Fungal calcium channels as therapeutic targets for AIDS-associated opportunistic

真菌钙通道作为艾滋病相关机会性感染的治疗靶点

• 批准号: 8015377
• 负责人: ANGIE GELLI
• 金额: $ 37.17万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8015377
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adjuvant; Anabolism; Antifungal Agents; Attenuated; Biological; Biological Assay; Calcium Channel; Candida albicans; Cell Line; Cells; Complex; Coupled; Cryptococcal Meningitis; Cryptococcus neoformans; Cryptococcus neoformans infection; Development; Doctor of Philosophy; Drug Combinations; Drug Compounding; Economics; Endoplasmic Reticulum; Environment; Equilibrium; Ergosterol; Event; Fluorescence Resonance Energy Transfer; Fungal Meningitis; Growth; Health; Image; Immune response; Immunosuppression; In Vitro; Industrial fungicide; Lead; Life; Maintenance Therapy; Mammalian Cell; Mediating; Meningoencephalitis; Modeling; Molecular; Mus; Mycoses; NIH Program Announcements; Opportunistic Infections; Patch-Clamp Techniques; Patients; Pharmaceutical Preparations; Physiological Processes; Physiology; Play; Preclinical Drug Evaluation; Role; Screening procedure; Signal Transduction; Structure of thyroid parafollicular cell; T-Lymphocyte; Testing; Tissues; Triazoles; Virulence; base; channel blockers; drug development; endoplasmic reticulum stress; high throughput screening; immune function; in vivo; inhibitor/antagonist; mortality; mutant; novel; novel strategies; patch clamp; pathogen; prevent; small molecule; small molecule libraries; synergism; therapeutic target; tool

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.

描述（由申请人提供）：我们开发了一种简单可靠的测定法来鉴定 Cch1-Mid1 Ca2+ 通道的小分子抑制剂。这种复合物促进了真菌生理学的许多关键方面。在新型隐球菌、白色念珠菌和光滑念珠菌中，Cch1-Mid1 信号传导在耐受和存活由麦角甾醇生物合成抑制剂（例如三唑抗真菌药物）诱导的内质网应激条件中发挥着至关重要的作用。新型隐球菌是艾滋病患者和其他缺乏足够的 T 细胞依赖性免疫功能的患者中真菌性脑膜炎的主要原因。小分子调节剂将为研究 ER 应激期间 Ca2+ 介导的信号事件提供关键工具，甚至可能为重要佐剂的开发奠定基础，这些佐剂可能与现有抗真菌药物协同作用，增强对危及生命的真菌感染的治疗。然而，目前还没有 Cch1 信号传导的抑制剂。我们的检测利用了模型真菌病原体新型隐球菌，它需要 Cch1-Mid1 Ca2+ 通道才能在低 Ca2+ 环境中生存。在低 Ca2+ 培养基上，如果存在阻断 Cch1 活性并阻止 Cch1 介导的信号传导的小分子，新型隐球菌的生长将会受到抑制。因此，该测定使用简单的读数（生长与生长停滞）来识别抑制 Cch1-Mid1 通道的小分子。由于该测定具有重复性且读数简单，因此该测定非常适合高通量筛选。我们将使用计数器筛选来确定先导化合物的优先级，并通过膜片钳技术和基于 FRET 的 Ca2+ 成像来验证每个分子的生物学相关性。我们预计，使用这种测定法进行的筛选可能会在理解 Cch1-Mid1 通道在关键 Ca2+ 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迈出关键的一步。该项目是对开发高通量药物筛选检测方法的计划公告的响应。公共健康相关性：本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目标的可行性的手段。由于真菌感染对于免疫反应受到抑制的患者来说会危及生命，因此开发更有效、耐受性更好的药物至关重要。

项目成果

Flucytosine resistance in Cryptococcus gattii is indirectly mediated by the FCY2-FCY1-FUR1 pathway.

格特隐球菌中的氟胞嘧啶抗性是由 FCY2-FCY1-FUR1 途径间接介导的。

• DOI: 10.1093/mmy/myx135
• 发表时间: 2018
• 期刊: Medical mycology
• 影响因子: 2.9
• 作者: Vu,Kiem;Thompson3rd,GeorgeR;Roe,ChandlerC;Sykes,JaneE;Dreibe,ElizabethM;Lockhart,ShawnR;Meyer,Wieland;Engelthaler,DavidM;Gelli,Angie
• 通讯作者: Gelli,Angie