DOI
https://doi.org/10.25772/9FKF-GP77
Defense Date
2014
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Umesh Desai
Abstract
Chemoenzymatically synthesized low molecular weight lignin polymers have been previously found to be potent inhibitors of a number of serine proteases via allosteric mechanisms targeting heparin binding sites. Herein, we describe the creation of synthetic sulfated β-O4 lignin (SbO4L) polymer, which is more homogenous compared to previous lignins with respect to its inter-monomeric linkage. SbO4L is a selective inhibitor of thrombin and plasmin. SbO4L was found to act via a unique mechanism targeting thrombin exosite 2 in a manner similar to platelet glycoprotein Ibα (GPIbα). Advanced hemostasis and thrombosis assays demonstrated that SbO4L acts via a dual mechanism: as an anticoagulant, by allosteric inhibition of thrombin catalysis; and as an antiplatelet agent, by competing with platelet GPIbα. These mechanisms are comparable in potency to low molecular weight heparins currently used in the market, indicating that targeting exosite 2 may yield clinically useful drugs in the future. Since the β-O4 type lignin was found to be selective for thrombin and plasmin, we hypothesized that other scaffolds from lignins could be potent inhibitors of other serine proteases. In particular, we screened a library of synthetic sulfated small molecules against factor XIa – an emerging target for prophylactic anticoagulation. Our search identified a sulfated benzofuran trimer (a mimic of β-5 type linkage found in lignins) as a potent inhibitor of factor XIa. Surprisingly, this inhibitor did not compete with heparin. A plausible binding site in the A3 domain of factor XIa was proposed by using molecular modeling techniques. The binding pose demonstrated good correlation with the structure activity data from in vitro studies. Further confirmation that the apple domains were required was proved by testing the trimer against recombinant catalytic domain. A 40-fold decrease in activity was observed. A temperature-dependant perrin plot demonstrated that factor XIa undergoes a large conformational change in the presence of the trimer, which is possibly converting the enzyme back into the zymogen-like shape. In general, the synthetic sulfated lignins can act as a useful foundation to develop anticoagulant, antiplatelet, and anti-inflammatory molecules in the future.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-11-2014