Thrombin active site

Mechanism of action - Argatroban is a synthetic, direct thrombin inhibitor that reversibly binds to the thrombin active site. It inhibits thrombin-catalyzed or induced reactions, including fibrin formation activation of coagulation factors V, VIII, and XIII protein C and platelet aggregation. 

K. Muller, F.A. Diedrich, Fluorine interactions at the thrombin active site Protein backbone fragments H-C -C = 0 comprise a favorable C-F environment and interactions of C-F with electrophiles, ChemBioChem 5 (2004) 666-675. 

The direct thrombin inhibitors (DTIs) exert their anticoagulant effect by directly binding to the active site of thrombin, thereby inhibiting thrombin s downstream effects. This is in contrast to indirect thrombin inhibitors such as heparin and LMWH (see above), which act through antithrombin. Hirudin and bivalirudin are bivalent DTIs in that they bind at both the catalytic or active site of thrombin as well as at a substrate recognition site. Argatroban and melagatran are small molecules that bind only at the thrombin active site. 

Stereo view of the three lowest energy minima of benzene obtained with the modified force field and the lowest energy minimum of benzamidine (thick lines for heavy atoms and thin lines for polar hydrogens) in the thrombin active site (thin lines). 

Bivalirudin is a direct thrombin inhibitor that has found utility for reducing the rate of acute reocclusion in patients treated with PCI. It is preferential to heparin in PCI when HIT is present. This drug is a derivative of hirudin, which is a dedicated thrombin inhibitor with no other in vivo activities of significance. The molecule is semisynthetic the C-terminal of hirudin is linked by a polyglycine spacer to the tetrapeptide region of the N-terminal that reacts with the thrombin active site (22). It is monitored by the activated clotting time test. Its pharmacologic properties are shown in Table I. 

Schumacher WA, Heran CL, Steinbacher TE (1996a) Low-molecular-weight heparin (Fragmin) and thrombin active-site inhibitor (argatroban) compared in experimental arterial and venous thrombosis and bleeding time. J Card Pharmacol 28 19-25... 

Figure 2. Example site points defined for the thrombin active site.

The difficulties associated with weak binders are illustrated in Figure 7 with thrombin as a target. The 7500 compound subset of the WDI mentioned above was docked into the thrombin active site together with three sets of 100... 

Recognition at the Thrombin Active Site Structure-Based Design and Synthesis of Potent and Selective Thrombin Inhibitors and the X-Ray Crystal Structures of Two Thrombin-Inhibitor Complexes. 

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.)...

Das J, Kimball SD. Thrombin active site inhibitors. Bioorg Med Chem 1995 3 999-1007. 

Fig. 7.1 Thrombin active site regions as defined by the binding of fibrinopeptide of sequence D-F-L-A-E-G-G-G-V-R (from [Ibbr]).

Fig. 7.2 Thrombin active site regions as defined by the binding of D-Phe-Pro-Arg analogues, e.g., PPACK. The recognition pocket (SI) is clear. The proximal (P) hydrophobic pocket binds the proline side chain and thus corresponds to S2. The distal (D) hydrophobic pocket binds the D-Phe side chain. For PPACK 1 Rl= CO.CHjCI.

Thrombin inhibition is a fruitful source of raw data for the study of molecular recognition. Several groups have determined, published, and deposited coordinates for sets of high-resolution X-ray crystal structures. In combination with binding and kinetic data, it is now possible to map the thrombin active site in some detail in terms of both structural changes and the energies of interactions. 

Hg. 29.27 Evolution of bicyclic p-strand scaffolds and application to inhibit the thrombin active site. 

Numerous variations of p-strand templates have been reported as thrombin active-site inhibitors capable of presenting PI and P3 groups in proper orientations while maintaining proper hydrogen-bonding capabilities. Several examples of monocyclic p-strand templates (28.1), ° (28.2), - ° ... 

Obst, U., Banner, D.W, Weber, L., and Diederich, E. (1997) Molecular recognition at the thrombin active site structure-based design and synthesis of potent and selective thrombin inhibitors and the X-ray structures of two thrombin inhibitors. Chemistry el Biology, 4, 287-295. 

The authors used the information gleaned from these studies to design the closely related chloromethyl ketone inhibitor of thrombin FPAM (68). Molecular modeling of this proposed inhibitor docked in the thrombin active site indicated that interactions similar to those important to fibrinopeptide binding could be obtained. Subsequent X-ray crystal structure analysis of the 68-thrombin complex was supportive of the peptidomimetic hypothesis,... 

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