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We describe here the design, synthesis, and activity of a novel class of
alpha-thrombin inhibitors named hirunorms. They were rationally designed to
interact through their N-terminal end with the alpha-thrombin active site in a
nonsubstrate mode and to specifically bind the fibrinogen recognition exosite.
An appropriate spacer that is able to properly orient the N-terminal end in the
active site was also selected. This spacer allowed the size of the inhibitors to
be reduced to about one-third of the amino acid residues in the hirudin
sequence. Hirunorms specifically inhibit the amidolytic action of human
alpha-thrombin toward a small chromogenic substrate. The most active compounds
of the series, hirunorms IV and V, inhibit alpha-thrombin catalyzed hydrolysis
of Tos-Gly-Pro-Arg-p-nitroanilide with K(i) = 0.09 and K(i) = 0.21 nM,
respectively. Comparison of the anticoagulant properties of hirunorms, natural
hirudin from the European leech Hirudo medicinalis, and the synthetic analog
hirulog-1 revealed that hirunorm IV is about 10-fold and 3-fold more active, on
a molar base, than hirudin and hirulog-1 in increasing the aPTT, PT, and TT of
normal human plasma. The peculiar structure of hirunorms makes them stable to
the amidolytic action of thrombin without the introduction of any peptide bond
modification. These molecules display long-lasting activity in human plasma, due
to the presence of several unnatural amino acids in susceptible positions.
Hirunorms are potential candidates for injectable anticoagulants, due to their
potency, specificity of action, long-lasting activity, and safety profiles.
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