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

• 批准号: 8482105
• 负责人: Todd B Reynolds
• 金额: $ 38.07万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8482105
• 关键词: 2-cyclopentyl-5-(5-isoquinolylsulfonyl)-6-nitro-1H-benzo(D)imidazole; 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; 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' 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; public health relevance; scale up; screening; small molecule; tool; trafficking

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）：这是一项提议，以开发高吞吐量筛选，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）合酶酶的小分子。这些化合物既可以作为抗真菌药物发育的治疗剂，也可以用作研究真菌模型生物中磷脂酰丝氨酸（PS）的生物学的探针。 CHO1P PS PS合酶抑制剂将使用选择性筛选，仅在抑制PS合成的情况下才能生长，因为否则该真菌将被PS结合毒素（Papuamide A（Papa））杀死。该屏幕基于以下观察结果：帕帕（Papa）杀死具有野生型PS的白色念珠菌细胞，但是PS合成缺陷的突变体对毒素具有抗性。 有2种鉴定PS合酶抑制剂的理由：（a）治疗前的抗真菌分子：需要新的抗真菌剂，因为这些药物中只有3类用于治疗浸润性mycoses，以及治疗毒性，耐药性，耐药性和较差的口腔利用能力的组合。 CHO1P PS合成酶抑制剂将是抗真菌发育的极佳铅化合物，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）在哺乳动物中找不到CHO1P，因此抑制剂不应对人类有毒。 3）CHO1P在整个真菌中都保存，因此抑制剂应影响其他真菌。 （b）PS综合探针：CHO1P抑制剂将是对了解更充分理解的强大工具 PS在模型真菌中的作用。 CHO1P PS合成酶抑制剂将引入一种在体内滴定PS水平的方法，以实时测量其影响。 PS对于 哺乳动物和酵母中的内体运输和信号转导，以及维持真菌细胞壁，真菌细胞壁是主要的抗真菌药物靶标。但是，PS影响这些过程的机制尚不清楚。 屏幕将以三个目的进行：特定目标1：为抑制白色念珠菌中CHO1P PS合酶的化合物开发主要筛选。试点筛选已经开始，该数据将用于优化主要屏幕，以选择抑制PS合酶并使细胞在384井板格式中对PAPA具有抗性的化合物。一旦进行了优化，它将扩展到圣裘德儿童研究医院可用的约500,000种化合物。特定的目标2：在主要筛选中鉴定的“ hit HIT”化合物将通过一系列的二级，第三纪和化学膜筛选进一步测试，以识别那些是实际的PS合酶抑制剂的分子。特定目标3：计数器筛选将用于评估PS合酶抑制剂作为治疗前药物的潜力。 AIM 1和2中鉴定出的HIT化合物将通过测量直接抗真菌作用和针对人类细胞系的毒性和真菌病毒力的宿主模型来进一步评估作为抗真菌铅化合物的潜力。 Galleria模型还将用于测试化合物是否可以减少活动物中的真菌毒力。