PubMed · 8444116
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V Tilsner. 1993-03-05. [Thrombosis prophylaxis].. https://pubmed.ncbi.nlm.nih.gov/8444116/
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Study of surface representations of the inhibitor-bound thrombin P-1 pocket revealed a lipophilic recess in this pocket which is not occupied by any known inhibitor. Solid-phase synthesis was used to generate benzylamides of D-diphenylAlaPro by aminolysis of Boc dipeptide Kaiser resin. The resulting amides inhibited thrombin in the range IC50 = 3-13,000 nM, and the structure-activity relationships and molecular modeling suggest a unique fit of the benzyl side chain into P-1 with the meta substituent occupying the recess.
Four kinds of monomers carrying a thrombin inhibitor, (2R,4R)-4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)sulfon yl]-L-arginyl]-2-piperidinecarboxylic acid (argatroban), were synthesized. These monomers were copolymerized with acrylamide to yield water-soluble polymeric conjugates possessing the argatroban moiety in the side chain. Their antithrombogenic activities were determined from the inhibitory effect on thrombin action and the prolongation effect on blood clotting time. The monomeric conjugates of 2-hydroxyethyl acrylate (HEA), 2-hydroxyethyl methacrylate (HEMA), and 4-hydroxybutyl acrylate (HBA) linked with argatroban through an ester bond were potent inhibitors of thrombin, prolonging the blood-clotting time, whereas a conjugate of amino methyl styrene (AMS) and argatroban through an amide bond was a less potent inhibitor than argatroban. None of the copolymers could prolong blood clotting when assessed just after preparation of their aqueous solutions, but the antithrombogenic activity of the aqueous solutions increased after incubation for 7 days at 37 degrees C for the polymeric conjugates through an ester bond. Free argatroban was detected in the aqueous solutions of polymeric conjugates after incubation, suggesting that argatroban was released by hydrolysis of the ester bond during incubation.
A novel class of synthetic, multisite-directed thrombin inhibitors, known as hirunorms, has been described recently. These compounds were designed to mimic the binding mode of hirudin, and they have been proven to be very strong and selective thrombin inhibitors. Here we report the crystal structure of the complex formed by human alpha-thrombin and hirunorm V, a 26-residue polypeptide containing non-natural amino acids, determined at 2.1 A resolution and refined to an R-factor of 0.176. The structure reveals that the inhibitor binding mode is distinctive of a true hirudin mimetic, and it highlights the molecular basis of the high inhibitory potency (Ki is in the picomolar range) and the strong selectivity of hirunorm V. Hirunorm V interacts through the N-terminal tetrapeptide with the thrombin active site in a nonsubstrate mode; at the same time, this inhibitor specifically binds through the C-terminal segment to the fibrinogen recognition exosite. The backbone of the N-terminal tetrapeptide Chg1"-Val2"-2-Nal3"-Thr4" (Chg, cyclohexyl-glycine; 2-Nal, beta-(2-naphthyl)-alanine) forms a short beta-strand parallel to thrombin main-chain residues Ser214-Gly219. The Chg1" side chain fills the S2 subsite, Val2" is located at the entrance of S1, whereas 2-Nal3" side chain occupies the aryl-binding site. Such backbone orientation is very close to that observed for the N-terminal residues of hirudin, and it is similar to that of the synthetic retro-binding peptide BMS-183507, but it is opposite to the proposed binding mode of fibrinogen and of small synthetic substrates. Hirunorm V C-terminal segment binds to the fibrinogen recognition exosite, similarly to what observed for hirudin C-termninal tail and related compounds. The linker polypeptide segment connecting hirunorm V N-and C-terminal regions is not observable in the electron density maps. The crystallographic analysis proves the correctness of the design and it provides a compelling proof on the interaction mechanism for this novel class of high potency multisite-directed synthetic thrombin inhibitors.