DEVELOPMENT OF SITE SPECIFIC ANTISICKLING AGENTS

最有效、毒性最小的止痛药很可能是那些 在负责联系的地点与哈佛商学院做出具体反应 聚合反应。使用各种实验和计算方法 技术，我们最近开发了一种成功的设计策略 定点定向靶向HBS聚合抑制剂，提供 通过与A结合竞争性抑制HBS聚合 靠近2，3-BPG结合位点和侧向的特定区域 HBS分子上的“受体”接触部位。我们现在提议继续进行 设计和开发新型止痛剂的两种方法 特别是在这个地方。第一个是基于我们最近的 目前批准的几种药物的鉴定也是高度有效的 HBS聚合的活性阻聚剂。我们提出了新的结构 修改以包括我们的共价靶向策略。这两个 将对国产药物和共价衍生物进行研究 有效性和特异性。Secons方法将基于 一系列结构上与之相关的混合磷酸酸酐 BPG并被预测为对其结合位点具有高亲和力， 提供了一种可供选择的目标策略。的速度和程度 用共价剂修饰HbA和HbS将是决定性的。已修改 多肽将从球蛋白中分离出来，并进行测序以确定 具体的改造地点。将使用NMT和X射线结晶学 确定修饰的Hb的抑制剂结合几何构型。结果 将与我们的计算机模型预测的结果进行比较，以便 设计具有更强的亲和力和特异性的新试剂，并改进 模型的预测能力。改性后的功能特性 将表征血红蛋白：Oxgyen亲和力、BPG结合和 HBS和溶解度。有希望的代理人的行为将在一个 细胞水平。途径和摄取率，以及 血红蛋白修饰，将在完整的红细胞中进行测量 悬浮液和全血中以确定是否有显著的血浆 蛋白质修饰就会发生。膜蛋白，如阴离子 运输蛋白，将被研究以识别任何未繁殖的 修改。经过处理的红细胞的代谢特性将 研究：细胞内，pH，BPG和ATP水平，葡萄糖利用和 乳酸盐生产。经过处理的镰状细胞的物理性质将是 研究，包括红细胞牙密度分布情况和 氧离子上的镰状形态与发现的形态相对应的百分比 在动脉和静脉循环中。将使用细胞研究 开发具有替代结构和反应性基团的试剂 可以特异性，降低毒性，提高红细胞摄取率。

The most effective and least toxic antisicking are likly to be those that react with Hbs specifically at the contact sites responsible for polymerization. Using a variety of experimental and computational techniques, we have recently developed a sucessful design strategy for site-direted targeting of Hbs polymerization inhibitors that provides competitive inhibiton of Hbs polymerization through binding to a specific region close to the 2,3-BPG binding site and the lateral "acceptor" contact site on the Hbs molecule. We now propose to pursue two approaches to design and develop new antisicking agents that act specifically at this site. The first is based on our recent identification of several currently approved drugs that are also hihly active inhibitors of Hbs polymerization. We propose new structural modifictions to include our covalent targeting strategy. Both the native drug and the covalent derivatives will be studied for effectiveness and specificity. The secons approach will be based on a series of mixed phosphate anhydrides that are structurally related to BPG and are predicated to have a high affinity for its bindin site, providing an alternative targeting strategy. The rates and extent of HbA abd HbS modification by covalent agents will be determies. Modified peptidues will be isolated from globins, and sequenced to determine the speicfic modification sites. NMT and X-ray crystallography will be used to determine the inhibitors binding geometries of modified Hb. Results will be compared to those predicated by our computer model, in order to design new agents having enhanced affinity and specifity, and to improve the predictive power of the model. Functional properties of modified hemoglobins will be characterized: oxgyen affinity, BPG binding, and Hbs, and solubility. Actions of promising agents will be studies at a cellular level. Routes and rates of uptake, as well as the rates of hemoglobin modification , will be measured both in intact erythrocyte suspensions, and in whole blood to determine wheather significant plasma protein modification occurs. Memebrance proteins, such as the anion transport protein, will be studied to identify any undersired midifications. Metabolic proterties of trated erythrocytes will be studies: intracelluar, pH, BPG and ATP levels, glucose utilization and lactate production. Physical properties of treated sickle cells will be studies, including red cell dentisity distribution profiles and the percent of sickled forms at oxygen teneions corresponding to those found in the arterial and venous circulation. Cellular studies will be used to develop agents with alternative structurs and reactive groups that may specificity, reduce toxicity, and ehance erythrocyte uptake.