Selective boron-containing thrombin inhibitors--X-ray analysis reveals surprising binding mode.

Based on the structural comparison of the S1 pocket in different trypsin-like serine proteases, a series of Boc-D-trimethylsilylalanine-proline-boro-X pinanediol derivatives, with boro-X being different amino boronic acids, have been synthesized as inhibitors of thrombin. Among the novel compounds, a number of derivatives were synthesized which appeared to have side-chain variants too big to fit into the S1 pocket. Nevertheless, these compounds inhibited thrombin in the nM range. The X-ray structure of one of these inhibitors bound to the active side of thrombin reveals that a new binding mode is responsible for these surprising results.

Design, synthesis and biological evaluation of selective boron-containing thrombin inhibitors.

Based on the structural comparison of the S-1 pocket in different trypsin-like serine proteases, a series of Boc-D-trimethylsilylalanine-proline-boro-X pinanediol derivatives, with boro-X being different amino boronic acids, have been synthesised as inhibitors of thrombin. The influence of hydrogen donor/acceptor properties of different residues in the P-1 side chain of these inhibitors on the selectivity profile has been investigated. This study confirmed the structure-based working hypothesis: The hydrophobic/hydrophilic character of amino acid residues 190 and 213 in the neighbourhood of Asp 189 in the S-1 pocket of thrombin (Ala/Val), trypsin (Ser/Val) and plasmin (Ser/Thr) define the specificity for the interaction with different P-1 residues of the inhibitors. Many of the synthesised compounds demonstrate potent antithrombin activity with Boc-D-trimethylsilylalanine-proline-boro-methoxypropylglycine++ + pinanediol (9) being the most selective thrombin inhibitor of this series.

Thrombin inhibition in an ex vivo model of porcine heart xenograft hyperacute rejection.

Prominent components of vascularized xenograft rejection such as platelet activation and microvascular thrombosis may be dependent upon thrombin generation in vivo. To study potential therapeutic benefits of a synthetic low-molecular-weight thrombin inhibitor, SDZ MTH 958, in hyperacute porcine heart rejection by human blood ex vivo, a working model of hyperacute rejection of porcine by fresh, heparinized (6 microM/ml) human blood with or without 1 microM SDZ MTH 958 was used. Thrombin-antithrombin complexes (TAT) and prothrombin fragment F1.2 levels as markers of thrombin activation were determined, and biopsies from rejected hearts were analyzed by immunohistopathology. Control porcine hearts (n=8) underwent a rapid and consistent decline in cardiac output, ceasing function by 60 min. Experimental cardiac output values of 14 ml/g (SEM 1.2) were significantly higher than seen in controls (5 ml/g SEM 0.6) after 5 min of cardiac work, and prolonged survival times up to 120 min were noted (P<0.05). Activity of SDZ MTH 958 was confirmed by functional assays throughout perfusion. Levels of TAT and F1.2 increased consistently in control samples when compared with plasma samples containing SDZ MTH 958. Immunohistopathological examination confirmed diminished fibrin deposition, reduced leukocyte adherence to endothelium, impaired diapedesis and less tissue necrosis in the hearts perfused with SDZ MTH 958. SDZ MTH 958, in this xenoperfusion model, prolonged survival, enhanced function of the explanted organ, and improved histological features at the time of rejection. Effective and specific antagonism of thrombin may be useful as an adjunct therapy to complement inhibition for xenograft rejection

Antithrombotic activity in vivo of SDZ 217-766, a low-molecular weight thrombin inhibitor in comparison to heparin.

Antithrombotic potency of SDZ 217-766, a potent inhibitor of thrombin and other trypsin-like serine proteases, was studied in comparison with heparin in rat models of thrombin induced lung platelet accumulation, of thrombosis in arterio-venous shunt, and of venous thrombosis induced by tissue factor. Thrombin-induced platelet accumulation in the lung was inhibited dose-dependently by SDZ 217-766 following intravenous (i.v.) administration of 0.03 mg/kg to 0.3 mg/kg as well as by intraduodenal (i.d.) administration of 3 mg/kg and 10 mg/kg. Comparable inhibitory effects were observed with heparin at 30 IU/kg and 100 IU/kg. In the rat arterio-venous shunt, following i.v. administration of SDZ 217-766, thrombus formation was inhibited by 40% at 0.1 mg/kg, by 60% at 0.3 mg/kg and was abolished at 1.0 mg/kg whilst APTT was prolonged 1.1 fold over the control value at 0.1 mg/kg and 2.7 fold at 1.0 mg/kg. Similar inhibitory effects were observed following i.d. administration of 10 and 30 mg/kg with only marginal (1.2 to 1.8 fold) APTT elevation. In the same model, heparin administered either i.v., 30-300 IU/kg, or subcutaneously, 100 and 300 IU/kg, inhibited thrombus formation dose dependently but in contrast to SDZ 217-766, the inhibitory effect was paralleled by 5-to > 10 fold APTT elevation over baseline. In the venous thrombosis model, SDZ 217-766 infused at 10 micrograms/kg/min and 20 micrograms/kg/min, reduced thrombus formation by 35% and 70%, respectively. In comparison, thrombus formation was decreased by 22% when heparin was infused at 1 IU/kg/min, and abolished at 3 IU/kg/min.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic low-molecular weight thrombin inhibitors: molecular design and pharmacological profile.

Thrombin is a multifunctional protein: in addition to its role in coagulation, thrombin has important biological effects on platelets, endothelial and smooth muscle cells, leukocytes, the heart and neurones. A detailed understanding of the structure of thrombin, of related serine proteases and of enzyme-inhibitor complexes has aided in the discovery of potent and selective new inhibitor molecules. Some of these novel thrombin inhibitors are active when administered orally and have shown remarkable efficacy as antithrombotic agents in animal models, offering a greater therapeutic potential than presently available drugs. This potential extends also to non-thrombotic indications where thrombin may be involved, namely inflammation, cancer and neurodegenerative diseases. The recent identification of specific thrombin receptors on different cells provides an alternative strategy for inhibiting thrombin's cellular actions, without necessarily compromising its role in haemostasis. In this review, Carlo Tapparelli and colleagues present a comprehensive update of these recent developments in the field of thrombin biology and pharmacology suggesting a new era of therapeutic drugs is on the horizon.

Platelet aggregation and fibrinogen binding in human, rhesus monkey, guinea-pig, hamster and rat blood: activation by ADP and a thrombin receptor peptide and inhibition by glycoprotein IIb/IIIa antagonists.

Platelet aggregation and fibrinogen binding in whole blood, induced either by ADP or by a 14 amino acid peptide mimicking an N-terminal region of the platelet thrombin receptor (TRP, thrombin receptor activating peptide), have been studied with blood from different species. Aggregation was assessed by counting the number of single platelets in blood before und after addition of the agonist with an automated cell counter. Both ADP (0.02-0.5 microM) and TRP (1-10 microM) were found to be potent agonists of platelet aggregation in human, rhesus monkey and guinea-pig blood, causing a near-maximal aggregatory response within 2 min of agonist addition. In contrast, hamster and rat platelets were much less responsive to both ADP and TRP in terms of the whole blood aggregation. Echistatin, RGDW and a synthetic glycoprotein (GP) IIb/IIIa antagonist, Ro 43-8857, inhibited fibrinogen binding to purified immobilized human GP-IIb/IIIa with IC50's of 1.6, 88 and 11.4 nM, respectively. In whole human blood, the respective IC50's (as determined by flow cytometric analysis of platelet fibrinogen binding) were 4.4, 1700 and 29.5 nM. The affinities of these three compounds for inhibiting fibrinogen binding in whole blood from rhesus monkeys and guinea-pigs were similar to the affinities for human platelets, but with rat blood echistatin, RGDW and Ro 43-8857 were all around 100-fold less potent. Ro 43-8857 was a potent inhibitor of ADP- or TRP-induced platelet aggregation in human, rhesus monkey and guinea-pig whole blood (IC50 of 69-320 nM) but was much less active in hamster blood.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo characterization of a neutral boron-containing thrombin inhibitor.

Peptide boronic acid derivatives have proven to be very potent inhibitors of serine proteases with boroarginine derivatives being particularly potent thrombin inhibitors. The importance of the charged side chain of arginine has been investigated by synthesizing a derivative in which this side chain has been replaced by a neutral one. This boronic acid derivative, D-benzyloxycarbonyl (Z)-Phe-Pro-methoxypropylglycine-pinanediol (MpgC10H16), inhibited thrombin by a competitive mechanism with an inhibition constant (Ki) of 8.9 nM. In comparison to boroarginine derivatives, Z-D-Phe-Pro-boroMpgC10H16 displayed higher selectivity for thrombin over trypsin (Ki = 1.1 microM) and plasmin (Ki = 15.7 microM). Prolongation of thrombin time and activated partial thromboplastin time were observed with micromolar concentrations of Z-D-Phe-Pro-boroMpgC10H16. In a thrombin-dependent in vitro aggregation assay with human platelets, Z-D-Phe-Pro-boroMpgC10H16 inhibited aggregation with an IC50 of 85 nM. When tested in a thrombin-dependent platelet accumulation model in the rat, a bolus injection of (Z)-D-Phe-Pro-boroMpgC10H16 (0.3-3 mg/kg) inhibited platelet accumulation. Thus, the substitution of the charged guanidino group in the P1 side chain by the neutral methoxy group resulted in a potent and highly selective thrombin inhibitor with an interesting pharmacological profile with in vitro as well as in vivo models.

Benzyloxycarbonyl-D-Phe-Pro-methoxypropylboroglycine: a novel inhibitor of thrombin with high selectivity containing a neutral side chain at the P1 position.

Thrombin, the blood-clotting enzyme, is a serine proteinase with trypsin-like specificity and is able to cleave Arg-Xaa peptide bonds but only in a very limited number of substrates (and sites therein). For the prevention and treatment of thrombosis the control of thrombin activity is a key target, and a variety of synthetic inhibitors have been introduced recently, all of which have a positive charge at the P1 site. We report the synthesis of the first example of a new class of inhibitor containing a neutral side chain at the P1 site, the peptide benzyloxycarbonyl-D-Phe-Pro- methoxypropylboroglycine. The peptide is a potent inhibitor of thrombin [Ki (limiting) = 7 nM] and is highly selective for its target enzyme in respect of other serine proteinases. This may be expected to confer considerable advantage in terms of specificity of action and reduced toxicity over conventional, positively charged, inhibitors.

1H NMR studies of echistatin in solution. Sequential resonance assignments and secondary structure.

Two-dimensional 1H-NMR methods have been used to obtain complete proton resonance assignments for the 49-residue protein echistatin from the viper Echis carinatus. The protein in solution contains only a small amount of regular secondary structure with four very short beta-strands. These beta-strands form two short segments of antiparallel beta-sheet, as evidenced by the observed cross-strand NOE. The first two strands are connected with a tight reverse turn, whereas the remaining two strands are linked together by an 11-residue loop forming a so-called hairpin. The tripeptide unit Arg-Gly-Asp, responsible for the binding of echistatin to the fibrinogen receptor glycoprotein GPIIb/IIIa, is located at the tip of this very hydrophilic loop.

Binding of a Bolton-Hunter substituted homostatine analog to affinity-immobilized human renin.

The binding of a Bolton-Hunter reagent substituted homostatine analog, SDZ 213-776, to human renin was investigated at pH 6.5 and 7.4. At both pH values, SDZ 213-776 bound to human renin in a reversible and saturable manner. The binding characteristics conformed to a one-site binding model. The dissociation constant Kd, obtained at equilibrium, was four-fold lower at pH 6.5 that at pH 7.4 (0.94 nM vs 3.7 nM). Under non-equilibrium conditions, only the association kinetic constant k+1 was affected by pH. The results of the binding assay at pH 6.5 correlated well with those obtained in enzymatic assay at the same pH.