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

描述（由申请人提供）：近几十年来，侵袭性真菌病急剧增加，包括危及生命的弥散性和使人衰弱的粘膜感染。受影响个体的不同情况，致病真菌种类的多样性，以及这些真核病原体与我们自身细胞之间的相似性，都为设计具有选择性毒性的有效治疗提出了重大挑战。目前的抗真菌疗法针对的真菌成分范围很窄，并且存在严重的局限性，包括患者毒性、疗效、可用配方、活性谱和/或耐药性的发展。我们的分子研究表明，破坏最普遍的人类真菌病原体白色念珠菌的液泡生物发生，严重损害其定植和入侵哺乳动物组织的能力。液泡功能也是远亲人类病原体新隐球菌在哺乳动物宿主内生存所必需的。在这两种真菌中，液泡功能的丧失导致对各种宿主相关应激的超敏反应和毒力属性的严重表达减少，表明发病机制在多个层面上受到深刻影响。哺乳动物细胞中缺乏一种密切相关的细胞器，这表明真菌液泡可能提供了一个宝贵的机会，可以用特定的化学剂选择性地靶向入侵的真菌病原体。本研究的目的是建立靶向真菌液泡作为抗真菌治疗新策略的有效性。具体来说，我们假设液泡可以用于开发有效的低宿主毒性的新型抗真菌疗法。我们已经采用了一种强大的高通量分析方法，使我们能够有效地筛选液泡破坏剂（VDAs）的大量化学文库。初步研究证实了该方法的有效性，并确定了库化合物作为vda的命中率为~0.44%。在目标1中，我们将应用筛选来识别各种化学上不同的化合物，这些化合物会破坏白色念珠菌和/或新生念珠菌的液泡。对任何一种真菌有活性的药剂将被认为具有“广谱”活性。然后将测试vda对哺乳动物细胞的毒性。在Aim 2中，将测试具有真菌特异性活性的vda对白色念珠菌和新生念珠菌耐受宿主相关应激的能力、毒力相关属性的表达，以及它们从宿主细胞相互作用的体外模型中清除真菌的能力。最后，在Aim 3中，我们将使用分子方法来定义白色念珠菌液泡在体内支持宿主定植和发病机制的精确功能。这将为选择具有最大抗真菌功效的vda建立标准。

DESCRIPTION (provided by applicant): In recent decades, there has been a dramatic increase in invasive mycoses, including life-threatening disseminated as well as debilitating mucosal infections. The varied condition of affected individuals, diversity of causative fungal species, and similarity between these eukaryotic pathogens and our own cells, all present significant challenges in devising effective therapies with selective toxicity. Current antifungal therapies target a narrow range of fungal components and have serious limitations including patient toxicity, efficacy, available formulations, spectrum of activity and/or the development of resistance. Our molecular studies have shown that disrupting vacuole biogenesis in the most prevalent human fungal pathogen, Candida albicans, severely impairs its capacity to colonize and invade mammalian tissue. Vacuolar function is also essential for the distantly related human pathogen, Cryptococcus neoformans, to survive within the mammalian host. In either fungus, loss of vacuolar function results in hypersensitivity to a variety of host related stresses and severely diminished expression of virulence attributes, indicating that pathogenesis is profoundly impacted on multiple levels. The absence of a closely related organelle in mammalian cells suggests that the fungal vacuole may provide an invaluable opportunity to selectively target the invading fungal pathogen, with specific chemical agents. The purpose of this study is to establish the validity of targeting the fungal vacuole as a novel strategy for antifungal therapy. Specifically, we hypothesize that the vacuole can be exploited to develop effective new antifungal therapies with low host toxicity. We have adapted a powerful high through-put assay that will enable us to efficiently screen vast chemical libraries for Vacuole Disrupting Agents (VDAs). Preliminary studies have validated this approach, and established a hit rate of ~0.44% of library compound as VDAs. In Aim 1 we will apply the screen to identify an assortment of chemically diverse compounds which disrupt the vacuole of C. albicans and/or C. neoformans. Agents active against either fungi will be considered to have 'broad spectrum' activity. VDAs will then be tested for toxicity to mammalian cells. In Aim 2, VDAs with fungal specific activity will be tested for their impact on C. albicans and C. neoformans capacity to endure host related stress, expression of virulence related attributes, and for their ability to clar either fungus from an in vitro model of host cell interaction. Finally, in Aim 3 we will use a molecular approach to define the precise functions of the C. albicans vacuole which support host colonization and pathogenesis in vivo. This will establish criteria for the selection of VDAs with the greatest antifungal efficacy.