Assay for GI Drug Discovery

描述（由申请人提供）：离子通道靶向药物的发现受到无法利用生理学和药理学相关测量来表征化合物活性的基于细胞的测定的阻碍。本项目将开发一种测定细胞内氯离子和pH值的方法，作为直接生理测量，以询问氯离子通道和质子转运蛋白，用于GI药物发现。该提案的第一阶段将侧重于使用T84细胞合成、表征和验证氯离子传感指示剂。本提案的第II阶段将重点关注pH传感指示剂的开发，pH传感测定与Cl测定的整合;通过描述GI药物发现的[Cl-]i和pHi的细胞内偶联动力学，使用T84细胞验证联合氯化物和pH测定;以及扩大商业化测定的生产规模。公共卫生相关性：氯离子通道和质子转运是治疗腹泻、便秘和胃反流的两个主要药物发现胃肠道（GI）靶点。Cytoptics公司提议开发新一代氯离子和pH传感指示剂，以同时测量细胞内氯离子浓度、[Cl-]i和pHi，用于基于细胞的GI药物发现。该I期提案将重点关注1）氯离子传感指示剂的合成和光谱表征; 2）指示剂加载方案的开发;以及3）T84细胞中[Cl-]i的生理药理学测量。这将使氯离子传感测定试剂盒在I期完成后易于上市用于GI药物发现和开发。本提案的第II阶段将重点关注pH传感指示剂的开发，pH传感测定与Cl测定的整合;通过描述GI药物发现的[Cl-]i和pHi的细胞内偶联动力学，使用T84细胞验证联合氯化物和pH测定;以及扩大商业化测定的生产规模。

DESCRIPTION (provided by applicant): Ion channel targeted drug discovery has been hampered by the unavailability of cell-based assays that utilize physiological and pharmacological relevant measurements to characterize compound activity. This project will develop an assay to measure intracellular chloride ion and pH as direct physiological measurements to interrogate the chloride ion channels and proton transporter(s) for GI drug discovery. The Phase I of this proposal will focus on the synthesis, characterization and validation of the chloride ion sensing indicator using T84 cells. The Phase II of this proposal will focus on the development of the pH sensing indicator, integration of the pH sensing assay with the Cl assay; validation of the joint chloride and pH assay using T84 cells by delineating the intracellular coupling dynamics of [Cl-]i and pHi for GI drug discovery; and scaling up the manufacturing of the assays for commercialization. PUBLIC HEALTH RELEVANCE: Ion channels of chloride and proton transports 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 to measure intracellular chloride ion concentrations, [Cl-]i, and pHi simultaneously for cell-based GI drug discovery. This Phase I proposal will focus on the 1) synthesis and spectral characterization of chloride ion sensing indicator; 2) development of the loading protocols of the indicator; and 3) physio-pharmacological measurements of [Cl-]i in T84 cells. This will enable a chloride ion sensing assay kit to be readily marketed for GI drug discovery and development following the completion of the Phase I. The Phase II of this proposal will focus on the development of the pH sensing indicator, integration of the pH sensing assay with the Cl assay; validation of the joint chloride and pH assay using T84 cells by delineating the intracellular coupling dynamics of [Cl-]i and pHi for GI drug discovery; and scaling up the manufacturing of the assays for commercialization.