Plasmin inhibitor reduces T-cell lymphoid tumor growth by suppressing matrix metalloproteinase-9-dependent CD11b(+)/F4/80(+) myeloid cell recruitment.

Activation of the fibrinolytic system during lymphoma progression is a well-documented clinical phenomenon. But the mechanism by which the fibrinolytic system can modulate lymphoma progression has been elusive. The main fibrinolytic enzyme, plasminogen (Plg)/plasmin (Plm), can activate matrix metalloproteinases (MMPs), such as MMP-9, which has been linked to various malignancies. Here we provide the evidence that blockade of Plg reduces T-cell lymphoma growth by inhibiting MMP-9-dependent recruitment of CD11b(+)F4/80(+) myeloid cells locally within the lymphoma tissue. Genetic Plg deficiency and drug-mediated Plm blockade delayed T-cell lymphoma growth and diminished MMP-9-dependent CD11b(+)F4/80(+) myeloid cell infiltration into lymphoma tissues. A neutralizing antibody against CD11b inhibited T-cell lymphoma growth in vivo, which indicates that CD11b(+) myeloid cells have a role in T-cell lymphoma growth. Plg deficiency in T-cell lymphoma-bearing mice resulted in reduced plasma levels of the growth factors vascular endothelial growth-A and Kit ligand, both of which are known to enhance myeloid cell proliferation. Collectively, the data presented in this study demonstrate a previously undescribed role of Plm in lymphoproliferative disorders and provide strong evidence that specific blockade of Plg represents a promising approach for the regulation of T-cell lymphoma growth.

Structure-inhibitory activity relationship of plasmin and plasma kallikrein inhibitors.

Based on the structure of Tra-Tyr(O-Pic)-octylamide, a portion of the octylamine was replaced with moieties bearing hydrophobic, basic or acidic groups. Replacement of the C-terminal residue with a moiety bearing a hydrophobic group gave the proper affinity of the inhibitor to both plasmin (PL) and plasma kallikrein (PK). While addition of a basic residue did not improve the affinity of the inhibitor, a carboxylic acid attached to the phenyl ring increased the PK selectivity of the inhibitor.

Isolation of plasma kallikrein by high efficiency affinity chromatography and its characterization.

We established a simple method for the purification of human plasma kallikrein (PK) by affinity chromatography and characterized it by analytical reverse phase-HPLC and Time of Flight Mass Spectroscopy (TOF-MS). The affinity resin (PKSI-Toyopearl) was synthesized using a selective synthetic inhibitor of plasma kallikrein (PKSI-527) as a ligand. The resin was found to have the highest efficiency in PK purification when the coupling ratio of PKSI-527 per resin was 9-14 micromol/g. PK was purified 466-fold with a yield of 83% from acetone-activated human plasma by affinity chromatography. The purity of PK thus obtained was confirmed by reverse phase-HPLC with a linear gradient of acetonitrile. The molecular weight of the purified PK was determined to be 86,151 by TOF-MS.

Binding diversity of a noncovalent-type low-molecular-weight serine protease inhibitor and function of a catalytic water molecule: X-ray crystal structure of PKSI-527--inhibited trypsin.

PKSI-527 is a noncovalent-type low-molecular-weight inhibitor. The X-ray crystal structure of the trypsin-PKSI-527 complex revealed a binding mode (Form II) different from the previously reported one (Form I) [Nakamura, M. et al. (1995) Biochem. Biophys. Res. Commun. 213, 583--587]. In contrast to the previous case, the electron density of the inhibitor revealed the whole structure clearly. Each structural part of the inhibitor in Forms I and II was differently located at the active site, although the modes of binding of the terminal amino group to the Asp189 carboxyl group were similar. This binding diversity, which is a characteristic of the noncovalent-type low-molecular-weight inhibitor, provides a suitable example for estimating the possible mechanism toward the enzymatic inhibition, together with the structural basis necessary for a specific inhibitor. The mode of binding in Form II reflects the inhibitor-specific situation and is in contrast with the substrate-mimetic binding mode for Form I. Based on the generally accepted catalytic mechanism for serine protease, we propose that a water molecule located at the catalytic site plays an important role in blocking the catalytic function of the reactive Ser193 OH group.

Development of plasmin and plasma kallikrein selective inhibitors and their effect on M1 (melanoma) and HT29 cell lines.

trans-4-Aminomethylcyclohexanecarbonyl-Tyr(O-Pic)-octylamide (YO-2) inhibited plasmin (PL) selectively, while trans-4-aminomethylcyclohexanecarbonyl-Phe-4-carboxymethylanili de (YO-1) inhibited plasma kallikrein (PK). YO-2 induced apoptosis of M1 (melanoma) cell line and HT29 colon carcinoma cells during 24 h through activation of caspase-3, while YO-1 did not affect either cell line even during 48 h.

Development of plasmin-selective inhibitors and studies of their structure-activity relationship.

Various compounds were synthesized by combining three components at positions P1, P1' and P2'. Of these, N-(trans-4-aminomethylcyclohexanecarbonyl)-Tyr(O-2-bromobenzylo xycarbonyl)- octylamide inhibited plasmin selectively with IC50 values of 0.80 and 0.23 microM towards S-2251 and fibrin, respectively. This compound also inhibited plasma kallikrein, urokinase, thrombin and trypsin with IC50 values of 10, > 50, > 50 and 1.6 microM, respectively.

Development of potent and selective plasmin and plasma kallikrein inhibitors and studies on the structure-activity relationship.

Based on structure-activity relationship studies, we designed and synthesized plasmin (PL) and plasma kallikrein (PK) inhibitors. Trans-(4-aminomethylcyclohexanecarbonyl)-Tyr(O-Pic)-octylamide inhibited PL, PK, urokinase (UK) and thrombin (TH) with IC50 values of 0.53, 30, 5.3 and > 400 microm, respectively. Trans-(4-aminomethylcyclohexanecarbonyl)-Tyr(O-2-Pyrim)-4-carboxyanilide inhibited PL, PK, UK and TH with IC50 values of 36, 0.56, 440 and > 1,000 microM, respectively.

Amino acids and peptides. LIII. Synthesis and biological activities of some pseudo-peptide analogs of PKSI-527, a plasma kallikrein selective inhibitor: the importance of the peptide backbone.

Pseudo-peptide analogs of trans-4-aminomethylcyclohexanecarbonyl-L-phenylalanyl-4-aminopheny l acetic acid (PKSI-527, plasma kallikrein selective inhibitor), in which an amide bond (peptide bond) has been replaced by a CH2-NH bond, i.e., trans-4-aminomethylcyclohexanecarbonyl-L-phenylalanyl-psi (CH2-NH)-4-aminophenyl acetic acid (I), trans-4-aminomethylcyclohexanecarbonyl-psi (CH2-NH)-L-phenylalanyl-4-aminophenyl acetic acid (II) and trans-4-aminomethylcyclohexanecarbonyl-D-phenylalanyl-psi (CH2-NH)-4-aminophenyl acetic acid (III) were synthesized. These pseudo-peptide analogs did not exhibit any detectable inhibitory activity against plasma kallikrein (PK), plasmin (PL), urokinase (UK), thrombine (TH) or trypsin (TRY). These results indicate that both carbonyl groups in the PKSI-527 are important for the manifestation of potent inhibitory activity against plasma kallikrein.

Development of plasma kallikrein selective inhibitors.

During the course of the development of active center-directed plasmin inhibitors, it was found that N-(trans-4-aminomethylcyclohexanecarbonyl)-lysine-4-methoxycarb onylanilide inhibited plasma kallikrein more potently than other enzymes such as plasmin, urokinase, and thrombin, although the inhibitory activity was not as potent and enzyme selectivity not as high. Based on studies of structure-activity relationship, we designed and synthesized the plasma kallikrein selective inhibitor, N-(trans-4-aminomethylcyclohexanecarbonyl)-phenylalanine-4-carboxy methyl- anilide (Tra-Phe-APAA). Tra-Phe-APAA inhibited plasma kallikrein with a Ki value of 0.81 microM, while it inhibited glandular kallikrein, plasmin, urokinase, tissue plasminogen activator, factor Xa, factor XIIa, and thrombin with Ki values of > 500, 390, 200, > 500, > 500 > 500, and > 500 microM, respectively. We designated Tra-Phe-APAA as PKSI-527. Using PKSI-527 as an affinity ligand, we synthesized a new affinity gel (PKSI-Toyopearl) and employed it for the rapid purification of plasma kallikrein from human plasma. Human plasma activated with kaolin after acid treatment was applied to a PKSI-527-Toyopearl column. Adsorbed protein was eluted with 50 mM glycinehydrochloric acid buffer (pH 3.0). Plasma kallikrein was purified 181-fold with a yield of 85% from the kaolin-activated plasma.

Design of plasma kallikrein inhibitors: functional and structural requirements of plasma kallikrein inhibitors.

The synthetic plasma kallikrein (PK) inhibitor trans-4-aminomethylcyclohexanecarbonylphenylalanine-4-carboxyme thylanilide (PKSI-527) consists of three parts. Each part was replaced by analogues in an attempt to improve the potency and the selectivity of PKSI-527. Among the peptides examined, trans-4-aminomethylcyclohexanecarbonylphenylalanine-4-carboxyan ilide (peptide 16) inhibited PK with a high selectivity and an IC50 value of 2.7 microM, being as potent as PKSI-527.

Purification method for human plasma kallikrein by a new affinity chromatography.

We synthesized a new affinity gel (PKSI-Toyopearl) using a selective synthetic inhibitor of plasma kallikrein (PKSI-527) as an affinity ligand, and employed it for the rapid purification of plasma kallikrein from human plasma. Human plasma activated with kaolin after acid treatment was applied to a PKSI-Toyopearl column. Adsorbed protein was eluted with 50 mM glycine-hydrochloric acid buffer (pH 3.0). Plasma kallikrein was purified 181-fold with a yield of 85% from the kaolin-activated plasma. Further purification was performed by chromatography on a DEAE-Toyopearl 650M column. Plasma kallikrein was finally purified 1720-fold with a 63% yield by these procedures. On sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, a band was observed at approximately 88 kDa. These findings indicate that PKSI-Toyopearl is a valuable tool for the purification of plasma kallikrein from human plasma.

Enzyme-controlling medicines: introduction.

A short history of the research work of S. Okamoto and co-workers, for the previous 50 years, is briefly described. In the 1950s, when the physiologic role of fibrinolysis had not been established, they began to seek for compounds that inhibit the action of plasmin. They examined approximately 200 lysine derivatives and discovered epsilon aminocaproic acid (EACA) and tranexamic acid (t-AMCHA). In the 1970s, we selected thrombin as the target enzyme to be controlled; structure-activity relationship studies, taking arginine as the skeleton structure, led to the discovery of the selective thrombin inhibitor No. 205 (4-ethyl-1-[N2-(5-dimethylamino-1-naphthalenesulfonyl)-L-arginyl]- 1-piperidine), and further attempts to minimize the toxicity finally led to No. 805 (argatroban, MD-805, (2R,4R)-4-methyl-1-(N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-sulfo nyl]-L-arginyl)-2-piperidine carboxylic acid). Argatroban, without any cofactor, inhibits thrombin competitively. The high selectivity of the action of argatroban is promising for treating thrombosis in clinical practice. More recently, taking advantage of our knowledge obtained through previous studies, active center-directed plasmin inhibitors and a selective inhibitor of kallikrein have been found.

Effects of a highly selective synthetic inhibitor of plasma kallikrein on disseminated intravascular coagulation in rats.

We found a new, highly selective plasma kallikrein inhibitor, trans-4-aminomethyl-cyclohexanecarbonylphenylalanine 4-carboxymethylanilide hydrochloride, called PKSI-527 in our laboratories. This study was conducted to evaluate PKSI-527, on thromboplastin (TP)- and endotoxin (LPS)-induced disseminated intravascular coagulation (DIC) in rats. PKSI-527 was infused intravenously at 0.1 mg/kg/min for 250 min. Three of the parameters of the coagulation and fibrinolysis system, fibrinogen level, platelet counts and fibrin(ogen) degradation products (FDP) level were assayed. PKSI-527 prevented the change in the coagulation and fibrinolysis system in LPS-induced DIC, however it was not clearly effective in TP-induced DIC. The parameters of organ failure, such as serum glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatine phosphokinase (CPK), lactate, blood urea nitrogen and beta-glucuronidase, were assayed. Although the changes in the fibrinogen level, platelet counts and FDP level were almost the same in both models, the parameters of organ failure apparently increased in LPS-induced DIC more so than in TP-induced DIC. PKSI-527 significantly suppressed the increases in GOT and GPT in LPS-induced DIC. These results indicate that plasma kallikrein may play a significant role in LPS-induced DIC. Therefore, PKSI-527, as a synthetic plasma kallikrein inhibitor may be a valuable tool to explore the mechanism of DIC and the accompanying organ failure.

Use of an active center-directed plasmin inhibitor elucidates the multiplicity of plasmin actions.

In our studies, designed to synthesize an active center-directed plasmin (PL) inhibitor, N-(4-aminomethylbenzoyl)-4-(3-picolyloxy)-L-phenylalanine n-hexylamide dihydrochloride (PASI-535) was found. We characterized PASI-535 and analyzed the actions of PL, comparing with those of PASI-535 and tranexamic acid (t-AMCHA). (1) PASI-535 strongly inhibited not only fibrinolysis (IC50: 2.9 x 10(-6) M) but also amidolysis (Ki value: 2.9 x 10(-6) M) and fibrinogenolysis (IC50: 4.5 x 10(-6) M) induced by PL. While t-AMCHA which strongly inhibited fibrinolysis (IC50: 6.0 x 10(-5) M), rarely inhibited amidolysis (Ki value: 4.0 x 10(-2) M) and fibrinogenolysis (IC50: 1.0 x 10(-2) M). (2) PL is able to liberate kinins by degrading kininogen. This kinin-generation by PL was inhibited by 2 x 10(-5) M PASI-535. However, it was little inhibited even by 1 x 10(-3) M t-AMCHA. (3) The inhibitory effect of PASI-535 and t-AMCHA on tumor growth was studied. In sarcoma-180 bearing mice, ascites retention and the increase of tumor cells were markedly suppressed by subcutaneous injection of PASI-535, either 30 mg/kg/day or 50 mg/kg/day, for 5 days, and the inhibitory effect was dose-dependent. Although t-AMCHA also reduced both ascites retention and the increase of tumor cells, it needed approximately 40 times (2 g/kg/day) the amount of PASI-535 to exert these effects. PASI-535 may be a useful tool in analyzing the multiplicity of PL actions. Moreover, PASI-535 can be used as an antifibrinolytic drug which has a mechanism of function different from that of t-AMCHA.

Design of noncovalent trypsin inhibitor based on the X-ray crystal structure of the complex.

The inhibitory mechanism of trans-4-aminomethylcyclohexanecarbonyl-L-phenyl-alanine-4-carbo xymethylanilide (1), a noncovalent serine protease inhibitor synthesized based on previous structure-activity studies, was clarified based on the X-ray crystal structure of the complex (2.2 A resolution, R = 0.175), where the amino group of the aminomethylcyclohexane moiety was bifurcately hydrogen-bonded to the carboxyl oxygens of Asp 189 side group (specificity pocket), and the hydrogen bonds of the cyclohexanecarbonyl oxygen to NHs of Gly 193 and Ser 195 residues (oxyanion hole) and of Phe NH to Ser 195 O gamma atom (catalytic triad) were observed. In contrast, the Phe benzene moiety and terminal carboxymethylanilide of 1 were not well located on the electron density map, suggesting the conformational freedom of these P1' and P2' sites at the binding pocket. Based on these insights, trans-4-aminomethylcyclohexanecarbonyl-4-nitro-L-phenylalanine-4-+ ++benzoylanilide (2) was designed, in which the P1' and P2' sites were modified so as to effectively interact with the amino acid residues of trypsin binding pocket via hydrogen bonding and van der Waals interactions, respectively. Consequently, 2 showed 40 times higher inhibitory activity against trypsin than 1.

Development of active center-directed plasmin and plasma kallikrein inhibitors and studies on the structure-inhibitory activity relationship.

The molecule of trans-4-aminomethylcyclohexanecarbonylphenylalanine 4-carboxymethylanilide (8), which is a potent and selective inhibitor of plasma kallikrein, can be divided into three parts (P1, P1' and P2'), each of which contains one of the rings. In order to study the role of each part in the manifestation of potent and selective inhibitory activity and the relationship between the structure and inhibitory activities toward plasmin, plasma kallikrein, urokinase and thrombin, each part was substituted with various other moieties to give many kinds of analogs and their inhibitory activities against the above enzymes were examined. Among them, trans-4-aminomethylcyclohexanecarbonyl-O-2-bromobenzyloxycarbon yltyrosine 4-acetylanilide (12) inhibited plasmin and plasma kallikrein with IC50 values of 2.3 x 10(-7) M and 3.7 x 10(-7) M, and K(i) values of 1.2 x 10(-7) M and 1.3 x 10(-7) M, respectively.

Effects of a highly selective plasma kallikrein inhibitor on collagen-induced arthritis in mice.

A study was conducted to evaluate the effects of a new, highly selective plasma kallikrein inhibitor, PKSI-527, on collagen-induced arthritis (CIA) in mice. PKSI-527 or indomethacin was administered daily intraperitoneally from day 20 postimmunization. Clinical evaluation was performed, and two of the components of the kallikrein-kinin system, high-molecular-weight (HMW) kininogen and plasma prekallikrein, were assayed. PKSI-527, as well as indomethacin, reduced the severity of arthritis significantly. PKSI-527, but not indomethacin, restored consumed components of the kallikrein-kinin system. These results suggest that PKSI-527 suppresses CIA by modifying the kallikrein-kinin system and PKSI-527 as a synthetic plasma kallikrein inhibitor would be a valuable tool to study the mechanism of inflammation of arthritic diseases.