Anticoagulation therapy with heparin- and low-molecular-weight (LMW) heparins, used in thrombotic and vascular disorders, has reached an annual cost of nearly $ 4 billion for the drugs alone. Yet heparin usage suffers from several problems, including bleeding complications, that may prove fatal. Consensus is growing that LMW heparins also suffer from similar problems, although the frequency and severity of incidences may be lower. Fondaparinux, a heparin-based pentasaccharide, was introduced in 2001 to treat some thrombotic conditions. Preliminary indications are that this five-residue sequence may have bleeding problems. In addition, its synthesis is difficult and effort intensive. Heparin, LMW heparin and fondaparinux accelerate the inhibition of critical enzyme(s) of the clotting cascade by antithrombin, a plasma serine proteinase inhibitor. Advances made on the molecular mechanism of heparin activation of antithrombin by others and the PI led to the rational design of small, highly sulfated, synthetic, non-heparin organic antithrombin activators. These rationally designed molecules were the first small, organic molecules found to activate antithrombin. We have now advanced our rational approach by designing a second generation activators, including IQ3A67S-B345S, based on our earlier fundamental studies. Preliminary studies suggest that the second generation activator IQ3A67S-B345S binds and activates antithrombin ~9-fold better than the first generation activator. A patent application has been submitted for this activator and its derivatives. This proposal focuses on I) developing a high-yielding synthesis and purification method for the second generation activator and its derivatives; II) studying the interaction of the activator (and its structural analogs) with antithrombin at a molecular level; and III) testing the efficacy of the organic antithrombin activator (and its derivatives) in in vitro and ex vivo models of thrombosis. Our approach of computerized molecular design guided by mechanistic biochemical knowledge and ex vivo efficacy is a unique feature directing this work. The proposed work is critical for determining the organic activator to focus advanced phase II efforts.
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