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.

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.

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.

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.

Preservation of in vitro function of platelets stored in the presence of a synthetic dual inhibitor of factor Xa and thrombin.

Background: The use of citrate anticoagulant limits the clinical significance of platelet function tests. Thrombin inhibitors cannot prevent thrombin-induced platelet activation completely. We examined the influence of benzylsulfonyl-d-Arg-Pro-4-amidinobenzylamide (BAPA), a dual inhibitor of Factor Xa (FXa) and thrombin, on platelet responsiveness to agonists when measured between 2 and 24 h after venipuncture.

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.

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.

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.

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.

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.

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.

Synthetic urokinase inhibitors as potential anti-invasive drugs.

In carcinogenesis, proteinases play an important role in metastasis of solid malignant tumors. Aside from several matrix metalloproteinases and cathepsins, the urokinase-plasmin system seems to be one of the main players within the cell surface-associated proteolytic activity, facilitating tumor cell migration and invasion. Upon binding to a specific receptor, the enzymatic activity of urokinase is focused to the cell surface.

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.

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.

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.

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.

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.

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.

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.

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.

Blood coagulation and fibrinolysis after long-duration treadmill exercise controlled by individual anaerobic threshold.

For rehabilitation training it is recommended that the intensity of exercise should be clearly below the individual anaerobic threshold (IAT). We investigated blood coagulation, particularly endogenous thrombin potential (ETP) and fibrinolysis following a standardized treadmill (TR) ergometer test at 90% IAT for 60-120 min. Sixteen healthy male non-smokers underwent the TR test.

ZZ made EZ: influence of inhibitor configuration on enzyme selectivity.

Selectivity of drug targeting is necessary in order to forestall undesired side-effects. Here, we examine the structural grounds for the configuration-dependent selectivity of 2,7-bis(4-amidinobenzylidene)-cycloheptan-1-one (1) for factor Xa and trypsin: Previous studies showed that factor Xa is preferentially inhibited by the (Z,Z) configuration isomer of (1), whilst trypsin binds equally well to both (E,Z) and (Z,Z) forms. Using engineered trypsin variants, we find similar overall binding modes for the (E,Z) and (Z,Z) isomers.

Blood coagulation and fibrinolysis after extreme short-term exercise.

Introduction: Maximal exercise may be a trigger for cardiovascular events. The aim of the study was to investigate changes in blood coagulation and fibrinolysis following maximal short-term exercises with different durations up to 90 s.

Methods: A total of 15 healthy nonsmokers underwent three isokinetic maximal tests on an SRM cycle ergometry system with durations of 15, 45, and 90 s.