Small Molecule Therapeutics for Sickle Cell Anemia

项目摘要 镰状细胞病(SCD)是一种常见的遗传疾病，影响着大约10万非洲人 美国的遗产，全球超过400万人。这种疾病是由单一氨基酸替代引起的。 在b-珠蛋白中，它促进红细胞(RBC)的刚性镰刀状表型，并最终导致 多种临床表现，如血管闭塞危象、溶血、周围组织损伤、肾脏 功能不全和过早死亡。各种治疗方法已获批准或正在开发中 治疗SCD，包括骨髓移植、基因治疗、诱导胎儿血红蛋白和抗病 探员们。临床批准的治疗方法往往疗效有限、副作用大，或者过于昂贵。 以覆盖大多数SCD患者。SCD的治疗将通过开发新的和 有效的抗病药物。我们的实验室已经开发出一种创新的成像分析方法，利用自动 显微镜和检测红细胞镰状表型的定制算法。这项化验已经被 小型化至1536孔板格式，适用于高通量筛选。与以前的方法不同， 鉴定抗镰状化合物--通常仅限于直接与血红蛋白相互作用的药物-- 新的检测方法以非常高的吞吐量检测红细胞的形态变化，并提供抗- 针对调节血红蛋白聚合或红细胞形态的途径的镰刀状药物，包括 不涉及与血红蛋白直接相互作用的机制。这允许阐明新的类别 SCD的潜在治疗方法以及研究血红蛋白潜在机制的新工具 聚合和红细胞呈镰状。这项提议的R61阶段的总体目标是产生反镰刀 未来临床前和临床研究的候选人。我们将大规模、高通量地开展药物研发。 筛选以确定防止患病红细胞镰状的分子。由此产生的命中将在剂量上进行验证 反应和一系列二次检测。先导化合物的选择将基于生理上的 血红蛋白氧亲和力和红细胞膜变形性的可接受变化。进一步的优化将 在R33阶段通过药物化学进行。作为R33的一部分，将合成铅支架 并对抗镰刀效力、血红蛋白氧亲和力和改善红细胞膜变形性进行了评估 以确定最适合动物安全性和有效性研究的制剂。表达人珠蛋白的Townes HbSS小鼠 基因将被用来测试短期致死率、抗病活性、红细胞氧亲和力和红细胞半衰弱。 活体内的生命。作为本提案的最终交付方案，3种或更多安全有效的候选药物 小鼠模型将被选择用于未来的临床前研究。

Project Summary Sickle Cell Disease (SCD) is a common genetic condition that affects approximately 100,000 people of African heritage in the US, and more than 4 million worldwide. The disease is caused by a single amino acid substitution in b-globin that promotes a rigid sickle shape phenotype of red blood cells (RBCs) and eventually leads to numerous clinical manifestations such as vaso-occlusive crises, hemolysis, peripheral tissue damage, renal insufficiency, and premature death. Various therapeutic approaches have been approved or are in development for SCD, including bone marrow transplantation, gene therapy, induction of fetal hemoglobin, and anti-sickling agents. Clinically approved therapies often have limited effectiveness, significant side effects, or are too costly to reach the majority of SCD patients. Treatment of SCD would be advanced by development of novel and effective anti-sickling drugs. Our laboratory has developed an innovative imaging assay utilizing automated microscopy and customized algorithms that detect the sickled phenotype of RBCs. This assay has been miniaturized to the 1536-well plate format suitable for high throughput screening. Unlike previous approaches to identifying anti-sickling compounds – which are often limited to drugs that interact directly with hemoglobin – the new assay detects morphological changes in RBCs at very high throughput and provides identification of anti- sickling agents targeting pathways modulating hemoglobin polymerization or RBC morphology, including mechanisms that do not involve direct interaction with hemoglobin. This allows elucidation of new classes of potential therapeutics for SCD, as well as novel tools for investigating the underlying mechanisms of hemoglobin polymerization and RBC sickling. The overall goal of the R61 phase of this proposal is to generate anti-sickling candidates for future pre-clinical and clinical studies. We will carry out large scale, high throughput drug screening to identify molecules that prevent sickling of diseased RBCs. The resulting hits will be verified in dose response and a series of secondary assays. Lead compounds will be selected based on physiologically acceptable changes of hemoglobin oxygen affinity and RBC membrane deformability. Further optimization will be carried out in the R33 phase by medicinal chemistry. As part of the R33, lead scaffolds will be synthesized and evaluated for anti-sickling potency, hemoglobin oxygen affinity, and improved RBC membrane deformability to identify agents best suited for animal safety and efficacy studies. Townes HbSS mice expressing human globin genes will be used to test short term lethality, efficacy of anti-sickling activity, RBC oxygen affinity, and RBC half- life in vivo. As the final deliverable of this proposal, 3 or more drug candidates that are safe and efficacious in the mouse model will be selected for future pre-clinical studies.