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，包括危及生命的播散性和使人衰弱的粘膜感染。受影响个体的不同状况，致病真菌物种的多样性以及这些真核病原体与我们自己细胞之间的相似性，都对设计具有选择性毒性的有效疗法提出了重大挑战。目前的抗真菌疗法针对范围狭窄的真菌组分，并且具有严重的局限性，包括患者毒性、功效、可用制剂、活性谱和/或耐药性的发展。我们的分子研究表明，破坏空泡生物发生在最普遍的人类真菌病原体，白色念珠菌，严重损害其能力，殖民和入侵哺乳动物组织。液泡功能对于远亲人类病原体新型隐球菌在哺乳动物宿主中生存也至关重要。在任一真菌中，空泡功能的丧失导致对多种宿主相关应激的超敏反应和毒力属性的表达严重减少，表明发病机制在多个水平上受到深刻影响。在哺乳动物细胞中缺乏一个密切相关的细胞器表明，真菌液泡可能提供了一个宝贵的机会，选择性地针对入侵的真菌病原体，与特定的化学试剂。本研究的目的是建立一个新的抗真菌治疗策略的有效性，针对真菌空泡。具体来说，我们假设空泡可以被利用来开发有效的新的抗真菌疗法，具有低宿主毒性。我们已经采用了一种强大的高通量测定，这将使我们能够有效地筛选大量的化学库中的干扰素（VDA）。初步研究已经验证了这种方法，并建立了约0.44%的文库化合物作为VDA的命中率。在目标1中，我们将应用筛选来鉴定各种各样的化学上不同的化合物，这些化合物破坏了C. albicans和/或C.新人类对任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。在目标2中，将测试具有真菌比活性的VDA对C.白色念珠菌和C.新型真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任一真菌的能力。最后，在目标3中，我们将使用分子方法来定义C的精确功能。白色念珠菌空泡，支持宿主定植和体内致病。这将建立选择具有最大抗真菌疗效的VDA的标准。

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.