DEVELOPMENT OF SITE SPECIFIC ANTISICKLING AGENTS

最有效和毒性最小的抗粘剂可能是那些 与血红蛋白发生反应，特别是在接触部位， 聚合法 使用各种实验和计算 技术，我们最近开发了一种成功的设计策略， Hbs聚合抑制剂的位点定向靶向， 通过结合到一个 靠近2，3-BPG结合位点的特异性区域和侧链 Hbs分子上的“受体”接触位点。 我们现在建议 设计和开发新的抗粘剂的两种方法， 特别是在这个网站上。 第一个是基于我们最近的 鉴定几种目前批准的药物， Hbs聚合的活性抑制剂。 我们提出了新的结构 包括我们的共价靶向策略。 两者 天然药物和共价衍生物将被研究， 有效性和特异性。 Secons方法将基于 一系列混合的磷酸酐，结构上与 BPG和被预测对其结合位点具有高亲和力， 提供了一种替代的瞄准策略。 的比率和程度 将测定共价试剂对HbA和HbS的修饰。改性 肽将从球蛋白中分离出来，并测序以确定 特定的修饰位点。 将使用NMT和X射线晶体学 以确定修饰Hb的抑制剂结合几何形状。 结果 将与我们的计算机模型预测的结果进行比较，以便 设计具有增强的亲和力和特异性的新试剂，并改善 模型的预测能力。 改性的功能特性 血红蛋白将被表征：氧亲和力，BPG结合，和 Hbs和溶解度。 有前途的代理人的行动将在一个研究 细胞水平。 吸收途径和吸收率，以及 血红蛋白修饰，将在完整的红细胞中测量 悬浮液和全血中，以确定是否存在显著的血浆 发生蛋白质修饰。 膜蛋白质，例如阴离子 运输蛋白，将进行研究，以确定任何不必要的 歪曲事实 红细胞的代谢特性， 研究：细胞内、pH、BPG和ATP水平、葡萄糖利用和 乳酸盐生产 经处理的镰状细胞的物理性质将是 研究，包括红细胞密度分布概况和 对应于发现的那些的镰状形式的百分比 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基团的试剂， 可能具有特异性、降低毒性和增强红细胞摄取。

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.