| Sulfated low molecular weight lignins (LMWLs), CDSO3 and FDSO 3, designed recently as macromolecular mimetics of heparin, were found to exhibit potent anticoagulant activity. Small molecules based on the same scaffold, SBD and SBT, showed promising thrombin inhibition. All studied molecules were found to allosterically inhibit thrombin as inferred from Michaelis-Menten kinetics. Absence of competition with hirugen, an exosite 1 ligand, and competition with polymeric heparin points to exosite 2 as the site of interaction for these inhibitors. Site-directed mutagenesis of all positively charged exosite 2 residues showed that Arg93 and Arg175 are the major residues involved in CDSO3 binding. FDSO3 showed a progressively greater defect in inhibition with double point mutations, the triple mutant Arg93,97,101Ala displayed a 50 fold drop in inhibition. A single mutant, Arg173Ala, displayed 22-fold reduction in IC50 of SBD, while Arg233Ala was the only mutation that impaired SBT inhibition. Salt-dependence studies showed that CDSO3 had fewer ionic contacts with thrombin, with most of its binding energy derived from non-ionic interactions. FDSO3 on the other hand had a balanced contribution of ionic and non-ionic forces. Thermodynamic studies showed that both polymers have a positive ΔCp of binding, which proves the involvement of electrostatic forces and signals the burial of the polar residues on thrombin exosite 2. The presence of sodium was found to enhance the binding of FDSO3 at exosite 2. The results identify novel binding sub-sites within exosite 2 that are energetically coupled to thrombin's catalytic function and linked to the sodium binding site. The design of high affinity small molecules based on LMWLs scaffold presents major opportunities for developing clinically relevant, allosteric modulators of thrombin. |
