Life Lipids Maintenance Mammals Measures Methods Modeling Moths Mycoses NIH Program Announcements Oral Pathogenesis Pathway interactions Patients Pharmaceutical Preparations Phenotype Phosphatidylethanolamine Phosphatidylserine Synthase Phosphatidylserines Phospholipids Process Resistance Role Saccharomyces cerevisiae Saint Jude Children&apos s Research Hospital Series Signal Transduction Testing Therapeutic Time Toxic effect Toxicology Toxin Virulence Waxes Yeasts assay development auxotrophy base drug development essential phospholipids fungus high throughput screening in vitro Assay in vivo inhibitor/antagonist killings mutant pathogen scale up screening small molecule tool trafficking

项目摘要

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.

描述（由申请人提供）：这是一项开发高通量筛选以鉴定抑制白色念珠菌（人类最常见的真菌病原体）中真菌磷脂酰丝氨酸（PS）合酶的小分子的提案。这些化合物可以作为抗真菌药物开发的预治疗剂和/或用于研究真菌模式生物中磷脂酰丝氨酸（PS）生物学的探针。 Cho 1 p PS合成酶抑制剂将使用选择性筛选来鉴定，其中C.只有当PS合成被抑制时，白色念珠菌才会生长，因为否则真菌将被PS结合毒素papuamide A（PapA）杀死。这个屏幕是基于PapA杀死C的观察。白色念珠菌细胞与野生型水平的PS，但突变体的PS合成缺陷是耐毒素。鉴定PS合成酶抑制剂有2个基本原理：（A）治疗前抗真菌分子：需要新的抗真菌药物，因为只有3类这些药物用于治疗侵袭性真菌病，并且毒性、耐药性和口服利用度差的组合限制了它们的有效性。Cho 1 p PS合成酶抑制剂将成为抗真菌药物开发的优良先导化合物，因为：白色念珠菌Cho 1/突变体在动物模型中不会引起疾病。2)Cho 1 p在哺乳动物中没有发现，因此抑制剂对人类应该没有毒性。3)Cho 1 p在整个真菌中是保守的，因此抑制剂应该会影响其他真菌。(B)PS合成探针：Cho 1 p抑制剂将是更全面了解 PS在模式真菌中的作用。Cho 1 p PS合成酶抑制剂将引入一种暂时滴定体内PS水平的方法，以便在真实的时间内测量其影响。PS对于哺乳动物和酵母中的内体运输和信号转导，以及真菌细胞壁的维持，这是一个主要的抗真菌药物靶标。然而，PS影响这些过程的机制还不清楚。本研究的目的主要有三个：第一个目标：初步筛选出能抑制C.白色念珠菌。中试筛选已经开始，这些数据将用于优化初步筛选，以选择抑制PS合酶并使细胞在384孔板格式中对PapA具有抗性的化合物。一旦优化，它将扩大到筛选圣裘德儿童研究医院提供的约50万种化合物。具体目标二：在初级筛选中鉴定的“命中”化合物将进一步通过一系列二级、三级和化学信息学筛选进行测试，以鉴定那些是实际PS合酶抑制剂的分子。具体目标3：计数器筛选将用于评估PS合成酶抑制剂作为预治疗剂的潜力。将通过测量对人类细胞系和大蜡螟（蜡螟幼虫）宿主真菌毒力模型的直接抗真菌作用和毒性，进一步评估目标1和2中确定的命中化合物作为抗真菌先导化合物的潜力。Galleria模型还将用于测试这些化合物是否可以降低活体动物中的真菌毒力。