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）测定来生理和药理学表征化合物活性，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）指标，用于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）靶向胃肠道药物发现。这些荧光显微镜和基于细胞的HTS分析利用可调尺寸，非重叠发射波长，离子选择性QD来同时测量T84WT /T84C1C2以及HEK293WT/HEK293C1C-2细胞中的[Cl-]i和pHi。利用量子点（QD）作为Forester共振能量转移（FRET）供体，结合Cl-和H+的化学传感器作为受体，这两种纳米传感器Cl-QD和pH-QD根据各自的Cl-和H+传感器结合的Cl-和H+的浓度淬灭其QD的荧光强度。我们已经完成了第一阶段的所有目标，即：1)氯离子传感发光量子点Cl-QD的合成和光谱表征；2)建立Cl-QD在T84细胞内的加载方案；3)使用Cl- QDTM测量T84WT和T84C1C-2细胞的生理药理学[Cl-]i反应。在第二阶段，Cytoptics将整合并扩展第一阶段的成果，生产六种针对基于细胞的胃肠道药物靶点发现市场的检测方法。它们是(1)荧光显微镜的Cl-QDTM测定法；(2)基于细胞的HTS的Cl-QDTM检测；(3)荧光显微镜pH-QD法；(4)细胞基HTS的pH-QD检测；(5)基于细胞的HTS的Cl-QDTM和pH-QD联合检测；(6)利用稳定的T84C1C-2和HEK293C1C-2细胞靶向C1C-2通道的Cl-QDTM和pH-QD联合检测用于胃肠道药物发现。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的药物发现研究。

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.