Assay for GI drug discovery

描述（由申请人提供）：由于缺乏基于细胞的高通量筛选（HTS）试验来生理学和生物学表征化合物活性，离子通道药物的发现受到阻碍。Cytoptics公司（Cytoptics）提出开发新一代氯离子和pH传感发光量子点（QD）指示剂，以测量细胞内氯离子浓度、[Cl-]i和pHi，用于荧光显微镜和基于细胞的高通量筛选（HTS）靶向GI药物发现。这些荧光显微镜和基于细胞的HTS测定利用尺寸可调、非重叠发射波长、离子选择性QD来同时测量T84 WT/T84 C1 C2以及HEK 293 WT/HEK 293 C1 C-2细胞中的[Cl-]i和pHi。使用量子点（QD）作为Forester共振能量转移（FRET）供体和与QD缀合的相应Cl-和H+化学传感器作为受体，这两种纳米传感器Cl-QD和pH-QD根据与其相应Cl-和H+传感器结合的Cl-和H+的浓度猝灭其QD的荧光强度。我们已经完成了所有提出的I期目标，即1）氯离子传感发光QD（Cl-QD）的合成和光谱表征; 2）开发Cl-QD到T84细胞中的加载方案;和3）使用Cl-QDTM测量T84 WT和T84 C1 C-2细胞中的生理药理学[Cl-]i响应。在第二阶段，Cytoptics将整合并扩展第一阶段的成就，以生产六种针对基于细胞的GI药物靶点发现市场的检测方法。它们是（1）用于荧光显微镜的Cl-QDTM测定法;（2）用于基于细胞的HTS的Cl-QDTM测定法;（3）用于荧光显微镜的pH-QD测定法;（4）用于基于细胞的HTS的pH-QD测定法;（5）用于基于细胞的HTS的Cl-QDTM和pH-QD整合测定法;和（6）使用稳定的T84 C1 C-2和HEK 293 C1 C-2细胞的靶向C1 C-2通道的Cl-QDTM和pH-QD整合测定用于GI药物发现。这些新的检测方法解决了基于细胞的HTS药物发现的关键需求，也将广泛影响离子信号转导中的药物发现研究。 公共卫生相关性：氯离子通道和质子转运蛋白是治疗腹泻、便秘和胃反流的两个主要药物发现胃肠道（GI）靶点。Cytoptics公司提出开发新一代氯离子和pH传感指示剂（分别为Cl-QD和pH-QD），以首次同时测量上皮细胞中细胞内氯离子浓度（[Cl-]i）和pH（pHi）的动态。这些纳米传感器将用于询问Cl-通道和H+-转运蛋白，作为GI药物发现的直接生理和药理学测定。使用具有不同发射波长的量子点（QD）用于这两种纳米传感器，Cl-QD和pH-QD发射不同的颜色，其非干扰荧光强度根据各自的Cl-和H+浓度而变化，Cytoptics将利用这两种新型基于QD的纳米传感器进行六种检测，以瞄准基于细胞的GI HTS药物发现市场。这些新的测定也将提供离子细胞信号转导机制，迄今为止是不可能的无与伦比的见解。

DESCRIPTION (provided by applicant): Ion channel drug discovery has been hampered by the lack of cell-based high throughput screening (HTS) assay to physiologically and pharmacologically characterize compound activities. Cytoptics Corporation (Cytoptics) proposes to develop a new generation of chloride ion and pH sensing luminous quantum dots (QD) indicators to measure intracellular chloride ion concentrations, [Cl-]i, and pHi for fluorescence microscopy and cell-based high throughput screening (HTS) targeting GI drug discovery. These fluorescence microscopy and cell-based HTS assays utilize the size-tunable, non- overlapping emission wavelengths, ion-selective QD to measure [Cl-]i and pHi simultaneously in T84WT /T84C1C2 as well as in HEK293WT/HEK293C1C-2 cells. Using the quantum dots (QD) as Forester Resonance Energy Transfer (FRET) donors and the respective Cl- and H+ chemical sensors conjugated to the QD as acceptors, these two nanosensors, Cl-QD and pH-QD, quench their fluorescence intensities of the QD according to the concentrations of the Cl- and H+ bound to their respective Cl- and H+ sensors. We have accomplished all the proposed Phase I objectives, namely, 1) synthesis and spectral characterization of chloride ion sensing luminous QD, Cl-QD; 2) development of a loading protocol of the Cl-QD into T84 cells; and 3) measurements of the physio-pharmacological [Cl-]i responses in T84WT and T84C1C-2 cells using the Cl- QDTM. In Phase II, Cytoptics will incorporate and extend the accomplishments of Phase I to produce six assays to target the cell-based GI drug target discovery market. They are (1) the Cl-QDTM assay for fluorescence microscopy; (2) the Cl-QDTM assay for cell- based HTS; (3) the pH-QD assay for fluorescence microscopy; (4) the pH-QD assay for cell-based HTS; (5) the Cl-QDTM and pH-QD integrated assay for cell-based HTS; and (6) the Cl-QDTM and pH-QD integrated assay targeting C1C-2 channels using the stable T84C1C-2 and HEK293C1C-2 cells for GI drug discovery. These novel assays address the critical needs of cell-based HTS drug discovery and will also broadly impact drug discovery research in ionic signal transduction. PUBLIC HEALTH RELEVANCE: Chloride channels and proton transporters are the two predominant drug discovery gastrointestinal (GI) target sites for the treatments of diarrhea, constipation and gastric reflux. Cytoptics Corporation proposes to develop a new generation of chloride ion and pH sensing indicators (Cl-QD and pH-QD, respectively) to simultaneously measure the dynamics of the intracellular chloride ion concentrations ([Cl-]i) and pH (pHi) in epithelial cells for the first time. These nanosensors will be utilized to interrogate the Cl-channels and H+-transporters as direct physiological and pharmacological assays for GI drug discovery. Using the quantum dots (QD) with different emission wavelengths for these two nanosensors, Cl-QD and pH-QD emit separate colors with their non-interfering fluorescence intensities varied according to their respective concentrations of Cl- and H+, Cytoptics will produce six assays utilizing these two novel QD-based nanosensors to target the cell-based GI HTS drug discovery market. These novel assays will also provide unparallel insights in the ionic cellular signal transduction mechanisms that heretofore not been possible.