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）：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化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，在这一时期内可以评价的不同化合物的总数肯定相对较少。最近，自动化高通量筛选（HTS）已经使得快速筛选数十万种不同小分子的文库以发现有希望的候选药物或用于药物开发的可能的新靶点成为可能。 目前，还没有非常适合HTS的直接作用抗镰状化剂的测定。使用血红蛋白S溶液的测定简单且易于自动化，但不能解决RBC中的药物摄取或任何其他可能的靶点，而使用完整RBC的形态学镰状化测定缓慢且难以自动化和标准化。本研究的目的是开发一个简单和强大的HTS兼容的384孔筛选分析的基础上简化模型的血管闭塞。该测定法测量了在Sephacryl柱中的珠之间形成的狭窄通道中脱氧RBC的捕获，并且具有通过光学成像读取的简单且稳定的终点。初级筛选将检测RBC是否被捕获（阴性结果）或通过凝胶（阳性“抗镰状化”结果）。 次级试验将测量活性（剂量-反应）并检查每次“击中”的作用机制。开发阶段包括：检查所有重要测定变量的贡献，特别是供试RBC变异的影响;开发稳健、可重现和灵敏的最佳测定和方案;开发质量控制程序，以确保供试RBC的可重现性能;最后，在>1500种化合物的半自动筛选过程中测试和进一步改进测定法和程序，以模拟在HTS环境中使用测定法。