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

描述（由申请人提供）：V-ATP酶是细胞和全身pH稳态所必需的主动转运蛋白。V-ATP酶功能的抑制扰乱细胞pH;并且因为pH影响几乎所有的细胞过程，大多数细胞在没有功能性V-ATP酶的情况下死亡。V-ATP酶泵的抑制可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-ATP酶依赖性分选机制。本研究旨在鉴定在体内高效抑制酵母V-ATP酶的小分子。具有抗真菌活性的V-ATP酶抑制剂有望阻断毒力。 我们提出了高通量筛选（HTS）的分子库探针中心网络（MLPCN）库的小分子（约330，000化合物）。为了实现这一目标，我们与新墨西哥州大学分子发现中心合作开发了两种HTS流式细胞术测定。这些测定使用pH依赖性荧光探针和HyperCyt来监测模型酵母S的胞质溶胶（初级测定）和液泡腔（次级测定）中的pH变化。啤酒。对于一级测定，将捕获在胞质溶胶中的pH敏感性GFP（pHLuorin）用于监测胞质溶胶酸化。由于通过抑制V-ATP酶泵使细胞溶质酸化与液泡腔碱化同时发生，因此在二级试验中将对降低细胞溶质pH值的化合物进行质疑，以评估其对液泡pH值的影响。对于二级试验，将化合物加入到用截留在液泡中的BCECF染色的细胞中。V-ATP酶抑制剂可使BCECF液泡pH升高，荧光强度增强。抑制剂将在直接测量液泡膜中ATP水解的中等通量三级测定中确认。在三级试验中，通过比较具有正常V-ATP酶功能的野生型膜和具有对巴夫洛霉素和康卡霉素耐药的V-ATP酶的突变型膜，将增强抗真菌选择性。将鉴定模拟康卡霉素和巴弗洛霉素（抑制酵母和哺乳动物V-ATP酶）的抑制剂和通过其他机制起作用的抑制剂。进一步的研究将确定抑制剂对酶动力学和组装的影响。在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-ATP酶抑制剂也将有助于了解pH调节和真菌疾病的分子机制。 公共卫生相关性：本研究提出了MLPCN文库的高通量筛选，以鉴定在体内高效抑制真菌V-ATP酶泵的小分子。V-ATP酶泵是真菌毒力所必需的蛋白质，预期酵母V-ATP酶抑制剂可阻断毒力。在这项研究中确定的V-ATP酶抑制剂将提供机会，开发新的治疗工具，以治疗念珠菌和数百个其他新兴的机会性真菌感染。他们还将有助于了解真菌疾病的分子机制。

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.