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，还有数百万人受到使人衰弱的粘膜和皮下真菌病的折磨。目前的抗真菌治疗存在严重的缺陷，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。开发有效的新抗真菌药物的主要障碍是这些真核病原体和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。本提案的目的是证实靶向真菌空泡的化疗药物的抗真菌疗效。在哺乳动物细胞中缺乏一个密切相关的细胞器，这表明液泡可能提供了一个宝贵的机会，选择性地针对感染性真菌。我们的分子研究表明，破坏空泡的完整性，在流行的人类病原体，白色念珠菌，严重损害其能力，殖民哺乳动物组织或造成致命的感染小鼠。嗜中性粒细胞功能也是新生隐球菌在哺乳动物宿主中生存并引起脑膜脑炎所必需的。在任一真菌中，空泡功能的丧失导致多种发病机制相关的表型，包括对各种应激的超敏反应和毒力属性的表达严重减少。因此，我们假设真菌空泡可以被利用来开发有效的新的抗真菌疗法，因为它是真菌发病机制所必需的，并且与哺乳动物溶酶体显著不同。为了测试这一点，我们设计了一种高通量筛选试验，迄今已确定了82个潜在的干扰素化学剂（VDA）。在本研究的目的1中，我们将表征这些VDA对真菌空泡以及等效哺乳动物溶酶体的活性，并选择具有强效和真菌选择性活性的VDA。在目标2中，我们将选择具有最大体外抗真菌活性的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靶向真菌空泡作为治疗危及生命的真菌感染的策略的真正潜力，建立一个可以形成这种干预措施基础的“铅”化合物管道，以及识别和验证化学上易处理的目标。

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.