A simple vaso-occlusion model for SCD drug discovery

用于 SCD 药物发现的简单血管闭塞模型

• 批准号: 7067050
• 负责人: TIMOTHY C FISHER
• 金额: $ 12.19万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7067050
• 关键词: African; African American; blood disorder chemotherapy; blood vessel occlusion; cardiovascular disorder prevention; chemoprevention; clinical research; disease /disorder model; drug discovery /isolation; drug screening /evaluation; erythrocytes; high throughput technology; human subject; method development; molecular /cellular imaging; patient oriented research; sickle cell anemia

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.

描述（由申请人提供）：在发现负责镰状细胞疾病（SCD）的分子缺陷（SCD）以来的55年中，研究人员已经搜索了反校园剂，以防止这种疾病的并发症，但成功却很少。但是，由于可用技术的局限性，在此期间可以评估的不同化学化合物的总数必须相对较小。最近，自动化的高通量筛选（HTS）使得可以快速筛选成千上万个不同的小分子的库，以找到有希望的候选药物或可能的药物开发目标。 目前，尚无适合HTS的直接作用反踢剂的测定法。使用血红蛋白溶液的测定很简单且可以自动化，但不解决RBC中的药物摄取或任何其他可能的靶标，而完整的RBC的形态疾病测定法是缓慢且难以自动化和标准化的。这项研究的目的是基于简化的血管批准模型开发一种简单且可靠的HTS兼容384孔筛选测定法。该测定法测量了在Sephacryl柱中珠子之间形成的狭窄通道中的脱氧RBC的捕获，并且具有简单稳定的端点，该端点是通过光学成像读取的。主要屏幕将检测到RBC是被困（负结果）还是通过凝胶（一个阳性的“反踢”结果）。 次要测定将测量活动（剂量反应），并检查每个“命中”的作用机理。发展的阶段将是：检查所有重要测定变量的贡献，尤其是测试RBC变异的影响；开发可靠，可重复和敏感的最佳测定法和协议；开发质量控制程序，以确保测试RBC的可重复性性能；最后，在半自动化的屏幕> 1500种化合物中测试和进一步改进了测定和过程，以模拟HTS环境中测定法的使用。