Discovery of New Inhibitors of Human Activated Protein C to Treat Hemophilia.

Background: Activated Protein C (APC) is a plasma serine protease with antithrombotic function. APC acts as an anticoagulant by promoting the degradation of factors Va and VIIIa, thus inhibiting the formation of thrombin. Specific inhibition of APC has been proposed to benefit hemophilia therapy.

Methods: We used chromogenic tripeptide substrate hydrolysis assay to screen a series of arginine and arginine-like containing small molecules to identify inhibitors of APC. Similar hydrolysis assays were used to determine selectivity against other serine proteases and blood-clotting enzymes. Molecular modeling was exploited to illustrate the binding of the most potent and selective inhibitor onto the putative binding site.

Results: We identified inhibitor 2 as a potent inhibitor with an IC₅₀ value of 1.1 μM. The molecule demonstrated >100-fold selectivity against thrombin, factor XIa, and neutrophil elastase, >50-fold selectivity against factor XIIIa, 10-fold selectivity against factor Xa, and 8-fold selectivity against human plasmin. Molecular modeling reveals that inhibitor 2 binds to the active site of APC with the best-docked structure, indicating that one protonated amidino group establishes a salt bridge to the side chain carboxylate of Asp189 residue. Another inhibitor was identified, yet it was not as selective to APC. Importantly, inhibitor 2 demonstrates favorable physicochemical, pharmacokinetic, and drug-likeness properties.

Conclusion: Inhibitor 2 is a selective and potent inhibitor of APC that serves as a powerful lead for the development of hemophilia therapy.