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III. Thrombin Inhibitors Directed at the Fibrinopeptide a Binding Pocket
Page 250
The majority of synthetic thrombin inhibitors interact at the fibrinopeptide A binding pocket, which
includes the catalytic residues Ser195 and His57, hydrogen-bonding capabilities within the oxyanion
hole, peptide backbone functional groups that hydrogen bond with the peptide backbone of the substrate,
and residues involved in amino acid recognition (Figure 3). Many of these binding determinants are
utilized by N-acetyl-(D-Phe)-Pro-Arg-chloromethylketone (PPACK [7]) and its boronic acid analog
(DUP714 [10]). The crystallographic structures of these molecules complexed with thrombin have both
served as starting points for structure-based drug design and as reference structures for comparison of
binding modes of other inhibitors.
The use of arginine boronate esters as transition-state mimetics results in potent peptidyl thrombin
inhibitors. These inhibitors, however, exhibit significant affinity for other serine proteases that have in
common a specificity for substrates with basic residues at P1 (e.g. trypsin, Factor Xa, and plasmin).
Earlier work demonstrated that neutral side chains of P1 boronate esters impart greater selectivity for
thrombin. The boropeptide shown in Figure 4 was investigated as the prototype of neutral side chain,
tripeptide thrombin inhibitors [11]. It had a K i against thrombin of 7 nM and shows selectivity relative to
other trypsin-like plasma proteases. Since these inhibitors have a neutral residue at the P1 site, Deadman
and coworkers [11] sought to demonstrate the mode of binding to thrombin in the absence of a salt
bridge with Asp189.
Figure 3
Schematic diagram of binding determinants within
the fibrinopeptide A binding pocket of thrombin and
their utilization by N-acetyl-(D-Phe)-Pro-boroArg-OH.
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