Inhibitors thrombin

Another important area of research in structure-based design is the elucidation of possible binding modes and the rationalization of observed structure-activity relationships. This aspect is highlighted by the examples described in sections 2.4 and 2.5. 

The three-dimensional structure of the transition state and the protein environment exhibit ideal complementarity to each other, which is the prerequisite for the observed rate enhancement of the hydrolytic reaction. 

The specificity for cleavage after arginine or lysine is mediated by a recognition pocket with an aspartate side chain at the bottom. This pocket accommodates the positively charged side chain by forming a salt bridge with the carboxylate of the aspartic acid. 

In thrombin, the specificity extends far beyond the residue preceding the cleavage site. NMR [4] and X-ray studies [3, 5] revealed that three hydrophobic residues in fibrinogen 2 two, eight and nine residues before the cleavage site, respectively, occupy unique hydrophobic pockets (Fig. 3). These hydrophobic pockets are formed by a unique additional loop Tyr-Pro-Pro-Trp above the active site. 

The binding mode of three previously known inhibitors MD805 3 and NAPAP 4 (Fig. 4) as revealed in the X-ray structure of their complexes with thrombin [2,3] has some unexpected 

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D, H W Hoeffken, D Crosse, J Stuerzebecher, P D Martin, B F P Edwards and W Bode 1992. Refined 2.3 Angstroms X-Ray Crystal Structure of Bovine Thrombin Complexes Formed witli he 3 Benzamidine and Arginine-Based Thrombin Inhibitors NAPAP, 4-TAPAP and MQPA A Starting Point for Improving Antithrombotics. Journal of Molecular Biology 226 1085-1099. 

Two small molecule DTIs are argatroban (Novastan, MW 527 Da) and the oral thrombin inhibitor, ximelagatran (Exanta, MW 474 Da) Ximelagatran is an inactive pro-drug after absorption, it is metabolized to the active DTI, melagatran [MW 430 Da]. Concerns regarding hepatotoxicity have prevented (xi)melagatran... 

L-Dihydroxyphenylalanine 4-Dihydroxyphenylethylamine Dimeric Transcription Factors Dioxins Dipeptidase Dipeptidylpeptidase Dipeptidylpeptidase IV Direct Thrombin Inhibitors Discharge of Neurons... 

The 2(lH)-pyrazinone system has received increased interest in the past two decades by both synthetic and biological research, due to its presence in a variety of natural and non-natural products as well as pharmacologically active compounds. The easy and diverse methods for the generation of this versatile scaffold make it a prime choice for the current pharmaceutical research hke thrombin inhibitors, substance P antagonists, etc. The rich 1,4-azadiene... 

Bohm HJ, Banner DW, Weber L. Combinatorial docking and combinatorial chemistry design of potent non-peptide thrombin inhibitors. J Comput Aided Mol Design 1999 13 51-6. 

Figure 24.4 The decision-analytic model shows the three strategies that were examined by Arnold and researchers [22] to evaluate the financial implications of the direct thrombin inhibitor argatroban for early treatment (<48 hours after thrombocytopenia onset), compared with delayed treatment, of heparin-induced thrombocytopenia (HIT) with or without thrombosis.

Arnold R, Kim R, Zhou Y, Tang B. Budgetary impact of heparin-induced thrombocytopenia with thrombosis and treatment with the direct thrombin inhibitor Argatroban (P401E). ASHP 39th Midyear Clinical Meeting. Orlando, FL, 2004. 

Four naturally occurring thrombin inhibitors exist in normal plasma. The most important is antithrombin III (often called simply antithrombin), which contributes approximately 75% of the antithrombin activity. Antithrombin III can also inhibit the activities of factors IXa, Xa, XIa, Xlla, and Vila complexed with tissue factor. a2-Macroglobulin contributes most of the remainder of the antithrombin activity, with heparin cofactor II and aj-antitrypsin acting as minor inhibitors under physiologic conditions. 

Hadjipavlou-Litina D (2006) QSAR and Molecular Modeling Studies of Factor Xa and Thrombin Inhibitors. 4 1-53... 

Zwitterionic character is notable in several therapeutic area series, e.g. in angiotensin-converhng enzyme inhibitors, quinolone anhbacterials and thrombin inhibitors. The aqueous solubiUty measurement of zwitterions is very pH dependent as might be expected. The relationship of aqueous solubiUty to ionization state is extraordinarily complex if the zwitterion is of the type capable of an equi-Ubrium between true zwitterion and formally neutral forms (e.g. as in a quinolone antibacterial). For these types of complex equilibria, salt effects on solubility may be unexpectedly large, e.g. solubility unexpectedly may track with the chaotropic character of the salt... 

Frederick, R. Robert, S. Charlier, C. de Ruyck, J. Wouters, J. Masereel, B. Pochet, L. Mechanism-based thrombin inhibitors design, synthesis, and molecular docking of a new selective 2-OXO-2/7- l-benzopyxan derivative. J. Med. Chem. 2007, 50, 3645-3650. 

Given that thrombin is the central mediator of coagulation and amplifies its own production, it is a natural target for pharmacologic intervention. Direct thrombin inhibitors (DTIs) bind thrombin and prevent interactions with its substrates (Fig. 7-7). Several injectable DTIs are approved for use in the United States including lepirudin, bivalirudin, arga-troban, and desirudin. Several oral DTIs are currently in... 

FIGURE 7-7. Mechanism of action of direct thrombin inhibitors. 

Small-molecule direct thrombin inhibitors have been structurally modified for oral administration. Approximately... 

Activated partial thromboplastin time aPTT is performed by adding calcium phospholipids and kaolin to citrated blood and measures the time required for a fibrin clot to form. In this manner, aPTT measures the activity of intrinsic and common pathways. Prolongation of aPTT may be due to a deficiency or inhibitor for factors II, V, VIII, IX, X, XI, and XII. It also may be due to heparin, direct thrombin inhibitors, vitamin K deficiency, liver disease, or lupus anticoagulant. 

The most potent thrombin inhibitor is hirudin, originally isolated from the salivary glands of the medicinal leech Hirudo medicinalis. Its inhibition constant is in the femtomolar (10-15 M) range (57). It is a 65-amino-acid tyrosine-sulfated single-chain polypeptide. Recombinant hirudin differs from native hirudin by the absence of the sulfate group on tyrosine 63 (Tyr-63) and is referred to as desulfato hirudin. The loss of this sulfate group reduces the thrombin inhibitory potency by 10-fold. 

Bivalent inhibitors of thrombin have been synthesized to bind the anion-binding exosite and active (catalytic) site of thrombin simultaneously. By coupling the carboxy terminal fragment of hirudin to a tripeptide (D-Phe-Pro-Arg) by including a spacer molecule, both the anion exosite and the catalytic site are blocked. An example of such a molecule is Hirulog, which has 20 amino acids and has a Kj of 2 nM (61). Its ability to block the active site has been questioned, since thrombin has been shown to cleave the Arg-Pro bond of Hirulog slowly in vivo (58). In addition to hirudin and hirudin-like compounds, three other classes of site-directed thrombin inhibitors deserve mention. 

Synthetic heterocyclic and modified amino acid derivatives have been grouped in a class of thrombin inhibitors called peptidomimetics. An example of such a compound is argatroban, with a molecular mass of 532 Da. It blocks thrombin s active catalytic site by binding to the adjacent apolar binding site. This selective reversible inhibitor of thrombin has a K of 19 nM and blocks thrombin s role in coagulation and fibrinolysis (62). 

A group of peptide derivatives such as peptide arginals and boronic acid peptide derivatives belong to another class of reversible thrombin inhibitors. One such inhibitor is PPACK (D-Phe-Pro-Arg chloromethyl ketone), which functions as a powerful irreversible thrombin inhibitor by alkylating the histidine residue at the catalytic site of thrombin (58). It, however, is unstable in neutral solution, as it undergoes cyclization and inactivation. However, the D-methyl derivative of D-Phe-Pro-Arg-H (D-Mephe-Pro-Arg-H) called efegatran, with a molecular mass of 515 Da, is a stable selective reversible inhibitor of thrombin with a K. of approximately 100 nM. The basic amino terminus in this compound is responsible for promoting the specificity toward thrombin (63). 

Direct thrombin inhibitors such as hirudin, Hirulog, the peptide aldehyde efegatran, and peptidomimetic compound argatroban have undergone clinical trials. Their application in the prevention and treatment of deep vein thrombosis contin-... 

Finally, with the use of direct thrombin inhibitors such as hirudin for anticoagulant therapy, laboratory tests such as APTT may not be sensitive enough to follow therapy. 

Lefkovits J., Topol E. J. Direct thrombin inhibitors in cardiovascular medicine. Circulation 1994 90, 1522-36. 

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