Fungal calcium channels as therapeutic targets for AIDS-associated opportunistic

描述(由申请人提供)：我们开发了一种简单可靠的方法来鉴定Cch1-MID1钙通道的小分子抑制剂。这种复合体促进了真菌生理学的许多关键方面。在新生隐球菌、白色念珠菌和光滑念珠菌中，Cch1-MID1信号在耐受和存活由麦角甾醇生物合成抑制剂(如三唑类抗真菌药物)诱导的内质网应激条件中起着至关重要的作用。新生芽孢杆菌是艾滋病患者和其他缺乏足够T细胞依赖免疫功能的患者的真菌性脑膜炎的主要原因。小分子调节剂将为研究内质网应激过程中钙离子介导的信号事件提供关键工具，甚至可能导致重要佐剂的开发，这些佐剂可能与现有的抗真菌药物协同作用，并加强对威胁生命的真菌感染的治疗。然而，目前还没有Cch1信号的抑制剂。我们的分析利用了模式真菌病原体新生葡萄球菌，它需要Cch1-MID1钙通道才能在低钙环境中生存。在低钙条件下，如果有小分子阻断Cch1的活性并阻止Cch1介导的信号传导，新生隐球菌的生长将被抑制。因此，该分析使用简单的读数(生长与生长停滞)来识别抑制Cch1-MID1通道的小分子。由于该检测方法重复性好，读数简单，因此该检测方法非常适合于高通量筛选。我们将使用计数器筛选来确定先导化合物的优先顺序，并通过膜片钳技术和基于FRET的钙成像来验证每个分子的生物相关性。我们预计，使用这种检测方法的筛选可能会在理解Cch1-MID1通道在关键的钙离子介导的信号事件中的作用以及开发治疗真菌感染的新策略方面向前迈出关键的一步。该项目是对高通量药物筛选检测方法开发项目公告的回应。公共卫生相关性：这项研究旨在了解钙通道在真菌病原体中控制基本生理过程的作用，以此作为评估其作为抗真菌药物开发靶点的可行性的一种手段。由于真菌感染对免疫反应受抑制的患者来说是危及生命的，因此开发更有效、更耐受的药物是至关重要的。

DESCRIPTION (provided by applicant): We have developed a simple and reliable assay to identify small molecule inhibitors of the Cch1-Mid1 Ca2+ channel. This complex promotes many critical aspects of fungal physiology. In Cryptococcus neoformans, Candida albicans and C. glabrata, Cch1-Mid1 signaling plays a crucial role in tolerating and surviving ER stress conditions that are induced by inhibitors of ergosterol biosynthesis, such as triazole antifungal drugs. C. neoformans is the leading cause of fungal meningitis in AIDS patients and other patients without adequate T-cell-dependent immune functions. Small molecule modulators would provide critical tools for studying Ca2+- mediated signaling events during ER stress and might even lead the way to the development of important adjuvants that might synergize with existing antifungals and enhance the treatment of life-threatening fungal infections. However, there are currently no inhibitors of Cch1 signaling. Our assay takes advantage of the model fungal pathogen C. neoformans, which requires the Cch1-Mid1 Ca2+ channel for its survival in low Ca2+ environments. On low Ca2+ media in the presence of small molecules that block Cch1 activity and prevent Cch1-mediated signaling, the growth of C. neoformans would be arrested. Thus the assay uses a simple readout (growth versus growth arrest) to identify small molecules that inhibit the Cch1-Mid1 channel. Because the assay is reproducible and has a simple readout, this assay is highly amenable to high-throughput screening. We will use a counter screen to prioritize the lead compounds and also validate the biological relevance of each molecule through patch clamp techniques and FRET- based Ca2+ imaging. We anticipate that a screen using this assay may provide a crucial step forward in understanding the role of the Cch1-Mid1 channel in critical Ca2+-mediated signaling events and in developing a novel strategy for treating fungal infections. This project is responsive to the program announcement for the development of assays for high-throughput drug screening. PUBLIC HEALTH RELEVANCE: This study aims to understand the role of a calcium channel that governs essential physiological processes in fungal pathogens as a means to assess its viability as a target for antifungal drug development. Because fungal infections are life threatening for patients with a suppressed immune response, the development of more efficacious and better-tolerated drugs is essential.