Author ORCID Identifier
Doctor of Philosophy
Umesh R. Desai, Ph.D.
Glycosaminoglycans (GAGs) are complex linear chain carbohydrate molecules found on virtually all animal cell surfaces. Owing to their negatively charged nature, GAGs interact with a number of different proteins. Thus, although they have great potential as therapeutic agents, their apparent promiscuous interactions increase their side effect risk. GAG mimetics, including GAG oligosaccharides and non-saccharide GAG mimetics (NSGMs) are viable approaches to address this. This work discusses sulfated benzofuran thrombin inhibitors with submaximal protease inhibition, sulfated diflavonoid inhibitors of plasmin and GAG oligosaccharides with selectivity for human neutrophil elastase (HNE).
Anticoagulants are very important for the treatment of thrombotic diseases. The adverse effects associated with current clinically used anticoagulants warrant the continuous search for new agents. Thrombin, being the central player in the coagulation cascade, remains a very important target for anticoagulant therapy, however drugs inhibiting its activity carry the risk of prolonged bleeding. Based on a previously identified sulfated benzofuran thrombin inhibitor, we have developed analogs with submaximal inhibition of the protease. These agents inhibit thrombin with efficacies approaching 50%, for both chromogenic and macromolecular substrates, ensuring a basal level of thrombin activity even at saturating inhibitor concentrations. The most potent of these compounds had a potency of 1.8 µM, 2-3 fold better than the lead. Additionally, these compounds utilize an allosteric mechanism for thrombin inhibition. Further, studies have revealed structural features responsible for submaximal thrombin inhibition.
Fibrinolysis is an important part of hemostasis and plasmin is the most important fibrinolytic enzyme. Anti-plasmin agents are thus important for conditions such as hemophilia; however, there are no clinically used direct plasmin inhibitors. By structural modifications of a previously identified sulfated diflavonoid plasmin inhibitor, we have achieved a compound with 12-fold better potency (IC50 = 6.3 ± 0.4 µM), and a selectivity index of at least 22 over closely related serine proteases. We have shown that this compound inhibits plasmin mediated clot lysis, and further demonstrated that its activity is reversible using protamine sulfate, indicating its potential as a lead for the development of clinical anti-plasmin agents.
HNE, a serine protease associated with inflammatory diseases is known to be inhibited by GAGs. However, the interactions at the molecular level have remained elusive. Using biochemical methods, and by studying the inhibitory potency of different GAGs and GAG oligosaccharides, we have shown that an octasaccharide may be the ideal GAG length for the achievement of potent HNE inhibition. Under our assay conditions, the inhibition of HNE by an octasaccharide species was only 5-fold less than that of unfractionated heparin, whereas the hexasaccharide species was 30-fold less active. The data also suggests that the inhibition of HNE by GAGs is via an allosteric mechanism and using molecular modeling, we have identified putative GAG binding sites on HNE and further identified GAG species with potential selectivity for anti-HNE activity
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Available for download on Friday, July 08, 2022