Thrombin binding pocket

Other non-covalent interactions such as C=0 F-C type, between a fluorine atom and the carbonyl of an amino acid, may take place for the stabilisation of enzyme-inhibitor supramolecular structures [28,30]. It is why the 4-fluorophenyl group is an important motif for binding pocket, as shown by the enhancement of one order of magnitude of the K by introducing one fluorine atom on thrombin inhibitor (Fig. 5) [30],... 

III. Thrombin Inhibitors Directed at the Fibrinopeptide a Binding Pocket... 

Schematic diagram of binding determinants within the fibrinopeptide A binding pocket of thrombin and their utilization by N-acetyl-(D-Phe)-Pro-boroArg-OH.

Thrombin is the pivotal trypsin-like protease for the regulation of thrombosis and hemostasis. Thrombin hydrolyzes its natural substrates by recognition of the Pro-Arg motif in the apolar S2- and the primary specificity SI pocket [42]. The molecular structure of the thrombin-CtA complex (Figure 1.11) showed that CtA was bound to the active site of the enzyme. The arginine side chain formed an electrostatic interaction with Aspl89, located at the bottom of the SI binding pocket. 

Substrate-competitive inhibition is a well known strategy for targeting enzymes, which has been applied successfully in enzyme classes such as the proteases. Nevertheless, its use for kinase inhibition has met with little success. One of the reasons is the rather stretched substrate pocket of kinases. Kinases are likely to use additional binding pockets, which are not located in the immediate environment of the active site [16, 17]. Therefore, kinases lack the specific hydrophobic pockets that could serve as targets for peptidomimetics, as occurs with HIV protease or thrombin. 

As in Sect. 2.2, the overall essence of the QSAR studies on thrombin inhibitors is the high dependence of the binding of substrates or inhibitors on the molar refractivity. Generally, there was high collinearity between MR and/or CMR and C log P. This led the researchers to assume that the binding pocket around the active site in thrombin is not typically hydrophobic. 

Figure 7.1 Examples of automatically identified ligand-binding pockets with inhibitors bound. For pocket detection, a grid-based approach was used (Pocket-Picker). Dots represent surface cavities identified by PocketPicker, colored by buriedness . Solvent-accessible pocket surfaces are indicated by a mesh (left) or as hard surface (right). Darker shading of the grid dots indicates greater buriedness. a) Thrombin active site (PDB identifier 2cf8, 1.3 A resolution with a lactam inhibitor), b) co-crystal structure of Factor Xa (PDB entry lezq, 2.2 A resolution with inhibitor RPR128515). The automatically extracted pocket does not match with the surface-exposed parts of the actual inhibitor binding pocket. (Adapted from ref. 3.)...

However, Boehringer Mannheim developed thrombin inhibitors (92) (Fig. 15.40) that lack these H-bonds (163). This idea was exploited by researchers at 3D Pharmaceuticals, who were able to crystallize (93) in the active site (164,165). In this example, the benzene ring acts as a scaffold to display the three different substituents to fill the three principal binding pockets. 

The serine endopeptidases include the chymotrypsin family (EC 3.4.21.1), trypsin (EC 3.4.21.4), elastase (EC 3.4.21.37), thrombin (EC 3.4.21.5), subtilisin (EC 3.4.21.62) and a-lytic proteases (EC 3.4.21.12). The enzymes are all endopeptidases. The substrate specificities of the individual members of this group are often quite different, which is attributed to different structures of the binding pockets. 

Increasingly nonpeptide substituents have been incorporated into the primary specificity pocket binding portion of the bivalent inhibitors. Higher affinity for thrombin was achieved by replacement of the (d-Phe)-Pro-Arg with either dansyl-Arg-(D-pipecolic acid) (3-17, [27]) or 4-tert-butylbenzenesulfonyl-Arg-(D-pipecolic acid) (3-18, [27]). While the arginine side chain of these and the (D-Phe)-Pro-Arg-containing inhibitors make similar interactions with the aspartic acid within the SI specificity pocket, the dansyl-Arg-(D-pipecolic acid) inhibitors bind in a nonsubstrate mode [27]. This initial result suggests that other nonpeptide thrombin inhibitors may be successfully incorporated into bivalent inhibitors. 

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