Flow Cytometry HTS of Small Molecules that Regulate V-ATPase Proton Transport in

调节 V-ATP 酶质子转运的小分子的流式细胞术 HTS

• 批准号: 8069470
• 负责人: Karlett J Parra
• 金额: $ 3.77万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8069470
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Alkalinization; Antifungal Agents; Biological Assay; Biological Factors; Candida; Candidiasis; Cell physiology; Cells; Chemicals; Collaborations; Cytosol; Detection; Disease; Dose; Endosomes; Enzyme Kinetics; Enzymes; Exhibits; Flow Cytometry; Fluorescence; Follow-Up Studies; Human; Immunoprecipitation; Knowledge; Libraries; Life; Mammalian Cell; Measures; Membrane; Methods; Molecular; Molecular Bank; Monitor; Mycoses; New Mexico; PHluorin; Protein Isoforms; Proteins; Proton Pump; Protons; Pump; Regulation; Research; Resistance; Saccharomyces cerevisiae; Sorting - Cell Movement; Staining method; Stains; Testing; Tissues; Universities; Vacuole; Vesicular Transport Proteins; Virulence; Yeast Model System; Yeasts; design; high throughput screening; in vitro activity; in vivo; inhibitor/antagonist; lysosome membrane; mutant; novel therapeutics; pH Homeostasis; pathogen; prevent; ratiometric; response; small molecule; small molecule libraries; tool; trafficking; vacuolar H+-ATPase

DESCRIPTION (provided by applicant): V-ATPases are active transporters necessary for cellular and systemic pH homeostasis. Inhibition of V- ATPase function disturbs cellular pH; and because the pH influences nearly all cellular processes, most cells die without functional V-ATPases. Inhibition of V-ATPase pumps prevents virulence by Candida and other pathogens because trafficking of virulence-related proteins rely on V-ATPase-dependent sorting mechanisms. This study is aimed at identifying small molecules that inhibit yeast V-ATPases with high potency in vivo. V- ATPase inhibitors with anti-fungal activity are expected to block virulence. We propose high-throughput screening (HTS) of the Molecular Libraries Probe Centers Network (MLPCN) library of small molecules (~330,000 compounds). To accomplish this aim we developed two HTS flow cytometry assays in collaboration with the University of New Mexico Center for Molecular Discovery. These assays use pH-dependent fluorescence probes and HyperCyt@ to monitor pH changes in the cytosol (Primary Assays) and vacuolar lumen (Secondary Assays) of the model yeast S. cerevisiae. For the Primary Assays the pH-sensitive GFP (pHLuorin) trapped in the cytosol is used to monitor cytosolic acidification. Since acidification of the cytosol by inhibition of V-ATPase pumps goes together with alkalinization of the vacuolar lumen, compounds that lower the cytosolic pH will be questioned in Secondary Assays to assess their effect on the vacuolar pH. For the Secondary Assays the compounds are added to cells stained with BCECF trapped in the vacuoles. V-ATPase inhibitors will increase the vacuolar pH and enhance the fluorescence intensity of BCECF. Inhibitors will be confirmed in medium-throughput Tertiary Assays that measure ATP hydrolysis directly in vacuolar membranes. Anti-fungal selectivity will be enhanced in the Tertiary Assays by comparing wild-type membranes, which have normal V-ATPase function, and mutant membranes, which have V-ATPases resistant to bafilomycin and concanamycin. The inhibitors that mimic concanamycin and bafilomycin (inhibit both yeast and mammalian V-ATPases) and the inhibitors that act by other mechanisms will be identified. Further studies will establish the effects that inhibitors have on the enzyme kinetics and assembly. Small molecules identified in this study will offer opportunities to develop new therapeutic tools against pathogens such as Candida and hundreds of emerging opportunistic fungal infections. V-ATPase inhibitors will also contribute knowledge to the understanding of the molecular mechanisms governing pH regulation and fungal diseases. PUBLIC HEALTH RELEVANCE: This study proposes high throughput screening of the MLPCN library to identify small molecules that inhibit fungal V-ATPase pumps with high potency in vivo. V-ATPase pumps are proteins necessary for fugal virulence and yeast V-ATPase inhibitors are expected to block virulence. V-ATPase inhibitors identified in this study will offer opportunities to develop new therapeutic tools to treat Candida and hundreds of others emerging opportunistic fungal infections. They will also contribute knowledge to the understanding of the molecular mechanisms governing fungal diseases.

描述(由申请人提供)：V-ATPase是细胞和系统pH动态平衡所必需的活性转运体。V-ATPase功能的抑制会扰乱细胞的pH；由于pH影响几乎所有的细胞过程，大多数细胞在没有V-ATPase功能的情况下死亡。V-ATPase泵的抑制可以防止念珠菌和其他病原体的毒力，因为毒力相关蛋白的运输依赖于V-ATPase的分选机制。本研究旨在鉴定在体内高效抑制酵母V-ATPase的小分子。具有抗真菌活性的V-ATPase抑制剂有望阻止毒力。我们提出了分子文库探测中心网络(MLPCN)小分子(~330,000化合物)文库的高通量筛选(HTS)。为了实现这一目标，我们与新墨西哥大学分子发现中心合作开发了两种HTS流式细胞术分析方法。这些检测方法使用pH依赖的荧光探针和HyperCyt@来监测模式酵母酿酒酵母的胞浆(初级分析)和液泡腔(二级分析)的pH变化。在初步分析中，胞浆中对pH敏感的绿色荧光蛋白(PHLuorin)被用来监测胞浆酸化。由于通过抑制V-ATPase泵使胞浆酸化伴随着液泡腔的碱化，降低胞液pH值的化合物将在二次试验中被质疑，以评估它们对液泡pH值的影响。对于二次化验，这些化合物被加入到被BCECF染色的细胞中，这些细胞被困在液泡中。V-ATPase抑制剂可提高BCECF的空泡pH，增强其荧光强度。抑制剂将在中通量第三级测试中得到确认，该测试直接测量液泡膜中的ATP水解度。通过比较具有正常V-ATPase功能的野生型膜和V-ATPase耐药的突变膜，将在第三次检测中提高抗真菌的选择性。将确定模仿刀豆素和巴菲罗霉素的抑制剂(抑制酵母和哺乳动物的V-ATPase)以及通过其他机制发挥作用的抑制剂。进一步的研究将确定抑制剂对酶的动力学和组装的影响。这项研究中发现的小分子将为开发新的治疗工具提供机会，以对抗念珠菌和数百种新出现的机会性真菌感染等病原体。V-ATPase抑制剂也将有助于理解控制pH调节和真菌疾病的分子机制。 公共卫生相关性：这项研究建议对MLPCN文库进行高通量筛选，以确定在体内高效性抑制真菌V-ATPase泵的小分子。V-ATPase泵是真菌毒力所必需的蛋白质，酵母V-ATPase抑制剂有望阻断毒力。这项研究中确定的V-ATPase抑制剂将提供机会开发新的治疗工具来治疗念珠菌和其他数百种正在出现的机会性真菌感染。他们还将为了解控制真菌疾病的分子机制贡献知识。