Synthesis and evaluation of peptidic thrombin inhibitors bearing acid-stable sulfotyrosine analogues.

Tyrosine sulfation is an important post-translational modification of peptides and proteins which underpins and modulates many protein-protein interactions. In order to overcome the inherent instability of the native modification, we report the synthesis of two sulfonate analogues and their incorporation into two thrombin-inhibiting sulfopeptides. The effective mimicry of these sulfonate analogues for native sulfotyrosine was validated in the context of their thrombin inhibitory activity and binding mode, as determined by X-ray crystallography.

Mechanism and inhibition kinetics of peptide P13 as thrombin inhibitor.

Excessive coagulation can easily lead to arterial and venous thrombosis, which is the main reason for the evolution of myocardial infarction and cerebrovascular accidents. As a key coagulation factor for the coagulation pathway, thrombin has become a remarkable target for the control of thrombosis. The synthesized peptide P13 with amino acid sequence of N-RGDAGFAGDDAPR was expected to be an inhibitor with higher antithrombotic activity. The results showed that the IC50 (50% inhibition of thrombin activity) of the peptide P13 was determined by colorimetric method to be 115 µM. And enzyme kinetic experiments showed that P13 was a competitive inhibitor of thrombin with Ki = 106 µM. Fluorescence spectra and three-dimensional fluorescence showed that P13 could alter the secondary structure of thrombin and the microenvironment of certain chromogenic amino acids. P13 can spontaneously bind with thrombin exosite 1 in the form of 1:1 mainly through hydrogen bonding and van der Waals force. And the optimal docking mode of P13 and thrombin was revealed by molecular docking with "-CDOCKER_Energy" of 178.679 kcal mol-1. This study revealed P13 may become a potential anticoagulant drug widely used after further studies in preclinical and clinical trials.

Novel dabigatran derivatives with a fluorine atom at the C-2 position of the terminal benzene ring: Design, synthesis and anticoagulant activity evaluation.

This manuscript describes the preparation of dabigatran derivatives and their inhibitory potentials toward human thrombin. Among the tested compounds, 7c, 7k, 7m and 7o, with IC50 values of 1.54, 0.84, 1.18 and 1.42 nM, exhibited comparable inhibitory activity to dabigatran (IC50 = 1.20 nM). The in vivo anti-thrombotic activity of compounds 7c and 7o in SD rats was studied. Results showed that intravenously administering the two compounds significantly inhibited the growth of thrombus with an inhibition rate of (84.24 ± 1.53)% and (84.57 ± 0.45)%, which were comparable to that of dabigatran (85.07 ± 0.61)%. Furthermore, the docking simulation of active compounds (7k and 7m) provided a potential binding model. Results indicated that these compounds could be further investigated to determine their anticoagulant activities.

Conformationally restricted analogs of the direct thrombin inhibitor FM 19.

The serine protease thrombin plays several key roles in the clotting cascade within the hemostatic system, such as in fibrin formation and platelet activation. Thus, development of an inhibitor that binds to the enzyme's active site (a direct thrombin inhibitor) offers an approach for the treatment of thrombus-associated diseases. Previous structure-activity relationship studies originally based on the bradykinin breakdown product Arg-Pro-Pro-Gly-Phe (RPPGF) led to the development of lead compound FM 19 (d-Arg-Oic-Pro-d-Ala-Phe(p-Me)-NH(2)). The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed sites of modification to potentially improve inhibition. In this study, we report the synthesis and biological characterization of nine peptides that replace only the d-Arg residue of the FM 19 sequence, investigating ways to add conformational restriction, modification of the basic moiety at the end of the side chain, and removal of the charge from the N-terminus. Two of these peptides, 6 and 7 (IC(50) values of 0.51 and 0.45 µM, respectively), show similar potency to the best compounds in the FM 19 series reported thus far.

Synthesis, antiplatelet and antithrombotic activities of new 2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4,3-d]pyrimidin-5-ones.

Atherothrombotic coronary artery disease, associated with deep vein thrombosis, is one of the most common causes of death worldwide. Recently, antiplatelet combination therapy using agents with different mechanisms of action, such as aspirin, dipyridamole, and thienopyridines, seems to be an attractive preventive approach. Moreover, several large, randomized clinical trials support combination therapy with aspirin plus warfarin in high-risk patients with atherosclerotic heart disease. Our research on the benzopyrano[4,3-d]pyrimidine system gave rise to the synthesis of a large number of compounds endowed with in vitro anti-aggregating activity. Several SAR considerations suggest that the benzopyranopyrimidine system is an appropriate scaffold to obtain molecules that are able to act simultaneously in different pathways of aggregation. Now, we report the synthesis of new 2-substituted benzopyrano[4,3-d]pyrimidin-4-cycloamines and 4-amino/cycloamino-benzopyrano[4,3-d]pyrimidin-5-ones and the results of the pharmacological study on haemostasis. Some tested compounds showed a large-spectrum antiplatelet activity in vitro, and are more potent than aspirin as antithrombotics in vivo but, at variance with aspirin, they do not increase bleeding. This paper describes novel antithrombotic compounds with an interesting pharmacological profile and a potentially attractive benefit/risk ratio, with their mechanism of action generally, but not exclusively, dependent on antiplatelet activity, deserving further investigations.

Thrombin inhibitors identified by computer-assisted multiparameter design.

Here, we present a series of thrombin inhibitors that were generated by using powerful computer-assisted multiparameter optimization process. The process was organized in design cycles, starting with a set of randomly chosen molecules. Each cycle combined combinatorial synthesis, multiparameter characterization of compounds in a variety of bioassays, and algorithmic processing of the data to devise a set of compounds to be synthesized in the next cycle. The identified lead compounds exhibited thrombin inhibitory constants in the lower nanomolar range. They are by far the most selective synthetic thrombin inhibitors, with selectivities of >100,000-fold toward other proteases such as Factor Xa, Factor XIIa, urokinase, plasmin, and Plasma kallikrein. Furthermore, these compounds exhibit a favorable profile, comprising nontoxicity, high metabolic stability, low serum protein binding, good solubility, high anticoagulant activity, and a slow and exclusively renal elimination from the circulation in a rat model. Finally, x-ray crystallographic analysis of a thrombin-inhibitor complex revealed a binding mode with a neutral moiety in the S1 pocket of thrombin.

[Synthesis and antithrombin activity of phosphoalanine-containing peptides]. / Sintez i uzuchenie antitrombinovoi aktivnosti pepidov, soderzhashchikh fosfoalanin.

Boc/Tos-L-Phe-L-Arg-Xaa tripeptides (where Xaa = L-Ala-OBut, L-Ala, or DL-AlaP (OC2H5)2) were synthesized by conventional methods of peptide synthesis in solution. Special features of their interaction with thrombin and trypsin were studied. Unlike trypsin, thrombin did not catalyze the hydrolysis of the L-Arg-L-AlaP-(OC2H5)2 bond. The Tos-L-Phe-L-Arg-DL-AlaP(OC2H5)2 peptide was the most active inhibitor of thrombin among all the compounds studied. The relationship between the structure and inhibitory action of the synthesized peptides is discussed. A part of this study was reported in the Augustusburg Conference of Advanced Science: Nucleic Acids--Targets and Tools, September 17-19, 2000, Germany.

Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures.

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1' and P3' residues of the linker with thrombin S1' and S3' subsites, respectively, were identified using the "Methyl Scan" method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494-13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(D-pipecolic acid)-Xaa-Gly-Yaa-Gly-betaAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala- (be ta-cyclohexylalanine)-(D-Glu)-OH, in which nonpolar P1' residue Xaa or P3' residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3' residue with D-phenylglycine or D-Phe improved the K(i) value to (9.5 +/- 0.6) x 10(-14) or 1.3 +/- 0.5 x 10(-13) M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (K(i) = (2.4 +/- 0.5) x 10(-11) M). Similarly, substitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanine lowered the K(i) values to (8.2 +/- 0.6) x 10(-14) or (5.1 +/- 0.4) x 10(-14) M, respectively. The linker Gly-Gly-Gly-betaAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvement of the K(i) values to (3.8 +/- 0.6) x 10(-14) or (1.7 +/- 0.4) x 10(-14) M, respectively. These K(i) values are equivalent to that of natural hirudin (2.2 x 10(-14) M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with L-norleucine or L-beta-(2-thienyl)alanine at the P1' residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human alpha-thrombin. The crystal structures of these complexes were solved and refined to 2.1 A resolution. The Lys(60F) side chain of thrombin moved significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.

The design of potent, selective, non-covalent, peptide thrombin inhibitors utilizing imidazole as a S1 binding element.

Modeling of neutral or mildly basic functional groups in the S1 site of thrombin led to the targeting of imidazole as a S1 binding element and correctly predicted the optimal chain length for connecting this group with the S2 and S3 binding elements. Derivatives of 4-(3-aminopropyl)-imidazole can be selective inhibitors of thrombin demonstrating potent anticoagulant activity.

Bifunctional peptide boronate inhibitors of thrombin: crystallographic analysis of inhibition enhanced by linkage to an exosite 1 binding peptide.

The affinity of the hirudin49-64 segment for exosite 1 of thrombin has been used previously to enhance the potency of simple competitive inhibitors [DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J. , Ni, F., Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703., and Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W., II (1990) Biochemistry 29, 7095-7087.]. Using a similar approach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching this segment via a novel reverse oriented linker to each of two tripeptide boronate inhibitors. At P1, compound 1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged, bromopropyl residue. Inhibition of human alpha-thrombin by compound 1 shows slow, tight-binding competitive kinetics (final Ki of 2.2 pM, k1 of 3.51 x 10(7) M-1 s-1, and k-1 of 1.81 x 10(-)4 s-1). The addition of hirugen peptide (20 microM) competes for exosite 1 binding and restores the k1 and k-1 to that of the analogous tripeptide, 0.29 x 10(7) M-1 s-1 and 0.13 x 10(-)4 s-1, respectively. Compound 1 has enhanced specificity for thrombin over trypsin with KiTry/KiThr of approximately 900 compared to the analogous tripeptide, with KiTry/KiThr of approximately 4. Compound 2 acts as a competitive inhibitor (KiThr of 0.6 nM) and is highly selective with no effect on trypsin. Crystallographic analysis of complexes of human alpha-thrombin with compound 1 (1.8 A) and compound 2 (1.85 A) shows a covalent bond between the boron of the inhibitor and Ser195 (bond lengths B-O of 1.55 and 1.61 A, respectively). The isothiouronium group of compound 1 forms bidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2 and S3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming no interactions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described for hirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583-601.]. Because of the similarity of binding at each of these sites to that of the analogous peptides added alone, this approach may be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors and may also be applicable to the design of inhibitors of other proteases.

Large-scale production of peptides using the solid phase continuous flow method. Part 2: preparative synthesis of a 26-Mer peptide thrombin inhibitor.

A preparative method for the preparation of large peptides is described. An advantageous theoretical weight of peptide/weight of starting resin ratio (tPw/Rw) of about 0.3 was successfully experimented. The esterification of the first amino acid was realized with a racemization of less than 1%. The study of the coupling conditions led to the use of a diluted acylating mixture that allowed a 56% consumption of the amino acid derivatives (percentage use of amino acids) introduced in the synthesis. The cost analysis of the synthesis showed that the recovery of the amino acid derivatives was not worthwhile.

Hirunorms, novel hirudin-like direct thrombin inhibitors.

1. Hirunorms are new synthetic peptides designed to interact with thrombin in a similar way to the natural inhibitor hirudin. 2. Hirunorms are specific and efficient in vitro inhibitors of thrombin activity. 3. Hirunorms are potent anticoagulant and antithrombotic agents in in vivo experimental models devoid of hemorrhagic effects at doses that are active in preventing thrombosis.

Synthesis, structure, and structure-activity relationships of divalent thrombin inhibitors containing an alpha-keto-amide transition-state mimetic.

A new class of divalent thrombin inhibitors is described that contains an alpha-keto-amide transition-state mimetic linking an active site binding group and a group that binds to the fibrinogen-binding exosite. The X-ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition-state-like binding of the alpha-keto-amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side-chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition-state-containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their alpha-keto-amide counterparts. The most potent alpha-keto-amide inhibitor, CVS995, with a Ki = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.

Synthesis and study on a novel inhibitor of thrombin containing no side chain at the P1 position of the polypeptide chain.

The presented work allows one to speculate that the hydrophobic contacts of the residues located at the P2 and P3 positions with the corresponding subsites of thrombin (S2 and S3) allow the synthesis of compounds that can react with thrombin specifically and probably may not interact with other trypsin-like serine proteases. Substitution of proline by m-Abz-residue may result in the development of novel substrates and inhibitors for thrombin.

Immobilization of human thrombomodulin onto poly(ether urethane urea) for developing antithrombogenic blood-contacting materials.

Thrombomodulin (TM) is a newly described endothelial cell-associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant. In this study, focussing on the application of TM for biomedical materials, recombinant human TM (hTM) was immobilized onto the polymers for medical use, and the evaluation of their antithrombogenicity and the interaction with platelets were investigated. As the base polymer for immobilization reaction, poly(ether urethane urea) (PEUU), which was reported to have good blood compatibility, was used. hTM-immobilized PEUU showed superior antithrombogenic activity, such as the prolongation of plasma recalcification time and the inhibition of thrombin-induced platelet aggregation, though the amount of immobilized hTM was very small (i.e. less than 1 microgram/cm2). Platelet adhesions onto hTM-immobilized PEUU were not observed. These results show that the immobilization of hTM does not change the native good blood compatibility of PEUU, but provides excellent anticoagulant activity.

Inhibition of thrombin's clotting activity by synthetic peptide segments of its inhibitors and substrates.

Synthetic peptides corresponding to segments of heparin cofactor II, fibrinogen, thrombomodulin, and hirudin were identified that inhibit thrombin's clotting of fibrinogen without blocking the enzyme's active site. Thrombin activity was inhibited 50% by the following peptide concentrations, with numbers in parenthesis indicating residues in the protein sequence: heparin cofactor II(54-75), 38 microM; heparin cofactor II(49-75), 28 microM; fibrinogen gamma B-chain(410-427), 130 microM; thrombomodulin(426-444), 140 microM; hirudin(54-65), 1.3 microM; hirudin(54-75)SO4, 0.17 microM. All of these peptides are likely to bind to thrombin's anion-binding exosite, suggesting that this site has broad sequence specificity such that it may participate in many of thrombin's interactions with physiological substrates and inhibitors.

Thrombin receptor antagonists. Structure-activity relationships for the platelet thrombin receptor and effects on prostacyclin synthesis by human umbilical vein endothelial cells.

Structure-activity studies on a series of analogues of N-(3-methyl-S-(1-pyrrolidinyl carbonyl) butyl)-D-alanine ethyl ester hydrochloride (SC42619) have defined the features of this dipeptide analogue required for observation of thrombin receptor antagonist activity on the human platelet. The affinity for SC42619, and for its structural analogue SC43583 is enhanced by pretreatment of the platelets with chymotrypsin. Endothelial cell prostacyclin (PGI2) synthesis induced by thrombin and trypsin is selectively inhibited by SC42619 provided that prolonged exposure to this antagonist is avoided. However inhibition of PGI2 synthesis by SC42619 is not overcome by increasing the thrombin concentration. The data provide further support for identification of SC42619 and certain of its analogues as selective antagonists at the platelet thrombin receptor but suggest that these compounds may have more complex, and possibly non-selective effects on the endothelial cell.

Prevention of heparin-resistant thrombotic occlusion of hollow-fiber hemodialyzers by synthetic antithrombin.

Because heparin anticoagulation during hemodialysis with hollow-fiber devices is associated with progressive loss of volume of fiber bundles subsequent to thrombotic occlusion, we examined the antithrombotic and antihemostatic effects of the irreversible synthetic thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl-chloromethylketone (FPRCH2Cl) during repeated exposure of cupramonium cellulose hollow-fiber hemodialyzers in an extracorporeal blood circuit in baboons. By contrast with full anticoagulating doses of heparin, FPRCH2Cl (100 nmol/kg/min) decreased both the loss of fiber bundle volume (19.1% +/- 7.0% vs 6.4% +/- 3.6% p less than 0.01) and deposition of 111In-labeled platelets within the dialyzer (15.7 +/- 5.9 x 10(9) vs 3.2 +/- 1.2 x 10(9) platelets; p less than 0.01). Additionally, blood markers of thrombus formation in vivo (i.e., plasma beta-thromboglobulin, platelet factor 4, and fibrinopeptide A) remained at low levels throughout infusion of FPRCH2Cl, whereas levels were elevated during heparin therapy (p less than 0.01 in each case). FPRCH2Cl, but not heparin, prolonged bleeding times (p less than 0.001) without affecting the capacity of platelets to aggregate in response to the presence of either collagen or adenosine diphosphate ex vivo. Complement activation by the dialyzer was not affected by FPRCH2Cl. We conclude that the progressive loss of dialyzer hollow fibers is a platelet-dependent, thrombin-mediated process that, although resistant to heparin, is interrupted by the synthetic antithrombin FPRCH2Cl.