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的提案，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。这些化合物既可以作为抗真菌药物开发的预治疗剂，也可以作为研究真菌模型生物中磷脂酰丝氨酸 (PS) 生物学的探针。 Cho1p PS 合酶抑制剂将通过选择性筛选进行鉴定，其中只有 PS 合成受到抑制，白色念珠菌才会生长，否则真菌将被 PS 结合毒素巴布酰胺 A (PapA) 杀死。该筛选基于以下观察结果：PapA 以野生型水平的 PS 杀死白色念珠菌细胞，但 PS 合成缺陷的突变体对该毒素具有抗性。鉴定 PS 合酶抑制剂有 2 个基本原理： (A) 治疗前抗真菌分子：需要新的抗真菌药物，因为这些药物中只有 3 类用于治疗侵袭性真菌病，并且具有毒性和耐药性，并且口服可用性差限制了它们的有效性。 Cho1p PS 合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/突变体不能在动物模型中引起疾病。 2) Cho1p在哺乳动物中不存在，因此抑制剂不会对人类产生毒性。 3) Cho1p 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：Cho1p 抑制剂将成为更全面了解 PS 在模型真菌中的作用。 Cho1p PS 合酶抑制剂将引入一种暂时滴定体内 PS 水平的方法，以便实时测量其影响。 PS对于哺乳动物和酵母中的内体运输和信号转导，以及真菌细胞壁（主要抗真菌药物靶标）的维护。然而，PS 影响这些过程的机制尚不清楚。该筛选将针对三个目标进行：具体目标 1：对抑制白色念珠菌中 Cho1p PS 合酶的化合物进行初步筛选。试点筛选已经开始，这些数据将用于优化初级筛选，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。优化后，它将扩大规模，筛选圣裘德儿童研究医院可用的约 500,000 种化合物。具体目标 2：在初级筛选中鉴定出的“命中”化合物将通过一系列二级、三级和化学信息学筛选进一步测试，以鉴定那些真正的 PS 合酶抑制剂分子。具体目标 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。通过测量针对人类细胞系和大蜡螟（蜡蛾幼虫）宿主真菌毒力模型的直接抗真菌作用和毒性，将进一步评估目标 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。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Influence of phosphatidylserine and phosphatidylethanolamine on farnesol tolerance in Candida albicans.

磷脂酰丝氨酸和磷脂酰乙醇胺对白色念珠菌法呢醇耐受性的影响。

DOI：
发表时间：
2018-04
期刊：
影响因子：
0
作者：
Hasim, Sahar;Vaughn, Elyse N;Donohoe, Dallas;Gordon, Donna M;Pfiffner, Susan;Reynolds, Todd B
通讯作者：
Reynolds, Todd B

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity.

一些白色念珠菌突变体中的β-(1,3)-葡聚糖暴露与细胞壁表面粗糙度的增加和细胞壁弹性的降低相关。

DOI：
发表时间：
2017-01
期刊：
影响因子：
3.1
作者：
Hasim, Sahar;Allison, David P;Retterer, Scott T;Hopke, Ale;Wheeler, Robert T;Doktycz, Mitchel J;Reynolds, Todd B
通讯作者：
Reynolds, Todd B

SB-224289 Antagonizes the Antifungal Mechanism of the Marine Depsipeptide Papuamide A.

SB-224289 拮抗海洋缩酚肽 Papuamide A 的抗真菌机制。

DOI：
发表时间：
2016
期刊：
影响因子：
3.7
作者：
Cassilly, Chelsi D;Maddox, Marcus M;Cherian, Philip T;Bowling, John J;Hamann, Mark T;Lee, Richard E;Reynolds, Todd B
通讯作者：
Reynolds, Todd B

Role of phosphatidylserine synthase in shaping the phospholipidome of Candida albicans.

磷脂酰丝氨酸合酶在塑造白色念珠菌磷脂组中的作用。

DOI：
发表时间：
2017-03-01
期刊：
影响因子：
3.2
作者：
Cassilly, Chelsi D;Farmer, Abigail T;Montedonico, Anthony E;Smith, Terry K;Campagna, Shawn R;Reynolds, Todd B
通讯作者：
Reynolds, Todd B

Exposure of Candida albicans β (1,3)-glucan is promoted by activation of the Cek1 pathway.

Cek1 途径的激活促进白色念珠菌β(1,3)-葡聚糖的暴露。