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

描述（由申请人提供）：估计每年有150万人死于侵袭性真菌感染，还有数百万人死于粘膜和皮下真菌病。目前的抗真菌治疗存在严重缺陷，包括疗效差、活性谱有限、患者毒性和耐药真菌的出现。因此，死亡率仍然高得令人不安。开发有效的新型抗真菌药物的主要障碍是这些真核病原体与其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。本提案的目的是证实针对真菌液泡的化疗药物的抗真菌功效。哺乳动物细胞中缺乏一种密切相关的细胞器，这表明液泡可能为选择性靶向感染性真菌提供了宝贵的机会。我们的分子研究表明，破坏流行的人类病原体白色念珠菌的空泡完整性，严重损害其定植哺乳动物组织或在小鼠中引起致命感染的能力。空泡功能也是新生隐球菌在哺乳动物宿主内存活并引起脑膜脑炎的必要条件。在这两种真菌中，液泡功能的丧失会导致许多与发病机制相关的表型，包括对各种胁迫的超敏反应和毒力属性的严重表达减少。因此，我们假设真菌液泡可以被开发出有效的新抗真菌疗法，因为它是真菌发病的必要条件，并且与哺乳动物溶酶体有明显的差异。为了验证这一点，我们设计了一种高通量筛选试验，迄今已鉴定出82种潜在的液泡破坏化学剂（vda）。在本研究的目的1中，我们将对这些vda在真菌液泡以及等效的哺乳动物溶酶体上的活性进行表征，并选择具有强效和真菌选择性活性的vda。在Aim 2中，我们将选择体外抗真菌活性最大的vda。最后，在Aim 3中，我们将确定最有效的VDAs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靶向真菌液泡作为治疗危及生命的真菌感染的策略的真正潜力，建立可以形成此类干预基础的“先导”化合物管道，以及确定和验证化学上可处理的靶标。

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.