Substantial non-electrostatic forces are needed to induce allosteric disruption of thrombin’s active site through exosite 2

Abstract

Sulfated beta-O4 lignin (SbO4L), a non-saccharide glycosaminoglycan mimetic, was recently disclosed as a novel exosite 2-directed thrombin inhibitor with capability of mimicking sulfated tyrosine sequences of glycoprotein Ibα resulting in dual anticoagulant and antiplatelet activities. SbO4L engages essentially the same residues of exosite 2 as heparin and yet induces allosteric inhibition. Fluorescence spectroscopic studies indicate that SbO4L reduces access of the active site to molecular probes and affinity studies at varying salt concentrations nearly 6 ionic interactions, similar to heparin, but much higher non-ionic contribution. The results suggest that subtle increase in non-electrostatic forces arising from SbO4L’s aromatic scaffold appear to be critical for inducing allosteric dysfunction of thrombin’s active site.

Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin

“…This appears to be especially true for the R93A and R101A mutants, and possibly also for others. Earlier work on sulfated low molecular weight lignins has also shown the importance of hydrophobicity in recognition of exosite 2 and support the conclusion that 3g binds in exosite 2 through primarily non-ionic forces 51 .…”

“…This is an unusual observation and opposes the expectation of a loss in potency. Yet, an analysis of thrombin crystal structure shows that a majority of these residues are located adjacent to hydrophobic sub-sites within/near exosite 2 50 51 . Thus, expansion of the hydrophobic sub-domain through Ala replacement of Arg/Lys may induce better recognition of 3g ’s aliphatic chain and/or aromatic rings resulting in enhancement of potency.…”

Allosterism‐based simultaneous, dual anticoagulant and antiplatelet action: allosteric inhibitor targeting the glycoprotein Ibα‐binding and heparin‐binding site of thrombin

“…This implies that structural features of SbO4L, although mimicking the highly sulfated feature of heparin, are important for inducing an allosteric conformational change. We have shown previously that SbO4L recognizes hydrophobic subdomains present within exosite 2, inducing allosteric structural changes . SbO4L targets overlapping binding sites of heparin and GPIbα, which advantageously elicits the dual anticoagulant and antiplatelet function.…”

Development of Orally Active Thrombin Inhibitors for the Treatment of Thrombotic Disorder Diseases

“…In pursuit of such allosteric regulators, Desai’s group had earlier designed sulfated low molecular weight lignins (LMWLs) as functional mimetics of heparin which targeted to exosite II-like regions [39,40] of coagulation enzymes, including thrombin [41]. Despite their excellent in vitro and in vivo anticoagulant properties, sulfated LMWLs were challenging because of their polydispersity and microheterogeneity, which paralleled that observed with the heparins.…”