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.
