A simple vaso-occlusion model for SCD drug discovery

描述（由申请人提供）：自镰状细胞病（SCD）的分子缺陷被发现以来的55年里，研究人员一直在寻找抗镰状细胞病的药物来预防疾病的并发症，但收效甚微。然而，由于现有技术的限制，在这段时间内可以评估的不同化合物的总数必须相对较少。最近，自动化高通量筛选（HTS）使得快速筛选数十万种不同小分子的文库成为可能，以发现有希望的候选药物或可能的药物开发新靶点。目前，还没有适合于HTS的直接作用抗镰状细胞凝集剂的检测方法。使用血红蛋白S溶液的检测方法简单且易于自动化，但不能处理红细胞中的药物摄取或任何其他可能的靶标，而使用完整红细胞的形态学镰状细胞检测速度慢且难以自动化和标准化。本研究的目的是基于简化的血管闭塞模型，开发一种简单而稳健的HTS兼容384孔筛选试验。该测定方法测量在头孢丙基柱中珠珠之间形成的狭窄通道中脱氧红细胞的捕获，并且具有通过光学成像读取的简单而稳定的终点。主屏幕将检测红细胞是否被捕获（阴性结果）或通过凝胶（阳性“抗镰状细胞”结果）。二级分析将测量活度（剂量反应）并检查每次“击中”的作用机制。发展阶段将是：检查所有重要分析变量的贡献，特别是测试红细胞变化的影响；开发可靠、可重复和敏感的最佳检测方法和方案；制定质量控制程序，以确保测试红细胞的可重复性；最后测试和进一步完善的分析和程序，在半自动化筛选bb0 1500化合物，以模拟在HTS环境中使用的分析。

DESCRIPTION (provided by applicant): In the 55 years since the molecular defect responsible for sickle cell disease (SCD) was discovered, researchers have searched for anti-sickling agents to prevent the complications of the disease, but with little success. However, the total number of different chemical compounds that could have been evaluated over this period must be relatively small, due the limitations of the available technology. Recently, automated high throughput screening (HTS), has made it possible to rapidly screen libraries of hundreds of thousands of different small molecules to find promising drug candidates or possible new targets for drug development. At present, there are no assays for direct-acting anti-sickling agents that are well-suited for HTS. Assays that use hemoglobin S solutions are simple and amenable to automation, but do not address drug uptake or any other possible targets in the RBC, while morphologic sickling assays with intact RBCs are slow and difficult to automate and standardize. The aim of this study is to develop a simple and robust HTS compatible 384-well screening assay based upon a simplified model of vaso-occlusion. The assay measures the trapping of deoxygenated RBCs in the narrow channels formed between the beads in a Sephacryl column, and has a simple and stable endpoint that is read by optical imaging. The primary screen will detect whether the RBCs are trapped (the negative result) or pass through the gel (a positive "anti-sickling" result). Secondary assays will measure the activity (dose-response) and examine the mechanisms of action for each "hit". The phases of development will be: Examination of the contribution of all important assay variables, in particular, the influence of variation in the test RBCs; developing optimal assays and protocols that are robust, reproducible and sensitive; development of quality control procedures to insure reproducible performance of the test RBCs; and finally testing and further refinement of the assays and procedures during a semi-automated screen of >1500 compounds to simulate the use of the assays in a HTS environment.