The First Example of Allosteric Partial Inhibition of a Monomeric Protease
Authors: Verespy Stephen , Mehta Akul Y. , Afosah Daniel , Al‐Horani Rami A. , Desai Umesh R. 
Published: November 2023

Purpose

Allosteric, partial inhibition of monomeric proteases, like thrombin, has not been achieved previously, but may offer a regulatory mechanism for maintaining homeostasis of diseased systems. We therefore sought to identify and study novel agents that induce allosteric, partial inhibition of thrombin.

Methods

A library of 36 sulfated coumarins was synthesized and screened against thrombin, factor Xa, and factor XIa; proteases of the coagulation cascade. Eleven were selected to establish their half maximal inhibitory concentration (IC50) and efficacy of inhibition (ΔY). To study binding interactions, the most active compound was chosen to assess binding affinity, Michaelis‐Menten kinetics, competition studies, and recombinant mutant studies. To assess conformational change in and around the active site, fluorescence quenching studies and chemical kinetic studies using the thrombin‐antithrombin system were performed. Assays probing the inhibitors ability to induce partial inhibition of thrombin's endogenous substrates was performed using hydrolysis assays and UPLC‐MS monitoring.

Results

The IC50s of ten thrombin‐selective compounds ranged from 0.19–58 μM, with ΔY values ranging from 22–73%, indicating that this scaffold induced submaximal activity of thrombin; see Figure. Compound 3g (IC50 = 187 ± 40 nM; ΔY ~47%; 150‐fold selective for thrombin) was found to have a KD of 143 nM. Kinetic studies showed a decrease in the VMAX and no change in the KM, indicating a non‐competitive or allosteric mechanism. 3g was found to compete ideally with heparin for exosite 2 of thrombin, and not compete with hirudin peptide for exosite 1. Thrombin mutant studies suggested a region of binding, but did not identify a unique residue responsible for interaction, indicating the sulfate may engage multiple basic residues. Quenching studies produced Stern‐Volmer relationships showing 3g caused a 49% change in the active site, while BF caused a 71% change; within standard error to their observed ΔY values. Chemical kinetic studies showed that 3g slows the thrombin‐antithrombin complex formation by ~12‐fold, suggesting it induced a conformational change to the active site serine. 3g was found to inhibit thrombin catalyzed fibrin formation with an IC50‐ of 514 nM, but with a residual activity of 94%.

Conclusion

This is the first time a phenomenon of allosteric, partial inhibition has been observed with any monomeric protease. Similar to partial agonism of receptors and oligomeric proteins, this work represents the first group of probes found to induce submaximal activity with relatively high potency against thrombin. They will be useful as powerful tools in developing future anticoagulants with higher potencies combined with the possibility of little‐to‐no risk of bleeding events; which may offer a major advantage over clinically used thrombin inhibitors.

Support or Funding Information

Financial support from the NIH in the form of R01 HL090586, HL125639 and P01 HL107152