New antifungals targeting the synthesis of fungal sphingolipids

针对真菌鞘脂合成的新型抗真菌药物

• 批准号: 9182873
• 负责人: Maurizio Del Poeta
• 金额: $ 59.45万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9182873
• 关键词: Affect; Alveolar; Animal Model; Animals; Antifungal Agents; Aspergillosis; Aspergillus; Aspergillus fumigatus; Blood Circulation; Brain; Candida; Candida albicans; Candidiasis; Carbon Dioxide; Cell Cycle Progression; Cells; Ceramide glucosyltransferase; Ceramides; Cryptococcus neoformans; Cryptococcus neoformans infection; Cytokinesis; Development; Disease; Drug Kinetics; Environment; Enzymes; Gene Mutation; Generations; Genes; Glucosylceramides; Goals; Growth; Human; In Vitro; Industrial fungicide; Infection; Laboratories; Lead; Libraries; Lipids; Lung; Mammalian Cell; Meningoencephalitis; Molds; Mus; Mycoses; Pathogenesis; Pathogenicity; Pathway interactions; Pharmaceutical Chemistry; Pneumocystis carinii Pneumonia; Production; Reporting; Research Personnel; Resistance; Resistance development; Solubility; Sphingolipids; Structure-Activity Relationship; Testing; Therapeutic; Time; Toxic effect; Transcript; Validation; Vesicle; Virulence; Virulence Factors; Yeasts; alkalinity; base; combinatorial; compound 30; cytotoxicity; fungus; improved; in vivo; in vivo Model; inhibitor/antagonist; insight; mutant; new therapeutic target; novel; novel therapeutics; public health relevance; pulmonary granuloma; screening; small molecule libraries; trafficking; treatment strategy

 DESCRIPTION (provided by applicant): The goal of this project is to assess the therapeutic potential of novel antifungal agents, identified by our laboratory via screening a ChemBridge library, that target the synthesis of fungal but not mammalian glucosylceramide (GlcCer). Recently, it has been reported by us and other investigators that fungal GlcCer is required for virulence of many fungi, including Cryptococcus neoformans (Cn),1,2 Candida albicans (Ca),3-5 Aspergillus fumigatus (Af)6 and others.7 In addition, GlcCer is detected only in the host infective form (yeast) and not in the environmental form (mold) of many dimorphic fungi.8-10 Furthermore, the synthesis of GlcCer seems to be important also for Pneumocystis pneumonia (PCP) as glucosylceramide synthase transcripts are highly elevated at the time of isolation of the fungus from a fulminate lung infection.11 Studies in our and other labs revealed that GlcCer promotes alkaline tolerance in fungi.6,12-14 Particularly, GlcCer regulates fungal cell replication by promoting cell cycle progression and cytokinesis in a neutral/alkaline but not acidic environment.6,12,14 Taken together, these studies suggest that GlcCer is most likely a pan-fungal virulence factor required during infection to promote fungal growth at neutral/alkaline environments (e.g. alveolar spaces and bloodstream), and as such, it is a promising novel drug target. Therefore, we looked for inhibitors of GlcCer synthesis by screening a ChemBridge library for compounds that inhibit fungal growth in an environment similar to the lung and bloodstream: neutral/alkaline pH, 37ºC and 5% CO2 using Cn as a model organism. We identified 2 compounds (BHBM and its derivative D0) that significantly decreased the synthesis of GlcCer in Cn but not in mammalian cells (Fig. 1).15 The compounds are fungicidal (Fig. 2) and able to improve mice survival during invasive cryptococcosis, candidiasis and pneumocystosis (Fig. 3).15 Mechanistic studies show that the compounds target SLA2, a gene controlling fungal vesicle trafficking (Fig. 6), which is how ceramide is transported for the synthesis of GlcCer. Therefore, we hypothesize that targeting the fungal GlcCer pathway will be an effective novel therapeutic strategy for impeding the development of fungal diseases. To test this hypothesis, we propose the following aims: Aim 1. Medicinal Chemistry. Employ combinatorial/parallel synthesis to create a first-generation library of fungal GlcCer inhibitors based on early lead compounds BHBM and D0. Obtain ca. 300 compounds for initial screening for solubility, antifungal activity, toxicity studies and generate an optimization library of ca. 30 compounds to identify a dozen advanced lead compounds, all possessing good in vitro antifungal activity and with a EC50 cytotoxicity/MIC80 efficacy ratio >100 (based on studies in Aim 2). These compounds will then be tested in vivo for pharmacokinetics (PK) and animal studies (Aim 3). Aim 2. Mechanism of action and resistance development. Confirm and validate the compounds synthesized in Aim 1 for ability to inhibit fungal but not mammalian GlcCer/SLA2. Determine whether cells can develop resistance to the candidate compounds. Iterate library based on observed Structure-Activity-Relationship (SAR). Aim 3. PK and in vivo toxicity and efficacy. Study PK of lead compounds selected from Aim 2 and assesses their effect in in vivo models of cryptococcosis, candidiasis, pneumocystosis and aspergillosis.

 描述（由申请人提供）：本项目的目的是评估新型抗真菌剂的治疗潜力，这些抗真菌剂是由我们实验室通过筛选ChemBridge文库鉴定的，靶向真菌而非哺乳动物葡萄糖神经酰胺（GlcCer）的合成。最近，我们和其他研究人员报道，真菌GlcCer是许多真菌的毒力所必需的，包括新型隐球菌（Cn）、1，2白色念珠菌（Ca）、3 -5烟曲霉（Af）6等。7此外，GlcCer仅在宿主感染性真菌中检测到。 8 -10此外，GlcCer的合成似乎对肺孢子虫肺炎（PCP）也很重要，因为在从暴发性肺部感染中分离真菌时，葡萄糖神经酰胺合酶转录物高度升高。11我们和其他实验室的研究表明，GlcCer促进真菌的碱性耐受性。6，12-14具体地，GlcCer调节真菌细胞复制 通过在中性/碱性而非酸性环境中促进细胞周期进程和胞质分裂。6，12，14总之，这些研究表明GlcCer最有可能是感染期间促进中性/碱性环境（例如肺泡空间和血流）中真菌生长所需的泛真菌毒力因子，因此，它是一种有前景的新型药物靶标。因此，我们使用Cn作为模式生物，通过在ChemBridge文库中筛选在类似于肺和血流的环境中抑制真菌生长的化合物来寻找GlcCer合成的抑制剂：中性/碱性pH，37ºC和5% CO2。我们鉴定了两种化合物（BHBM及其衍生物D 0），其显著降低Cn中而非哺乳动物细胞中GlcCer的合成（图1）15化合物是杀真菌的（图2）并能提高小鼠在侵袭性隐球菌病、念珠菌病和肺囊虫病期间的存活率（图3）15机理研究表明，化合物靶向SLA 2，一种控制真菌囊泡运输的基因（图6），这是神经酰胺如何运输以合成GlcCer。因此，我们假设靶向真菌GlcCer途径将是一种有效的新型治疗策略，用于阻止真菌疾病的发展。为了验证这一假设，我们提出了以下目标：目标1。药物化学采用组合/平行合成来创建基于早期先导化合物BHBM和D 0的真菌GlcCer抑制剂的第一代文库。获取ca。300个化合物进行溶解性、抗真菌活性、毒性研究的初步筛选，并生成ca. 30种化合物以鉴定十几种先进的先导化合物，所有化合物都具有良好的体外抗真菌活性，并且EC 50细胞毒性/MIC 80功效比>100（基于目标2中的研究）。然后将对这些化合物进行体内药代动力学（PK）和动物研究（目的3）。目标二。作用机制和抗性发展。确认并验证目标1中合成的化合物抑制真菌而非哺乳动物GlcCer/SLA 2的能力。确定细胞是否能对候选化合物产生耐药性。基于观察到的结构-活性-关系（SAR）的迭代库。目标3。PK和体内毒性和疗效。研究选自目标2的先导化合物的PK，并评估其在隐球菌病、念珠菌病、肺孢子虫病和曲霉病的体内模型中的作用。