Molecular and chemical validation of the vacuole as a new antifungal target

液泡作为新抗真菌靶点的分子和化学验证

• 批准号: 8849822
• 负责人: Glen Palmer
• 金额: $ 40.07万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-20 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8849822
• 关键词: Address; Antifungal Agents; Antifungal Therapy; Azoles; Biological Assay; Candida; Candida albicans; Candida glabrata; Cell Communication; Cessation of life; Chemical Agents; Chemicals; Cryptococcus neoformans; Defect; Development; Disseminated candidiasis; Figs - dietary; Functional disorder; Genetic; Health; Human; Hypersensitivity; In Vitro; Infection; Intervention; Lead; Life; Lysosomes; Mammalian Cell; Meningoencephalitis; Molecular; Mus; Mycoses; Organelles; Outcome; Pathogenesis; Pathway interactions; Patients; Phagocytes; Phenotype; Resistance; Severities; Social Welfare; Stress; Testing; Therapeutic Agents; Tissues; Toxic effect; Toxicity Tests; Vacuole; Validation; Virulence; base; chemical genetics; conventional therapy; fungus; genetic analysis; genome-wide; high throughput screening; improved; in vitro Model; in vivo; injured; innovation; mortality; mouse model; novel; pathogen; patient population; screening; subcutaneous; trait

DESCRIPTION (provided by applicant): An estimated 1.5 million people die each year from invasive fungal infections, and many millions more are afflicted by debilitating mucosal and subcutaneous mycoses. Current antifungal therapies have serious deficiencies including poor efficacy, limited spectrum of activity, patient toxicity and the emergence of resistant fungi. Consequently, mortality rates have remained disturbingly high. A major obstacle to developing effective new antifungals is the fundamental similarity of these eukaryotic pathogens and their mammalian host at the cellular level. This presents a challenge in devising therapeutic agents with pathogen selective toxicity. The objective of this proposal is to substantiate the antifungal efficacy of chemotherapeutics that target the fungal vacuole. The absence of a closely related organelle in mammalian cells suggests that the vacuole may provide an invaluable opportunity to selectively target infectious fungi. Our molecular studies have shown that disrupting vacuolar integrity in the prevalent human pathogen, Candida albicans, severely impairs its ability to colonize mammalian tissue or cause lethal infection in mice. Vacuolar function is also essential for Cryptococcus neoformans to survive within the mammalian host and cause meningoencephalitis. In either fungus, loss of vacuolar function causes a multitude of pathogenesis related phenotypes, including hypersensitivity to a variety of stresses and severely diminished expression of virulence attributes. Therefore, we hypothesize that the fungal vacuole can be exploited to develop effective new antifungal therapies because it is essential for fungal pathogenesis, and has diverged significantly from the mammalian lysosome. To test this we have devised a high-throughput screening assay that has so far identified 82 potential Vacuole Disrupting chemical Agents (VDAs). In Aim 1 of this study we will characterize the activity of these VDAs upon the fungal vacuole as well as the equivalent mammalian lysosome, and select those with potent and fungal- selective activity. In Aim 2, we will select VDAs with the greatest in vitro antifungal activity. Finally, in Aim 3 we will identify the moleculr targets or pathways upon which the most efficacious VDAs act, and use a mouse model of disseminated candidiasis to test if the inhibition of these targets is sufficient to cure an established in vivo infection. Completion of these studies will uncover the true potential of targeting the fungal vacuole as a strategy to cure life-threatening fungal infections, establish a pipeline of 'lead' compounds that can form the basis of such interventions, as well as identify and validate chemically tractable targets.

描述(申请人提供)：据估计，每年有150万人死于侵袭性真菌感染，还有数百万人患有衰弱的粘膜和皮下真菌病。目前的抗真菌治疗存在着严重的不足，包括疗效差、活性谱有限、患者毒性和出现耐药真菌。因此，死亡率一直居高不下，令人不安。开发有效的新抗真菌药物的一个主要障碍是这些真核病原体与它们的哺乳动物宿主在细胞水平上的基本相似性。这对设计具有病原体选择性毒性的治疗剂提出了挑战。这项建议的目的是证实以真菌液泡为靶点的化疗药物的抗真菌效果。哺乳动物细胞中没有密切相关的细胞器，这表明液泡可能为选择性地针对感染性真菌提供了一个宝贵的机会。我们的分子研究表明，破坏人类常见病原体白色念珠菌的空泡完整性，严重损害其在哺乳动物组织中的定植能力或导致小鼠致命感染。空泡功能对新生隐球菌在哺乳动物宿主内生存并引起脑膜脑炎也是必不可少的。在这两种真菌中，液泡功能的丧失导致了许多与病程相关的表型，包括对各种胁迫的超敏和毒力属性表达的严重减弱。因此，我们推测真菌液泡在真菌发病中是必不可少的，并且与哺乳动物的溶酶体有很大的不同，因此可以利用它来开发有效的新的抗真菌治疗方法。为了测试这一点，我们设计了一种高通量筛选试验，到目前为止已经确定了82种潜在的液泡破坏化学物质(VDA)。在这项研究的目标1中，我们将表征这些VDA对真菌液泡以及等效的哺乳动物溶酶体的活性，并选择那些具有强大的真菌选择活性的VDA。在目标2中，我们将选择体外抗真菌活性最强的VDA。最后，在目标3中，我们将确定最有效的VDA作用的分子靶点或途径，并使用播散性念珠菌病的小鼠模型来测试这些靶点的抑制是否足以治愈已建立的体内感染。这些研究的完成将揭示将真菌液泡作为治疗威胁生命的真菌感染的一种策略的真正潜力，建立一条可构成此类干预措施基础的“先导”化合物管道，以及确定和验证化学上易处理的目标。