Thrombin is a key enzyme of the coagulation cascade exhibiting important roles in both pro-coagulation and anti-coagulation processes. Most clinically used anticoagulant drugs, including polymeric heparin, warfarin, hirudin, argatroban and the recently approved dabigatran, aim to reduce thrombin activity. There are several binding domains on thrombin including the active site, anion-binding exosites I and II, and the sodium binding site. We hypothesized that thrombin may be better regulated through an allosteric process mediated by small molecules binding to either exosite I or II. An appropriately designed allosteric regulator that reduces the procoagulant signal in a finely tuned manner may maintain a delicate balance between procoagulant and anticoagulant signals in blood resulting reduced bleeding complications. In this work, we synthesized and studied a library of potent, small, aromatic molecules as allosteric inhibitors of thrombin. Of the 28 potential inhibitors, 11 molecules inhibited thrombin with reasonable potency. Structure activity relationship studies showed that sulfation at the 5-position of the benzofuran scaffold was essential for targeting thrombin. Michaelis-Menten kinetic studies indicated a non-competitive, allosteric mechanism of inhibition. Site-directed mutagenesis, competitive binding and molecular modeling studies led to the identification of the most plausible binding pose for a potent sulfated dimer. To further improve the potency, a small library of sulfated benzofuran trimers was synthesized and studied for thrombin inhibition. Further, to find new scaffold to inhibit thrombin allosterically, docking-based virtual screening approach was used. All these molecules were found to be moderately potent thrombin inhibitors and can serve as lead to develop allosteric inhibitor. Overall, this work presents the first small, synthetic, sulfated aromatic molecules as potent allosteric modulators of thrombin. Finally, this work also highlights the opportunity of exploring allosteric modulators of other coagulation enzymes, e.g., factors Xa, IXa and XIa, based on the sulfated benzofuran scaffold.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1294 |
| Date | 07 November 2011 |
| Creators | Sidhu, Preetpal |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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