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trypsin. Application of three-fold iterative strategy of design involving synthesis, x-ray crystallography,
and molecular modeling, this group elaborated the 1-amidinopiperidine from structures that inhibited in
the micromolar range to some inhibiting in the picomolar range. In doing so significant improvements in
the selectivity of thrombin relative to trypsin were also achieved. In the case of the D-amino acid series
(1–2), a “second inhibitor binding mode” that differed from that of Argatroban was identified. In this
new and unexpected binding mode, the S2 pocket is unoccupied and the napthalenesulfonyl group fills
the S3 site and overlaps the front of the S2 site. The benzyl group of the phenylalanine is oriented
toward the protein surface and is partially exposed to solvent. The Argatroban or “inhibitor binding”
mode was favored by the more potent L-amino acid series (1–3 and 1–4) where the piperidide (1–3) or Nbenzyl
(1–4) binds to the S2 site and the aryl groups are found in the S3 site.
IV. Bivalent Thrombin Inhibitors Directed at the Fibrinopeptide a Binding Pocket and the
Fibrinogen Recognition Site
A strategy to prepare highly selective thrombin inhibitors involves linkage of molecules capable of
interacting at distinct subsites. This approach should result in inhibitors more specific for thrombin:
while serine proteases possess common structural features related to catalysis and some serine
proteases—including the coagulation enzyme Factor Xa—also exhibit primary substrate specificity for
positively charged residues, only thrombin possesses recognition subsites for fibrinogen and effector
molecules such as thrombomodulin. Nature has used this strategy in the evolution of hirudin, the
anticoagulant protein produced by the medicinal leech. When this effective anticoagulant binds
thrombin [18–20], the N-terminal domain blocks the primary specificity pocket while the C-terminal
residues adopt an extended conformation and make multiple interactions within the fibrinogen
recognition exosite.
Guided by structural and biochemical information, small molecules capable of simultaneous interactions
with both the primary specificity pocket and the fibrinogen recognition exosite were designed and
synthesized. These bivalent inhibitors are composed of three regions: a group to block the primary
specificity pocket, a sequence to bind the fibrinogen recognition site, and a chemical linker. The bivalent
inhibitor approach was first executed with peptides [21–22]. In 1990, DiMaio et al. (3–3 [22]) used the
peptide sequence from hirudin to link (d-Phe)-Pro-Arg-Pro, known to bind at the primary specificity
pocket [23], with hirudin C-terminal residues, known to bind at the fibrinogen recognition site.
Polyglycine linkers were also used to connect these sequences (Maraganore
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