Structural basis of the thrombin selectivity of a ligand that contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl- piperidin-3-yl)-propanoic acid at P1

We have studied the thrombin and trypsin complexed structures of a pair of peptidomimetic thrombin inhibitors, containing different P1 fragments. The first has arginine as its P1 fragment, and the second contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl-piperidin-3-yl)-propano ic acid (SAPA), a fragment known to enhance thrombin/trypsin selectivity of inhibitors. On the basis of an analysis of the nonbonded interactions present in the structures of the trypsin and thrombin complexes of the two inhibitors, the calculated accessible surfaces of the enzymes and inhibitors in the four complexes, data on known structures of trypsin complexes of inhibitors, and factor Xa inhibitory potency of these compounds, we conclude that the ability of this arginine mimic to increase thrombin selectivity of an inhibitor is mediated by its differential interaction with the residue at position 192 (chymotrypsinogen numbering). Thrombin has a glutamic acid at residue 192, and trypsin has a glutamine. The analysis also suggests that this constrained arginine mimic, when present in an inhibitor, might enhance selectivity against other trypsin-like enzymes that have a glutamine at residue position 192.     

Novel potent and selective thrombin inhibitors based on a central 1,4-benzoxazin-3(4H)-one scaffold

Novel thrombin inhibitors with the central 1,4-benzoxazine-3(4 H)-one scaffold, benzamidine P1 arginine side chain mimetic and various P3 moieties are described. 3-(Benzyl(2-(4-carbamimidoylbenzyl)-4-methyl-3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7-yl)amino)-3-oxopropanoic acid (7b), the most potent compound in the series, exhibited a K i of 2.6 nM in vitro for thrombin and high selectivity against trypsin and factor Xa.     

In-depth study of tripeptide-based alpha-ketoheterocycles as inhibitors of thrombin. Effective utilization of the S1' subsite and its implications to structure-based drug design

Thrombin inhibitors are potentially useful in medicine for their anticoagulant and antithrombotic effects. We synthesized and evaluated diverse heterocycle-activated ketones based on the d-Phe-Pro-Arg, and related thrombin active-site recognition motifs, as candidate inhibitors. The peptide-based alpha-ketoheterocycles were typically prepared by either an imidate or a Weinreb amide route (Schemes 1 and 2), the latter of which proved to be more general. Test compounds were generally assayed for inhibition of human alpha-thrombin and bovine trypsin. From a structure-based design standpoint, the heterocycle allows one to explore and adjust interactions within the S1' subsite of thrombin. The preferred alpha-ketoheterocycle is a pi-rich 2-substituted azole with at least two heteroatoms proximal to the carbon bearing the keto group, and a preferred thrombin inhibitor is 2-ketobenzothiazole 3, with a potent K(i) value of 0.2 nM and ca. 15-fold selectivity over trypsin. 2-Ketobenzothiazole 13 exhibited exceedingly potent thrombin inhibition (K(i) = 0.000 65 nM; slow tight binding). Several alpha-ketoheterocycles had thrombin K(i) values in the range 0.1-400 nM. The "Arg" unit in the alpha-ketoheterocycles can be sensitive to stereomutation under mildy basic conditions. For example, 2-ketothiazoles 4 and 59 readily epimerize at pH 7.4, although they are fairly stable stereochemically at pH 3-4; thus, suitable conditions had to be selected for the enzymatic assays. Lead d-Phe-Pro-Arg 2-benzothiazoles 3, 4, and 68 displayed good selectivity for thrombin over other key coagulation enzymes (e.g., factor Xa, plasmin, protein Ca, uPA, tPA, and streptokinase); however, their selectivity for thrombin over trypsin was modest (<25-fold). Compounds 3, 4, and 68 exhibited potent in vitro antithrombotic activity as measured by inhibition of gel-filtered platelet aggregation induced by alpha-thrombin (IC(50) = 30-40 nM). They also proved to be potent anticoagulant/antithrombotic agents in vivo on intravenous administration, as determined in the canine arteriovenous shunt (ED(50) = 0.45-0.65 mg/kg) and the rabbit deep vein thrombosis (ED(50) = 0.1-0.4 mg/kg) models. Intravenous administration of 3, and several analogues, to guinea pigs caused hypotension and electrocardiogram abnormalities. Such cardiovascular side effects were also observed with some nonguanidine inhibitors and inhibitors having recognition motifs other than d-Phe-Pro-Arg. 2-Benzothiazolecarboxylates 4 and 68 exhibited significantly diminished cardiovascular side effects, and benzothiazolecarboxylic acid 4 had the best profile with respect to therapeutic index. The X-ray crystal structures of the ternary complexes 3-thrombin-hirugen and 4-thrombin-hirugen depict novel interactions in the S(1)' region, with the benzothiazole ring forming a hydrogen bond with His-57 and an aromatic stacking interaction with Trp-60D of thrombin's insertion loop. The benzothiazole ring of 3 displaces the Lys-60F side chain into a U-shaped gauche conformation, whereas the benzothiazole carboxylate of 4 forms a salt bridge with the side chain of Lys-60F such that it adopts an extended anti conformation. Since 3 has a 10-fold greater affinity for thrombin than does 4, any increase in binding energy resulting from this salt bridge is apparently offset by perturbations across the enzyme (viz. Figure 4). The increased affinity and selectivity of 2-ketobenzothiazole inhibitors, such as 3, may be primarily due to the aromatic stacking interaction with Trp-60D. However, energy contour calculations with the computer program GRID also indicate a favorable interaction between the benzothiazole sulfur atom and a hydrophobic patch on the surface of thrombin.     

Design of selective thrombin inhibitors based on the (R)-Phe-Pro-Arg sequence

Potent and selective inhibitors of thrombin were sought based on the (R)-Phe-Pro-Arg sequence. The objective was to generate similar binding interactions to those achieved by potent competitive inhibitors of the argatroban type, so eliminating the need for covalent interaction with the catalytic serine function, as utilized by aldehyde and boronic acid type inhibitors. Improving the S(1) subsite interaction by substitution of arginine with a 4-alkoxybenzamidine residue provided potent lead 2 (K(i) = 0.37 nM). Though an amide bond, which H-bonds to the active site, is lost, modeling indicated that a new H-bond is generated between the alkoxy oxygen atom and the catalytic Ser-195 hydroxyl group. Substitution of the benzamidine system by 1-amidinopiperidine then gave compound 4, which provided a further gain in selectivity over trypsin. However, previous work had shown that these compounds were likely to be too lipophilic (Log D +0.4 and +0.2, respectively) and to suffer rapid hepatic extraction, presumably via biliary elimination. Accordingly, both proved short-acting when administered intravenously to rats and showed poor activity when given intraduodenally. The aim was then to reduce lipophilicity below a log D of -1.2, which in a previously reported series had been effective in preventing rapid clearance. It was anticipated that compounds of this type would rely on the cation selective paracellular route of absorption from the gastrointestinal tract. Potent polar analogues with selectivity >1000 over trypsin were obtained. The best in vivo activity was shown by compound 12. However, in the final analysis, its oral bioavilability proved poor, relative to analogues with similar physicochemical properties derived from argatroban, consistent with the hypothesis that molecular shape is an additional important determinant of paracellular absorption.     

Arginine mimetic structures in biologically active antagonists and inhibitors

Peptidomimetics have found wide application as bioavailable, biostable, and potent mimetics of naturally occurring biologically active peptides. L-Arginine is a guanidino group-containing basic amino acid, which is positively charged at neutral pH and is involved in many important physiological and pathophysiological processes. Many enzymes display a preference for the arginine residue that is found in many natural substrates and in synthetic inhibitors of many trypsin-like serine proteases, e.g. thrombin, factor Xa, factor VIIa, trypsin, and in integrin receptor antagonists, used to treat many blood-coagulation disorders. Nitric oxide (NO), which is produced by oxidation of L-arginine in an NADPH- and O(2)-dependent process catalyzed by isoforms of nitric oxide synthase (NOS), exhibits diverse roles in both normal and pathological physiologies and has been postulated to be a contributor to the etiology of various diseases. Development of NOS inhibitors as well as analogs and mimetics of the natural substrate L-arginine, is desirable for potential therapeutic use and for a better understanding of their conformation when bound in the arginine binding site. The guanidino residue of arginine in many substrates, inhibitors, and antagonists forms strong ionic interactions with the carboxylate of an aspartic acid moiety, which provides specificity for the basic amino acid residue in the active side. However, a highly basic guanidino moiety incorporated in enzyme inhibitors or receptor antagonists is often associated with low selectivity and poor bioavailability after peroral application. Thus, significant effort is focused on the design and preparation of arginine mimetics that can confer selective inhibition for specific trypsin-like serine proteases and NOS inhibitors as well as integrin receptor antagonists and possess reduced basicity for enhanced oral bioavailability. This review will describe the survey of arginine mimetics designed to mimic the function of the arginine moiety in numerous peptidomimetic compounds (thrombin inhibitors, factor Xa inhibitors, factor VIIa inhibitors, integrin receptor antagonists, nitric oxide synthase inhibitors), with the aim of obtaining better activity, selectivity and oral bioavailability.     

Synthesis of novel thrombin inhibitors. Use of ring-closing metathesis reactions for synthesis of P2 cyclopentene- and cyclohexenedicarboxylic acid derivatives

The thrombin inhibitory tripeptide d-Phe-Pro-Arg has been mimicked using either cyclopentenedicarboxylic derivatives or a cyclohexenedicarboxylic derivative as surrogate for the P2 proline. In the P3 position, tertiary amides were optimized as d-Phe P3 replacements. The P1 arginine was, in all compounds, substituted with the more rigid and biocompatible 4-aminomethylbenzamidine. One of the novel inhibitors was cocrystallized with alpha-thrombin and subjected to X-ray analysis. From analysis of the X-ray crystal structure, new ligands were designed leading to significantly improved binding affinity, the lead candidate exhibiting an in vitro IC(50) of 49 nM.     

Structure-based approach for the discovery of bis-benzamidines as novel inhibitors of matriptase

Matriptase, a trypsin-like serine protease, which may be involved in tissue remodeling, cancer invasion, and metastasis. Potent and selective matriptase inhibitors not only would be useful pharmacological tools for further elucidation of the role of matriptase in these processes but also could have therapeutic potential for the treatment and/or prevention of cancers. We report herein the structure-based approach for the discovery of bis-benzamidines as a novel class of potent matriptase inhibitors. The lead compound, hexamidine (1), inhibits not only the proteolytic activity of matriptase, (K(i) = 924 nM) but also of thrombin K(i) = 224 nM). By testing several available analogues, we identified a new analogue (7) that has a K(i) = 208 nM against matriptase and has only weak inhibitory activity against thrombin (K(i) = 2670 nM), thus displaying a 13-fold selectivity toward matriptase. Our results demonstrated that structure-based database screening is effective in the discovery of matriptase inhibitors and that bis-benzamidines represent a class of promising matriptase inhibitors that can be used for further drug design studies. Finally, our study suggested that there is sufficient structural differences between matriptase and its closely related serine proteases, such as thrombin, for the design of potent and selective matriptase inhibitors.     

Application of fragment-based lead generation to the discovery of novel, cyclic amidine beta-secretase inhibitors with nanomolar potency, cellular activity, and high ligand efficiency

Fragment-based lead generation has led to the discovery of a novel series of cyclic amidine-based inhibitors of beta-secretase (BACE-1). Initial fragment hits with an isocytosine core having millimolar potency were identified via NMR affinity screening. Structure-guided evolution of these fragments using X-ray crystallography together with potency determination using surface plasmon resonance and functional enzyme inhibition assays afforded micromolar inhibitors. Similarity searching around the isocytosine core led to the identification of a related series of inhibitors, the dihydroisocytosines. By leveraging the knowledge of the ligand-BACE-1 recognition features generated from the isocytosines, the dihydroisocytosines were efficiently optimized to submicromolar potency. Compound 29, with an IC50 of 80 nM, a ligand efficiency of 0.37, and cellular activity of 470 nM, emerged as the lead structure for future optimization.     

Novel non-covalent azaphenylalanine thrombin inhibitors with an aminomethyl or amino group at the P1 position

Design, synthesis and biological evaluation of a series of novel non-covalent azaphenylalanine thrombin inhibitors are presented. Replacement of the basic benzamidine moiety in azaphenylalanine derivatives by benzylamine (P1 part of a molecule) and the introduction of a N-cyclopentyl-N-methylamine moiety in the P2 part of a molecule resulted in the thrombin inhibitor LK-733 with greatly increased selectivity against trypsin and an in vitro Ki of 31 nM.     

Synthesis and kinetic evaluation of peptide alpha-keto-beta-aldehyde-based inhibitors of trypsin-like serine proteases

New, synthetic peptide analogues bearing a C-terminal basic alpha-keto-beta-aldehyde moiety were prepared as novel inhibitors of the trypsin-like serine proteases. The compounds, Ac-Leu-Leu-Arg-COCHO, Ac-Arg-Gln-Arg-COCHO and Boc-Val-Leu-Lys-COCHO were evaluated kinetically against trypsin and three other trypsin-like serine proteases, tryptase, plasmin and thrombin, all of which are implicated as mediators of important disease processes. Results illustrate that alpha-keto-beta-aldehydes are potent inhibitors, with similar potency to comparable peptide aldehydes, and intriguingly, appearto act, in some instances, by a novel mechanism of action. Ac-Leu-Leu-Arg-COCHO, an analogue of the natural product leupeptin, is a potent, tight-binding inhibitor of trypsin (Ki(final) = 1.9 microM), plasmin (Ki(final) = 4.9 microM) and tryptase (Ki(final) = 1.2 microM) and an irreversible inactivator of thrombin (k2nd 4,500 M(-1).min(-1)). Boc-Val-Leu-Lys-COCHO was found to be a tight-binding inhibitor of its target protease plasmin (Ki(final) = 3.1 microM) and was inactive against thrombin. Ac-Arg-Gln-Arg-COCHO was a slow-binding inhibitor of tryptase (Ki(final) = 1.6 microM) and also irreversibly inactivated trypsin (k2nd = 8,920 M(-1) min(-1)). Peptides or peptidomimetics with a C-terminal basic alpha-keto-beta-aldehyde function thus provide a useful new molecular template for the development of new therapeutic agents against a wide range of disorders, such as coagulopathies and asthma, which may be mediated by the aberrant activity of trypsin-like serine proteases.     

Synthetic inhibitors of serine proteinases. Part 25: Inhibition of trypsin, plasmin and thrombin by amides of N alpha-substituted amidinophenylalanines and 3-amidinophenyl-3-aminopropionic acids (author's transl)

Amides of N alpha-substituted 3-amidinophenylalanine are potent inhibitors of the serine proteinases trypsin, plasmin and thrombin. They belong to the most potent inhibitors of these enzymes of the benzamidine type. In contrast, amides of 4-amidinophenylalanine possess weak inhibitory activity towards trypsin and plasmin. The cyclic amides of this group, however, are potent thrombin inhibitors. These derivatives are the first benzamidines with specific antithrombin activity. The isomeric compounds of 3-amidinophenyl-3-aminopropionic acids possess weak inhibitory effects on trypsin, plasmin and thrombin.     

Novel thrombin inhibitors with azaphenylalanine scaffold

In this paper the synthesis and antithrombotic activity of a series of novel thrombin inhibitors with azaphenylalanine scaffold are described. By systematic structural modifications for this series we have identified optimal groups for achieving nanomolar potency, that led to potent inhibitors of thrombin with Ki values up to 11 nM.     

Design and synthesis of isoxazoline derivatives as factor Xa inhibitors. 2.

Intravascular clot formation is an important factor in a number of cardiovascular diseases. Therefore, the prevention of blood coagulation has become a major target for new therapeutic agents. One attractive approach is the inhibition of factor Xa (FXa), the enzyme directly responsible for thrombin activation. Herein we report a series of isoxazoline derivatives which are potent FXa inhibitors. Optimization of the side chain at the quaternary position of the isoxazoline ring led to SK549 which showed subnanomolar FXa potency (K(i) 0.52 nM). SK549 shows good selectivity for FXa compared to thrombin and trypsin, potent antithrombotic effect in the rabbit arterio-venous thrombosis model, and improved pharmacokinetics relative to other compounds evaluated from this series.     

Design, synthesis, and activity of 2,6-diphenoxypyridine-derived factor Xa inhibitors.

A novel series of 2,6-diphenoxypyridines has been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. The evolution from the photochemically unstable bisamidine (Z,Z)-BABCH to potent bisamidine compounds with a pyridine heterocycle as the core scaffold has been achieved. The most potent compound in the series, 6h, has a Ki for human factor Xa of 12 nM. The selectivity of 6h against bovine trypsin and human thrombin was greater than 90- and 1000-fold, respectively. Two proposed modes of binding of 6h to factor Xa are made based on the crystal structures of 6h by itself and of 6h bound to bovine trypsin.     

Design and synthesis of thrombin inhibitors: analogues of MD-805 with reduced stereogenicity and improved potency

Mitsubishi's MD-805, a potent and selective inhibitor of thrombin which contains four stereogenic centers, has been the starting point for an optimization program. A systematic synthetic study resulted in thrombin inhibitors achiral at P2 and P3 but with a 10-fold increase in potency over the original lead. A number of 4-substituted piperidines were synthesized and examined as replacements for 2-carboxy-4-methylpiperidine at P2; 4-fluoroethylpiperidine (FEP) among others provided inhibitors (e.g. 45g) of increased potency. An enantioselective route was developed to 3(R)-methyl-1,2,3,4-tetrahydroquinolinesulfonyl chloride. Inhibitors containing this enantiomerically pure P3 (42d) had similar potency to the racemic material and provided support, with modeling studies, for the preparation of the gem 3,3-disubstituted compounds. A series of inhibitors containing the novel 3, 3-dimethyl-1,2,3,4-tetrahydroquinolinesulfonyl (DMTHQS) P3 (Table 5) were synthesized and showed a similar activity profile as the monomethyl series. The combination of P3-DMTHQS, P2-FEP, and P1-arginine (45g) had a K(i) of 6 nM (MD-805 K(i) = 85 nM). In animal models of both venous and arterial thrombosis, one inhibitor (42e) was shown to produce a dose-dependent inhibition of thrombus formation that in some situations was superior to that of MD-805.     

Azaphenylalanine-based serine protease inhibitors induce caspase-mediated apoptosis

Molecules regulating cell death constitute prominent therapeutic targets. The pro-apoptotic role of serine protease inhibitors prompted us to search for novel modulators of this process. We have tested some recently synthesized antithrombotic compounds for their potential to induce apoptotic cell death. Cell based analyses revealed that inhibitors built on the azaphenylalanine scaffold are, for B-cell lymphoma cells, severely cytotoxic, while other compounds tested were moderate or non-cytotoxic. These inhibitors induced the time and concentration dependent biochemical and morphological characteristics of apoptosis, such as DEVDase activation, loss of mitochondrial membrane potential, nuclear degradation and genomic DNA fragmentation. Most of the inhibitors proved to be selective for thrombin, with inhibition constants (K_i) in the nanomolar range. However, they could also inhibit at least one additional serine protease (trypsin, chymotrypsin and/or coagulation factor X) with K_i values in the nanomolar or low micromolar range. These serine protease inhibitors constitute novel apoptosis inducing compounds in B-cell lymphoma cells.     

Dual inhibitors of the blood coagulation enzymes

The search for an ideal anticoagulant has spanned decades and has resulted in several approaches and the identification of novel target molecules for preventing and treating thrombosis. The first group of new anticoagulant agents acting through direct inhibition of coagulation factors were inhibitors of thrombin, but factor Xa inhibitors and, most recently, factor VIIa inhibitors have become attractive candidates. The structures of thrombin, factor Xa and factor VIIa show similarities in their active sites and, for this reason, attempts have been made to develop synthetic agents containing in a single molecule inhibitory activity against two of the enzymes of the blood coagulation cascade. Such dual inhibitors are now in preclinical studies and are, potentially, new anticoagulant drugs with improved properties. The emphasis of this review will be placed on dual inhibitors of thrombin/factor Xa and factor Xa/factor VIIa. Comparison of the active sites of these enzymes is included for better understanding of the structural demands to be met in designing effective dual inhibitors.     

Isolation and preliminary characterization of a 22-kDa protein with trypsin inhibitory activity from toad Bufo andrewsi skin.

A novel trypsin inhibitor termed BATI was purified to homogeneity from the skin extracts of toad Bufo andrewsi by successive ion-exchange, gel-filtration and reverse-phase chromatography. BATI is basic single chain glycoprotein, with apparent molecular weight of 22 kDa in SDS-PAGE. BATI is a thermal stable competitive inhibitor and effectively inhibits trypsin's catalytic activity on peptide substrate with the inhibitor constant (K(i)) value of 14 nM and shows no inhibitory effect on chymotrypsin, thrombin and elastase. The N-terminal sequence of BATI is EKDSITD, which shows no similarity with other known trypsin inhibitors.     

Mechanism of thrombin-induced endothelium-dependent coronary vasodilation in dogs: role of its proteolytic enzymatic activity

We have recently reported that exogenous thrombin produced a dose- and endothelium-dependent coronary vasodilation in both intact open-chested dogs and in isolated dog coronary artery preparations. To determine whether the observed vasodilatory effect may be related to thrombin proteolytic enzymatic activity, effects of other proteases, such as trypsin, chymotrypsin, and pepsin, on the mechanical responses of isolated dog coronary arteries were studied. Among the four proteases evaluated, only thrombin (0.01-0.1 U/ml) and trypsin (0.03-0.67 U/ml) consistently produced a potent dose- and endothelium-dependent relaxation, that was reproducible with repeated testings. Addition of chymotrypsin (0.01-1.0 U/ml) produced only a minimal effect and was not reproducible, while addition of pepsin, as much as 10 U/ml, did not produce any effect. The specific soybean trypsin inhibitor and aprotinin, but not heparin and hirudin, competitively shifted the trypsin dose-response to the right, whereas heparin, hirudin, and antithrombin III proved to be more effective than trypsin inhibitors in inhibiting the thrombin-induced vasodilation. In all cases, the thrombin- and trypsin-induced vasodilation were equally sensitive to inhibition by the specific synthetic thrombin inhibitor, PPACK (D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone, 1-30 nM). PPACK, however, had no effect on the other endothelium-dependent coronary vasodilators, such as acetylcholine and adenosine triphosphate, in our isolated dog coronary artery preparations. Biochemical determinations of the amidolytic activity of thrombin, using Tosylglycyl-L-prolyl-L-arginine-p-nitroanilide as a chromogen, also indicated a similar PPACK and heparin-antithrombin III dose-dependent inhibition of the thrombin enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design, synthesis and molecular modelling of 1-amidinopiperidine thrombin inhibitors

Design, synthesis and biochemical evaluation of a series of novel non-covalent thrombin inhibitors with a 1-amidinopiperidine moiety are presented. Replacement of the planar benzamidine group in azaphenylalanine derivatives with 1-amidinopiperidine resulted in lower activity but higher selectivity for this type of compounds. The binding conformation of inhibitors in the active site of thrombin was revealed by molecular modelling studies.