Correlating structure and ligand affinity in drug discovery: a cautionary tale involving second shell residues.

A high-resolution crystallographic structure determination of a protein-ligand complex is generally accepted as the 'gold standard' for structure-based drug design, yet the relationship between structure and affinity is neither obvious nor straightforward. Here we analyze the interactions of a series of serine proteinase inhibitors with trypsin variants onto which the ligand-binding site of factor Xa has been grafted. Despite conservative mutations of only two residues not immediately in contact with ligands (second shell residues), significant differences in the affinity profiles of the variants are observed. Structural analyses demonstrate that these are due to multiple effects, including differences in the structure of the binding site, differences in target flexibility and differences in inhibitor binding modes. The data presented here highlight the myriad competing microscopic processes that contribute to protein-ligand interactions and emphasize the difficulties in predicting affinity from structure.

Inhibition of urokinase activity reduces primary tumor growth and metastasis formation in a murine lung carcinoma model.

RATIONALE: Lung cancer is the most common malignancy in humans. Urokinase (uPA) plays a crucial role in carcinogenesis by facilitating tumor cell invasion and metastasis. OBJECTIVES: We investigated the effect of the highly specific urokinase inhibitor CJ-463 (benzylsulfonyl-D-Ser-Ser-4-amidinobenzylamide) on tumor growth, metastasis formation, and tumor vascularization in the murine Lewis lung carcinoma (LLC) and a human small lung cancer model. METHODS: A quantity of 3 x 10(6) LLC cells were subcutaneously injected into the right flank of C57Bl6/N mice, uPA knock out, and uPA receptor knockout mice. Seven days later mice were randomized to receive intraperitoneally either saline (control group), CJ-463 (10 and 100 mg/kg, twice a day), or its ineffective stereoisomer (10 mg/kg, twice a day). Tumor volume was measured every second day and metastasis formation was monitored by volumetric-computed tomography. Twelve days after onset of treatment mice were killed and tumors were prepared for histologic examination. MEASUREMENTS AND MAIN RESULTS: Treatment with CJ-463 resulted in a significant inhibition of primary tumor growth, with the highest efficacy seen in the 100 mg/kg group. In addition, histological analysis of the lung revealed a significant reduction in lung micrometastasis in the 100 mg/kg group. Similarly, a reduced seeding of tumor cells into the lung after intravenous injection of LLC cells was observed in inhibitor-treated mice. In these mice, treatment with CJ-463 appeared not to significantly alter the relative extent of tumor vascularization. In vitro, proliferation of LLC cells remained unchanged upon inhibitor treatment. CJ-463 was found to similarly reduce tumor growth in uPA receptor knockout mice, but was ineffective in uPA knockout mice. CONCLUSIONS: Our results suggest that synthetic low-molecular-weight uPA-inhibitors offer as novel agents for treatment of lung cancer.

Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick.

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1) pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

Geometry of GPPE binding to picrate and to the urokinase type plasminogen activator.

Crystal structure of 2-(4-guanidynephenyl)-1-phenyl-ethanone (GPPE) in two different environments was determined in order to compare the binding geometry of these compound to a simple picrate anion and to protein, urokinase-type plasminogen activator (uPA), which may be treated as a target for anti-cancer drugs. It was shown that the conformation and the hydrogen-bonding formation by GPPE molecule are similar in both environments, but several important differences were discovered and described.

Highly potent and selective substrate analogue factor Xa inhibitors containing D-homophenylalanine analogues as P3 residue: part 2.

A series of highly potent substrate-analogue factor Xa inhibitors containing D-homophenylalanine analogues as the P3 residue has been identified by systematic optimization of a previously described inhibitor structure. An initial lead, benzylsulfonyl-D-hPhe-Gly-4-amidinobenzylamide (3), inhibits fXa with an inhibition constant of 6.0 nM. Most modifications of the P2 amino acid and P4 benzylsulfonyl group did not improve the affinity and selectivity of the compounds as fXa inhibitors. In contrast, further variation at the P3 position led to inhibitors with significantly enhanced potency and selectivity. Inhibitor 27, benzylsulfonyl-D-homo-2-pyridylalanyl(N-oxide)-Gly-4-amidinobenzylamide, inhibits fXa with a K(i) value of 0.32 nM. The inhibitor has strong anticoagulant activity in plasma and doubles the activated partial thromboplastin time and prothrombin time at concentrations of 280 nM and 170 nM, respectively. Compound 27 inhibits the prothrombinase complex with an IC(50) value of 5 nM and is approximately 50 times more potent than the reference inhibitor DX-9065a in this assay.

From selective substrate analogue factor Xa inhibitors to dual inhibitors of thrombin and factor Xa. Part 3.

Highly potent and selective substrate analogue factor Xa inhibitors were obtained by incorporation of non-basic or modestly basic P1 residues known from the development of thrombin inhibitors. The modification of the P2 and P3 amino acids strongly influenced the selectivity and provided potent dual factor Xa and thrombin inhibitors without affecting the fibrinolytic enzymes. Several inhibitors demonstrated excellent anticoagulant efficacy in standard clotting assays in human plasma.

New substrate analogue inhibitors of factor Xa containing 4-amidinobenzylamide as P1 residue: part 1.

The trypsin-like serine protease factor Xa (fXa) is located at the convergence point of the intrinsic and extrinsic coagulation cascade, and therefore has emerged as an attractive target for the design of novel anticoagulants. During the development of substrate-analogue urokinase inhibitors we have found that the protection of the P3-dSer side chain leads to a scaffold of potent fXa inhibitors with the general structure R1-SO2-dSer(R2)-Gly-4-amidinobenzylamide. The first lead (3) with an N-terminal benzylsulfonyl group and dSer(tBu) as P3 residue inhibits human fXa with a Ki of 14 nM. A variety of derivatives with modified P4, P3, and P2 residues have been investigated in terms of inhibition of fXa and related proteases and for their anticoagulant potency and elimination behaviour. Most inhibitors were rapidly cleared from the circulation of rats. However, compound 48 (Ki= 3.5 nM), one of the most potent and selective inhibitors containing a dArg as P3 residue was relatively slowly eliminated (t1/2 approximately 1 h). Inhibitor 48 doubled clotting times in human plasma at 0.32 microM (aPTT) and 0.28 microM (PT), and is approximately 10-fold more potent than the reference fXa inhibitor DX-9065a in the inhibition of the prothrombinase complex. The structures of two inhibitors in complex with human fXa were solved by X-ray crystallography.

Secondary amides of sulfonylated 3-amidinophenylalanine. New potent and selective inhibitors of matriptase.

Matriptase is an epithelium-derived type II transmembrane serine protease and has been implicated in the activation of substrates such as pro-HGF/SF and pro-uPA, which are likely involved in tumor progression and metastasis. Through screening, we have identified bis-basic secondary amides of sulfonylated 3-amidinophenylalanine as matriptase inhibitors. X-ray analyses of analogues 8 and 31 in complex with matriptase revealed that these inhibitors occupy, in addition to part of the previously described S4-binding site, the cleft formed by the molecular surface and the unique 60 loop of matriptase. Therefore, optimization of the inhibitors included the incorporation of appropriate sulfonyl substituents that could improve binding of these inhibitors into both characteristic matriptase subsites. The most potent derivatives inhibit matriptase highly selective with K(i) values below 5 nM. Molecular modeling revealed that their improved affinity results from interaction with the S4 site of matriptase. Analogues 8 and 59 were studied in an orthotopic xenograft mouse model of prostate cancer. Compared to control, both inhibitors reduced tumor growth, as well as tumor dissemination.

The crystal structures of 3-TAPAP in complexes with the urokinase-type plasminogen activator and picrate.

The urokinase-type plasminogen activator (uPA) is a protein involved in tissue remodeling and other biological processes. The inhibitors of uPA have been shown to prevent the spread of metastasis and tumor growth, and accordingly this enzyme is widely accepted as a promising anticancer target. In this work, we have investigated the conformation of the uPA inhibitor 3-TAPAP in two different crystalline environments of a picrate and a uPA complex. These structures were compared to the known structure of the 3-TAPAP in the complex with trypsin. In the complexes with the proteins, trypsin, and uPA, the binding mode of 3-TAPAP is similar. A larger difference in the conformation, in the comparison to these structures, has been observed by us in the 3-TAPAP picrate crystal. This observation contradicts the hypothesis that 3-TAPAP derivatives inhibit serine proteinases in preformed stable conformations.

Effective inhibition of experimental metastasis and prolongation of survival in mice by a potent factor Xa-specific synthetic serine protease inhibitor with weak anticoagulant activity.

Clinical and experimental evidence suggests that the blood coagulation system is involved in the dissemination of malignant tumors. Consequently, anticoagulant agents have been tested as metastasis suppressors in experimental models. Recently, we have found a close correlation between factor Xa (FXa)-specificity of a series of synthetic serine protease inhibitors and their anti-metastatic potential in a murine T-cell lymphoma metastasis model. Interference of such inhibitors with blood-coagulation may represent a major experimental and clinical obstacle. Here, we test anti-metastatic effects of a recently developed, highly specific 3-amidinophenylalanine-type FXa inhibitor, WX-FX4, with weaker anticoagulant activity when compared to well-established FXa inhibitors, such as DX-9065a, as measured by the activated partial thromboplastin time, prothrombin time, prothrombinase complex activity, and coagulation time. Treatment of mice with WX-FX4 (1.5 mg/kg twice daily) led to significant reduction of experimental liver metastasis of a syngeneic T-cell lymphoma in DBA/2 mice (> 90%), and of experimental lung metastasis of a human fibrosarcoma in CD1 nu/nu mice (> 60%). Due to its relatively low anticoagulant activity, daily treatment over 100 days was possible, leading to significant survival benefits without inducing bleeding anomalities. FXa-inhibitors with highly efficient anti-metastatic potential without coagulation-related side effects may represent important new tools as anticancer agents.

In vitro inhibition of matriptase prevents invasive growth of cell lines of prostate and colon carcinoma.

Matriptase, also known as membrane-type-serine-protease 1 (MT-SP 1), is a type II transmembrane serine protease involved in the activation of the precursor form of hepatocyte growth factor/scatter factor (pro-HGF/SF). Since HGF/SF is a well-known extracellular signal, which plays a key role in the control of invasive growth, we investigated the effects of matriptase inhibition in cell lines derived from colon (DLD-1) or prostate (PC-3) carcinomas. Biochemical analysis showed that matriptase was very efficient in the proteolytic conversion of the inactive HGF/SF precursor into HGF/SF. Inhibition of endogenous matriptase synthesis in DLD-1 or PC-3 cells by specific small interfering RNAs impaired the conversion of pro-HGF/SF into HGF/SF at the cell surface and inhibited cell scattering upon pro-HGF/SF stimulation. The same effect was observed after treatment of these cells with matriptase inhibitors of the 3-amidinophenylalanine-type, CJ-697 or CJ-730. Inhibition of matriptase significantly reduced invasion of the extracellular matrix as well. Interestingly, this reduction was observed even in the presence of pre-activated HGF/SF. It is concluded that matriptase plays a dual-role in the events unleashing the invasive phenotype, one 'upstream' from the HGF/SF signalling cascade and one 'downstream', most likely at the level of the plasminogen activation system. These data provide a proof of concept for the targeting of matriptase in the search for anti-invasive drugs.

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.

Pure eccentric exercise does not activate blood coagulation.

Eccentric exercise can cause skeletal muscle damage with ultrastructural disruption, inflammation and increased proteolytic enzyme activity. It may be possible that these changes are able to trigger blood coagulation in vivo. The aim of the study was to investigate changes in blood coagulation via the measurement of aPTT, the thrombin potential (total [TTP] and endogenous [ETP], both intrinsic [in] and extrinsic [ex]) and the thrombin generation (prothrombinfragment 1 + 2 [F1 + 2] and thrombin-antithrombin complex [TAT]) after pure eccentric exercise. Seventeen healthy non-smokers (28 +/- 6 years, VO2-peak 59 +/- 7 ml/min/kg) underwent pure eccentric down jumps (9 x 28 isolated down jumps in 90 min, drop from a height of 55 cm), a cycle exercise (90% of the individual anaerobic threshold for 60-90 min) and a control experiment on different days. Blood samples were drawn after a 30-min rest, immediately, and 2 h after exercise. After the cycle exercise, a clear shortening by 12% (P<0.001) in aPTT and an increase in TTPin (13%; P<0.05) and TAT (33%; P<0.05) in comparison to the control experiment were seen, while after eccentric exercise only minimal changes in aPTT and thrombin potential (TTPin, ETPin) and no thrombin generation (F1 + 2 and TAT) were found. In contrast to concentric dynamic exercise, e.g. cycle ergometry, only insignificant changes in thrombin potential and no thrombin generation could be observed after skeletal muscle damage induced by pure eccentric exercise. It can be concluded that the mechanical impact associated with eccentric exercise does not activate blood coagulation.

Suppression of rat breast cancer metastasis and reduction of primary tumour growth by the small synthetic urokinase inhibitor WX-UK1.

The serine protease uPA (urokinase-type plasminogen activator) and its receptor uPAR (CD87) are often elevated in malignant tumours, hence, inhibition of this tumour-associated plasminogen activation system provides an attractive target for therapeutic strategies. WX-UK1, a derivative of 3-aminophenylalanine in the L-conformation with inhibitory antiproteolytic properties, was tested for its specificity spectrum using specific chromogenic paranitroanilide peptide substrates. The corresponding D-enantiomer of WX-UK1 was used as a control. The anti-tumour and anti-metastatic (number of lung foci and weight of the axillary lymph nodes) properties were studied by subcutaneous administration of WX-UK1 to Brown Norwegian (BN) rats carrying orthotopically transplanted BN472 rat breast tumours. WX-UK1 selectively inhibited tumour-related proteases from rats and humans such as uPA, plasmin, or thrombin in the sub or low micromolar range. The activity was stereoselective as the D-enantiomer of WX-UK1 inhibited uPA and plasmin at approximately 70-fold higher Ki values than the active L-form. Chronical administration of the L-enantiomer of WXUK1 impaired primary tumour growth and metastasis of BN472 rat breast cancer in a dose-dependent manner. The minimum inhibitory dosage with maximal effect was between 0.15 and 0.3 mg/kg/day. The inactive D-enatiomer of WX-UK1 was not active in this respect. Daily treatment with WX-UK1 for up to 35 days was well tolerated as judged by the unchanged body and organ weight development. In conclusion, our results provide evidence that WX-UK1 as a single agent inhibits breast tumour growth and metastasis in vivo, and thus is a promising candidate drug to treat human cancer.

Progress in the development of synthetic thrombin inhibitors as new orally active anticoagulants.

The trypsin-like serine protease thrombin is a multifunctional key enzyme at the final step of the coagulation cascade and is involved in the regulation of hemostasis and thrombosis. An increased activation of coagulation can result in severe thromboembolic disorders, one of the major reasons responsible for mortality and morbidity in western world. Therefore, an effective, safe, and orally available thrombin inhibitor could be a useful anticoagulant drug for the daily prophylaxis of venous and arterial thrombosis and prevention of myocardial infarction for high-risk patients. Synthetic thrombin inhibitors have a long history; initial compounds were derived from electrophilic ketone- and aldehyde-analogs of arginine. First potent leads of non-covalent inhibitors were developed in the early eighties, which were further optimised in the nineties, after the X-ray structure of thrombin became available. In the meantime a huge number of highly active and selective inhibitors has been published, however, only a few of them have an appropriate pharmacokinetic and pharmacodynamic overall profile, which could justify their further development. Very recently, with Ximelagatran a first orally available thrombin inhibitor has been approved in France for the prevention of venous thromboembolic events in major orthopaedic surgery after successful clinical phase III. However, it still has to be awaited, whether the extensive clinical use of Ximelagatran can demonstrate for the first time that direct thrombin inhibitors offer a real benefit in terms of efficacy and safety over established antithrombotic therapies. This review summarizes the current status of synthetic thrombin inhibitors with a focus on more recently published and promising new compounds.

Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as antimetastatic agents.

The serine protease urokinase-type plasminogen activator (uPA) interacts with a specific receptor (uPAR) on the surface of various cell types, including tumor cells, and plays a crucial role in pericellular proteolysis. High levels of uPA and uPAR often correlate with poor prognosis of cancer patients. Therefore, the specific inhibition of uPA with small molecule active-site inhibitors is one strategy to decrease the invasive and metastatic activity of tumor cells. We have developed a series of highly potent and selective uPA inhibitors with a C-terminal 4-amidinobenzylamide residue. Optimization was directed toward reducing the fast elimination from circulation that was observed with initial analogues. The x-ray structures of three inhibitor/uPA complexes have been solved and were used to improve the inhibition efficacy. One of the most potent and selective derivatives, benzylsulfonyl-D-Ser-Ser-4-amidinobenzylamide (inhibitor 26), inhibits uPA with a Ki of 20 nm. This inhibitor was used in a fibrosarcoma model in nude mice using lacZ-tagged human HT1080 cells, to prevent experimental lung metastasis formation. Compared with control (100%), an inhibitor dose of 2 x 1.5 mg/kg/day reduced the number of experimental metastases to 4.6 +/- 1%. Under these conditions inhibitor 26 also significantly prolonged survival. All mice from the control group died within 43 days after tumor cell inoculation, whereas 50% of mice from the inhibitor-treated group survived more than 117 days. This study demonstrates that the specific inhibition of uPA by these inhibitors may be a useful strategy for the treatment of cancer to prevent metastasis.

Increase of anti-metastatic efficacy by selectivity- but not affinity-optimization of synthetic serine protease inhibitors.

Although tumors frequently show elevated protease activities, the concept of anti-proteolytic cancer therapy has lost momentum after failure of clinical trials with broad-spectrum matrix metalloproteinase inhibitors. Thus we need to adapt our design strategies for protease inhibitors. Here, we employed a series of seven structurally fine-modulated and pharmacokinetically closely related synthetic 4-amidinobenzylamine-based inhibitors with distinct selectivity for prototypical serine proteases in a murine T cell lymphoma liver metastasis model. This in vivo screening revealed efficacy of urokinase inhibitors but no correlation between urokinase selectivity or affinity and anti-metastatic effect. In contrast, factor Xa-selective inhibitors were more potent, demonstrating factor Xa or a factor Xa-like serine protease likely to be more determinant in this model. Factor Xa selectivity, but not affinity, significantly improved anti-metastatic efficacy. For example, factor Xa inhibitors CJ-504 and CJ-510 exert similar affinity for factor Xa (K(i)=14 nM versus 8.8 nM) but CJ-504 was 70-fold more selective for factor Xa. This correlated with higher anti-metastatic efficacy (58.8% with CJ-504; 28.2% with CJ-510). Our results show that among the protease inhibitors employed that have affinities in the nanomolar range, the strategy of selectivity-optimization is superior to further improvement of affinity to significantly enhance anti-metastatic efficacy. This appreciation may be important for the future rational design of new anti-proteolytic agents for cancer therapy.

Blood coagulation and fibrinolysis before and after exhaustive exercise in patients with IDDM.

Diabetes mellitus involves changes in haemostasis which leads to the opinion that diabetes mellitus is a hypercoagulable state. However, little is known about the relationship of exercise and haemostasis in diabetics. Therefore, first of all the aim was to investigate if differences in blood coagulation and fibrinolysis can be demonstrated in subjects with insulin-dependent diabetes mellitus (IDDM) compared to controls and secondly, if differences concerning exercise induced changes can be seen in diabetics. 16 moderately fit subjects with IDDM and 16 matched controls underwent a maximal step test. Blood samples were taken after a 30 min rest, immediately and 1h after exercise and in addition after 30 min rest 7 days later at the same time of day. The rest values (mean of the two rest samples) in extrinsic total thrombin potential (TTPex, P=0.049), tPA-activity (P=0.007) were significantly higher and in PAI-1-antigen (P=0.002) -activity (P=0.049) lower in the diabetic group. APTT, PT, TAT (only control), TTPin, tPA-activity and -antigen and PAP were increased immediately and D-dimer (only control) 1 h after exercise, whereas PAI-1-activity and -antigen (only control) decreased immediately or 1 h after exercise (all minimal P<0.05). The increase of tPA-antigen and decrease in PAI-1-antigen after exercise were both lower in the diabetics (P<0.05). IDDM led to higher extrinsic total thrombin and fibrinolytic potential at rest, and reducing the exercise provoked distribution of tPA-antigen and decrease of PAI-1-antigen. Nevertheless a higher thrombotic risk after maximal exercise has not been investigated in young IDDM patients without complications and in good metabolic control.

Inhibitors of benzamidine type influence the virulence properties of Porphyromonas gingivalis strains.

Synthetic inhibitors of benzamidine type have been found to have inhibiting effects on arginine specific cysteine proteinases of P. gingivalis. The purpose of our study was to assess the effects of these inhibitors on the virulence properties of two P. gingivalis strains, the reference strain ATCC 33277 and JH16-1, a clinical isolate obtained from a patient with severe periodontitis. The inhibitors tested were pentamidine, benzamidine, three bis-benzamidine derivatives with a pentamidine-related structure, one bis-benzamidine derivative with another structure, and one arginine derivative as a negative control, each in the concentrations of 2 microM and 20 microM. As virulence criteria the following parameters were determined: arginine-specific amidolytic activity, growth inhibition, hemagglutination of sheep erythrocytes, adherence to KB cells and immuno-phagocytosis including intracellular killing. Pentamidine and the bis-benzamidine derivatives with pentamidine-related structure showed the most remarkable effects on reduction of amidolytic activity by 35%, growth inhibition and reduced hemagglutination. Except for the arginine derivative all other inhibitors tested enhanced the phagocytosis capacities of granulocytes. No clear influence of the inhibitors on adherence of P. gingivalis to KB cells was seen. Although in vitro effects of the synthetic inhibitors of cysteine proteinases on virulence of P. gingivalis were observed further in vitro tests concerning immunomodulatory effects should be done before these substances are used for therapy in clinically controlled studies.