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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查的建议，以确定在白色念珠菌(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，也可以作为研究真菌模式生物中磷脂酰丝氨酸(PS)生物学的探针。将使用选择性筛选来鉴定Cho1p PS合成酶抑制剂，只有在PS合成受到抑制的情况下，白色念珠菌才会生长，因为否则真菌将被PS结合毒素--木瓜酰胺A(PAPA)杀死。这一筛选是基于这样的观察，即Papa杀死具有野生型PS水平的白色念珠菌细胞，但PS合成缺陷的突变株对毒素具有抵抗力。鉴定PS合成酶抑制剂有两个基本原理：(A)治疗前的抗真菌分子：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，毒性、耐药性和较差的口服利用度限制了它们的有效性。由于1)白色念珠菌cho1/突变体不能在动物模型中引起疾病，因此Cho1p PS合成酶抑制剂将是抗真菌开发的优秀先导化合物。2)在哺乳动物中没有发现Cho1p，因此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，因此抑制剂可能会影响其他真菌。(B)PS合成探测器：Cho1p抑制剂将是更全面了解 PS在模式真菌中的作用。一个Cho1p PS合成酶抑制剂将引入一种方法来在体内暂时滴定PS水平，以便实时测量其影响。PS对以下方面至关重要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，这是主要的抗真菌药物靶标。然而，PS影响这些过程的机制还不是很清楚。该筛选将在三个目标中进行：特定目标1：开发抑制白色念珠菌Cho1p PS合成酶的化合物的初步筛选。中试筛选已经开始，这些数据将用于优化初步筛选，以在384孔板格式中筛选抑制PS合酶并使细胞对PAPA产生抗性的化合物。一旦进行优化，它将被放大到圣犹大儿童研究医院提供的约50万个化合物。具体目标2：将通过一系列二级、三级和化学信息学筛选进一步测试在初级筛选中确定的“Hit”化合物，以确定那些真正是PS合成酶抑制剂的分子。具体目标3：计数器筛查将用于评估PS合成酶抑制剂作为治疗前药物的潜力。在AIMS 1和2中确定的HIT化合物将通过测量直接抗真菌作用和对人类细胞系和真菌毒力的Galeria mellonella(蜡蛾幼虫)宿主模型的毒性来进一步评估其作为抗真菌先导化合物的潜力。Galeria模型还将用于测试这些化合物是否可以降低活体动物的真菌毒力。