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Date of Award
Robert J. Kerns
Heparan sulfate mediates many biological processes and plays a role in many diseases, such as cancer, atherosclerosis, inflammation, alzheimers, and viral infection. The structurally similar polysaccharide heparin is a powerful clinical anticoagulant but causes numerous side effects and interacts with proteins that are involved in heparan sulfate-mediated biological processes. Numerous therapeutic applications other than anticoagulation have been proposed for heparin or heparin-like analogs and mimics that selectively bind a particular protein target (heparin-binding protein) and thus mediate a specific biological activity. However, due to the polyanionic nature of heparin and most heparin mimics, the discovery of compounds that selectively target individual heparin-binding proteins has been difficult. Explored here is a new strategy for preparing and identifying novel heparin derivatives that display increased affinity and selectivity for individual heparin-binding proteins. Using heparin as a scaffold for the generation of libraries of novel heparin analogs, differential N-desulfonation and N-deacetylation of heparin was performed to reveal free amine groups for the subsequent introduction of structural diversity. Structurally diverse non-anionic moieties to be incorporated into the heparin structure were identified based on their capacity to interact with protein through hydrogen bonds and/or hydrophobic interactions. Utilizing parallel synthesis, these structurally diverse moieties were incorporated into heparin via chemical coupling reactions, affording a library of heparin analogs. Utilizing competition binding assays and enzyme inhibition assays, the library was screened against various heparin and HS binding proteins (alpha-thrombin, SLPI, MPO, laminin, VEGF, FGF1, and FGF2 for binding assays; alpha-thrombin, elastase, and cathepsin G for enzyme inhibition assays). Unique analogs were identified as 'hit' ligands for alpha-thrombin binding and inhibition of thrombin protease activity. One of those analogs, a N-desulfonated heparin fraction substituted with 3-(4-hydroxyphenyl)propionyl, demonstrated increased binding affinity and selectivity toward alpha-thrombin and displayed direct inhibition of this enzyme. Based on the hit ligand, a focused library of heparin analogs was synthesized. Structure-activity relationship studies revealed that a 4-hydroxyphenyl moiety properly distanced from the saccharide core afforded the heparin analogs with the greatest affinity for thrombin and were the most potent inhibitors of thrombin activity.
Huang, Liusheng, "Parallel synthesis and screening of libraries of heparin-based biomimetics targeting heparan sulfate-mediated biological processes" (2003). Wayne State University Dissertations. 3401.