WO1994003488A1

WO1994003488A1 - Novel peptide

Info

Publication number: WO1994003488A1
Application number: PCT/JP1993/001103
Authority: WO
Inventors: Yoichi Sakata; Jyun Mimuro; Munekiyo Kaneko; Toshinobu Murakami; Michio Matuda
Original assignee: Sii Technoresearch, Inc.
Priority date: 1992-08-05
Filing date: 1993-08-05
Publication date: 1994-02-17
Also published as: JPH0770190A; AU4760693A

Abstract

A peptide having the amino acid sequence containing at least four residues of an amino acid having a guanidino or amino group as the functional group, a derivative thereof, and a salt thereof. The peptide inhibits specifically the inhibition of urokinase-type plasminogen activators (uPA) by plasminogen activator inhibitor 1 (PAI-1), and is useful for preventing and treating thrombosis, such as cerebral infarction, the reobstruction of the blood vessel after the reopening effected by a thrombolytic agent, and disseminated intravascular coagulation (DIC).

Description

Specification

Title of invention

New peptide technology field

The present invention relates to a novel peptide, and more particularly, to a synthetic peptide that specifically inhibits PAI-1 that inhibits thrombolytic enzymes such as perokinase. Background art

The fibrinolytic system plays an important role in various biological processes, such as thrombolysis in blood vessels, cancer metastasis, angiogenesis, inflammation or wound healing. Plasminogen activator inhibitor (PA I-1), a member of the fibrinolytic factor, is composed of tissue-type plasminogen activator (.t PA) and perokinase-type plasminogen activator (uPA). ), And is the most important regulator of the fibrinolytic system. In addition, plasminogen activator inhibitor 2 (PA I—

2; also has an inhibitory activity against perokinase (UK) and tPA, a type of uPA. However, the rate of inactivation of single-chain tPA by PAI-2 depends on PAI-1. Significantly lower than that, underscoring the physiological importance of PAI-1.

By the way, attempts to activate a fibrinolytic system by inhibiting a fibrinolytic enzyme inhibitor have been made for a long time, and it has been reported that, for example, anti-inflammatory agents and the like have such an effect. However, the effects of these compounds are weak or they work in vitro but not in vivo, and there is almost no dissociation between the concentration range showing pharmacological effects and the concentration range showing toxicity. It was not practical at all. As described above, there is no practically available inhibitor for fibrinolytic enzyme inhibitors, and PAI-1 and u No specific inhibitor for the reaction with PA has been reported so far.

By the way, in recent years, the kinetics of the enzyme inhibition between PA I-1 and tPA or uPA has been studied. Is reversibly bound to a specific site by a hydrogen bond. In addition, the inventors have found that in the inhibition of tPA by PAI-1, the amino acid sequence in tPA kringle domain 2 is used as the specific binding site.

Asp Arg Arg Arg Leu was found to be involved (Kaneko, Μ ·, Sakata, Y., Matuda, Μ. & Mimuro, J .: J. Biochem. Ill, 244-248 (1992); Kaneko, M., Sakata, Y., Matuda, M. & Mimuro, J. (1991) Biochem. Biophys. Res. Commun. 178, 1160-1166)-However, regarding the binding site of PAI-1 to uPA, Was not known.

Here, the classification of uPA is as follows. UPA is an abbreviation of perokinase-type plasminogen activator, and is a single-chain perkinase-type plasminogen activator (hereinafter referred to as scu PA), perokinase (hereinafter referred to as UK), It can be classified as low molecular perokinase (hereinafter LMWUK). Scu PA has no enzymatic activity, and its peptide bond is cleaved by plasmin or the like to produce a high molecular weight perokinase, commonly known as perokinase (UK), which has a kring domain and a growth factor domain in the enzyme molecule. Next, LMWUK is a form in which the degradation of the UK has progressed and the clinging domain and the growth factor domain have been removed from the enzyme molecule.The enzymatic activity at the in vivo mouth does not change much, but the accumulation of thrombus in the living body is UK. The UK and scu PA are clinically more important.

In recent years, with the development of genetic engineering, administration of tPA or itPA to a concentration several thousand times the physiological concentration dissolves thrombus in a short period of time, which causes patients to suffer severe myocardial infarction and other diseases. Attempts to save lives from various diseases are already a reality. Power, power, simultaneous In addition, the adverse effects of such high doses have also become a problem, as seen in reperfusion arrhythmias and bleeding. Especially with either tPA or uPA therapy, reocclusion is seen in a few of the cases that reopened. The cause of this reocclusion is an increase in PAI-11 after reopening. Prevention or treatment of re-occlusion by re-administration of tPA or uPA in such re-obstructed cases is dangerous because of the side effects associated with systemic fibrinolytic activation. In this case, it is appropriate to prevent reocclusion by other means such as reducing the elevated PAI-1 activity with a PAI-1 inhibitor.

Also, in sepsis caused by bacteria, bacterial endotoxin raises tissue factor (coagulant activator) and PAI-1 in the blood, thereby causing multiple thrombi in the body, resulting in so-called dissemination. Causes vascular coagulation syndrome (DIC). The thrombus thus formed is difficult to dissolve and causes severe ischemic organ damage.

Therefore, inhibiting PAI-1 activity is considered to be extremely desirable as a causative therapy from the viewpoint of both prevention and treatment of severe organ damage associated with DIC and the like.

By the way, as mentioned above, PAI-1 is a physiological inhibitor of the physiological thrombolytic agents tPA and uPA, and is contained in large amounts in the blood, preventing thrombus dissolution by these ferrous elements. . Therefore, administration of a substance that antagonizes the action of these enzyme inhibitors, that is, the administration of a PAI-1 inhibitor, indirectly increases the concentration of tPA or uPA in the blood, which is indirectly In addition, it exerts a thrombolytic effect that is more moderate. Such a PAI-1 inhibitor opens up the possibility of prevention or treatment for other thrombosis, such as cerebral infarction, which requires even higher safety.

Furthermore, PAI-1 has been shown to play various physiological roles, There are still many research questions that remain unclear. Thus, PA1-1 inhibitors can serve as a new tool for studying PA1-1 and can also help diagnose PA1-1 or uPA in blood.

In addition, if the PA I-1 inhibitor is a small molecule, it can be produced by a chemical technique without using genetic engineering techniques. Compared to the molecular enzyme protein, it can be mass-produced at a lower cost.

For the above reasons, an object of the present invention is to provide a reversible small molecule inhibitor of uPA inhibition by PAI-11.

Other objects, features and advantages of the present invention will become apparent from the following description with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the binding of [1251] PA I-1 to the UK N-terminal fragment. In FIG. 1, the part surrounded by a square shows the result of the scattered analysis. Other insets show the results of Western blotting and SDS electrophoresis of the UK monoclonal antibody Mo Ab JTU-A3 (1) and the UK fragment (2) with a molecular weight of 21,000 containing clingled mains.

FIG. 2 is a diagram showing the binding of [1251] PAI-1 to the IS ′ terminal fragment of UK in the presence of various synthetic peptides. The symbols in the figure are as follows.

C: U 99 (99-106 *) His Asn Tyr Cys Arg Asn Pro Asp •: ϋ 103 (103-110) Arg Asn Pro Asp Asn Arg Arg Arg □: U107 (107-1) 14) Asn Arg Arg Arg Pro Trp Cys Tyr ■: ϋ 373 (373-398) He Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val

Ser His Phe Leu Pro Trp

•: The number in parentheses indicates that the amino acid sequence matches the amino acid sequence of the amino acid sequence number from the N-terminus of UK indicated by the numerical value. FIG. 3 is a diagram showing the binding of [125 I 3 P AI-1 to various synthetic peptides. The symbols in the figure are as follows.

0: U 99

•: U103

□: U 1 07

FIG. 4 is a diagram showing the binding of [1251] PA I-1 to various synthetic peptides. The symbols in the figure are as follows.

Δ: U 373 Reference: U 99

FIG. 5 is a diagram showing the effect of a synthetic peptide on the formation of the UK / P AI-1 complex. The symbols in the figure are as follows.

□: ϋ 1 07 〇: U 99

Δ: U 373 ■ ϋ 180 (180-187) HRGG SVTY

•: U103

FIG. 6 shows the effect of synthetic peptides on UK / PA I-1 complex formation. The symbols in the figure are as follows.

□: U 1 07 0: ϋ 99

Δ: U 373 ■ U 180

•: U103 disclosure of invention

In order to solve the above problems, the present inventors have synthesized various peptides and made intensive research efforts. Force as a result, for example, peptides synthesized based on the amino acid sequences in UK is, inhibits the binding of PAI 1 of UK, and reduces the Kassoc (M- one ¹ X 10- ⁶⁾ Speed We completed this study.

That is, an object of the present invention is to provide a peptide comprising an amino acid sequence having at least four amino acids containing a guanidine group or an amino group as a functional group.

Another object of the present invention is to increase the number of radiolabeled plasminogen activator inhibitors by observing the frequency of binding of the radiolabeled plasminogen activator inhibitor after the compound is attached to the substrate to prevent nonspecific adsorption. An object of the present invention is to provide a screening method characterized by screening for a minogen activator-inhibitor inhibitor.

Still another object of the present invention is to prevent thrombosis such as cerebral infarction, reocclusion after reopening with a thrombolytic agent, and prevention of organ damage associated with disseminated intravascular coagulation (DIC) in patients with severe infection. And a pharmaceutical composition comprising the peptide having the amino acid sequence described above, which is used for therapy.

Still another object of the present invention is to provide thrombosis such as cerebral infarction, reocclusion after reopening with a thrombolytic agent, and diseases such as disseminated intravascular coagulation (DIC) and plasminogen reaction. Another object of the present invention is to provide a peptide having an amino acid sequence as described above, which is used as a pharmacological reagent for the study of one inhibitor.

Further, another object of the present invention is to provide a diagnostic agent for a perokinase-type plasminogen activator or a plasminogen-activator inhibitor in blood containing a peptide having the amino acid sequence described above. To provide.

The peptide according to the present invention has an amino acid sequence having at least four amino acids containing a guanidine group or an amino group as a functional group. In more detail The length of the amino acid sequence having at least 4 or more amino acids containing a guanidine group or an amino group as a functional group is 4 to 28, preferably 6 to 26. As is well known, the guanidine group and the amino group can be represented by the following chemical formulas, respectively.

NH ₂ (CNH) NH- NH ₂ -Guanidine group Amino group

In detail, the peptide according to the present invention includes a peptide synthesized based on the amino acid sequence in the UK kringle domain Asn Arg Arg Arg Pro Trp Cys Tyr (U107), in a UK catalytic domain. Peptide lie Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly

Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp (U37 3), peptide His Asn Tyr Cys Arg Asn Pro Asp (U99), peptide lie Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp (U373), Peptide (Asn Arg Arg Arg Pro Trp), Peptide (Asn Arg Arg Arg Pro Trp), Peptide (Asn Arg Arg Arg Pro Trp Cys), Peptide ( Arg Asn Pro Asp Asn Arg Arg Arg Pro)

Furthermore, the peptide according to the present invention also includes a derivative in which an amino group, a carboxyl group, a guanidine group, or the like is protected, or a salt with an inorganic acid such as hydrochloric acid or acetic acid or an organic acid such as lactic acid or acetic acid.

Further, the peptide according to the present invention or its derivative or salt can be used alone or in combination of two or more as a pharmaceutical composition or a diagnostic agent. The peptide of the present invention is administered at the time of treatment of a patient with myocardial infarction with either tPA or uPA, thereby suppressing an increase in PAI-11 activity after reopening and preventing reocclusion by this effect. have. Further, the peptide according to the present invention ―

PCT / JP93 / 01103

8

Prevents severely infectious patients from having elevated PAI-1 activity in the blood, which facilitates the spontaneous lysis of thrombus caused by bacterial endotoxins, thereby disseminating It can prevent or treat the development of ischemic organ damage associated with intravascular coagulation (DIC).

In addition, the peptide compound according to the present invention has an unexpectedly good property that it does not inhibit fibrinolytic activity despite containing many amino groups in the peptide sequence. found.

Therefore, the pharmaceutical composition containing the peptide according to the present invention has a high risk of bleeding, and is a patient with myocardial infarction who cannot administer tPA or uPA, or a brain which is also generally considered to have a high risk of bleeding. It can be used for the prevention and treatment of these diseases by administering it to an infarcted patient and indirectly producing a thrombolytic action indirectly and more mildly.

Furthermore, the peptide according to the present invention basically has an action of inhibiting the reaction between u-PA and PAI-1, but also inhibits the neutralization reaction of t-PA with PAI-1. It was also found out. That is, the peptides of the present invention, two Second order chain t-PA and PAI-1 rate constant 1.14 ± OO ^ M ^ sec- 1) X 10- 7 0.31 soil 0.03 (M- ^ ec- ¹⁾ 10 - ⁷ significantly decreased, and further the double-stranded t labeled with radioactive Yodo - PA and PAI - 1 is of the coupling the base peptide be lowered to dose-dependent, after a sample SDS-PAGE, the complex Observation by autoradiography revealed this.

In addition, high blood levels of PAI-1 after thrombolytic therapy with PA are considered to be one of the causes of post-thrombotic reocclusion (Sakata, Y., Tanaka, T .: Academic Press, Inc. 109-121, 1991). Therefore, if administered the base peptide during thrombolytic therapy, tp _A, u- PA PAI in administration - 1 of the inhibitory effect on together when attenuating, administration of peptide persisted after PA administration Increases the action of endogenous t-PA It seems to be effective in preventing reocclusion.

In addition, DIC, in which infectious diseases such as sepsis occur concurrently with the underlying disease, often have extremely high blood PAI-1 levels, which is clearly important as one of the causes of organ damage. (Saka Y., Murakami, T., Noro, A., Mori, K. and Matsuda, Μ ·: Blood 77, 1949-1957, 1991, Hiroshi Sakata: Illustration;

Mechanism of DIC, Japanese clinical practice 51: 2-4, 1993). Therefore, it is considered that administration of this peptide during DIC associated with sepsis is effective for organ damage associated with DIC.

A common feature of these peptides having PAI-1 inhibitory activity is that the total number of functional groups such as guanidine and amino groups found in Arg and Lys is four or more. Therefore, a peptide consisting of an amino acid sequence having at least four amino acids whose functional group is a guanidine group or an amino group naturally has a PAI-1 inhibitory activity and is included as a peptide compound according to the present invention. Things.

In addition, various excipients commonly used in this field can be used for the above-mentioned drugs, diagnostic agents, and reagents containing the peptide for the purpose of improving stability and the like. When the peptide according to the present invention is used as a medicine, it can be administered by a usual administration method such as oral administration and intravenous injection. The dosage is used alone or in combination within a range that can be varied depending on the mode of administration, usually in the range of 1 mg to 5 g, preferably 10 mg to 500 mg.

The peptide U 107 (Asn Arg Arg Arg Pro Trp Cys Tyr) is

Inhibits the binding to double-stranded high-molecular-weight protein kinase by binding to I-1, but shows no or weak inhibition of one-chain protein kinase (scuΡ) or small-molecule protein kinase . On the other hand, peptide U 3 7 3 (He Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp) binds to a different site from peptide U107 on PAI-1 (Asn Arg Arg Arg Arg Pro Trp Cys Tyr), but in this case it is also double stranded for small molecule perokinase. It also inhibits binding to molecular oral kinases and inhibits the action of PAI-1 to enhance fibrinolytic activity.

In addition, the peptide of the present invention is prepared by observing the binding frequency of radiolabeled plasminogen activator-inhibitor after performing a treatment for adhering the compound to the substrate to prevent nonspecific adsorption according to a conventional method. Can be used to screen for plasminogen activator inhibitor inhibitors. BEST MODE FOR CARRYING OUT THE INVENTION

Example 1

The peptide U 107 (Asn Arg Arg Arg Pro Trp Cys Tyr) was synthesized using a peptide synthesizer model 43OA (Applied Biosystems). The Cys residue was protected by acetamidomethylation. After purification by reverse-phase HPLC, each peptide preparation was shown to be homogeneous by reverse-phase HPLC analysis. After dissolving in Tris-HCl buffer, the pH was adjusted to 7.0-8.0 and used for the experiment.

Example 2

U 3 7 3 (lie Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp) is a peptide synthesizer model 43 A (Applied Biosystems) ). The Cys residue was protected by acetamidomethylation. After purification by reverse phase HPLC, each peptide preparation was shown to be homogeneous by reverse phase HPLC analysis. After dissolving in Tris-HCl buffer, the pH was adjusted to 7.0 to 8.0 and used for the experiment. P93 / 01103

11

Example 3

The peptide U99 (His Asn Tyr Cys Arg Asn Pro Asp) was synthesized using a peptide synthesizer model 430A (Applied Biosystems). The Cys residue was protected by acetamidomethylation. After purification by reverse-phase HPLC, each peptide preparation was shown to be homogeneous by reverse-phase HPLC analysis. After dissolving in Tris-HCl buffer, the pH was adjusted to 7.0-8.0 and used for the experiment. Example 4

Peptide U 103 (Arg Asn Pro Asp Asn Arg Arg Arg) was synthesized using a peptide synthesizer model 430A (Applied Biosystems). The Cys residue was protected by acetamidomethylation. After purification by reversed-phase HPLC, each prepared peptide was shown to be homogeneous by reverse-phase HPLC analysis. After dissolving in Tris-HCl buffer, the pH was adjusted to 7.0-8.0 and used for the experiment.

Example 5

Penad (Asn Arg Arg Arg Pro Trp), Peptide (Asn Arg Arg Arg Arg Pro Trp), Peptide (Asn Arg Arg Arg Arg Pro Trp Cys) and Peptide (Arg Asn

Pro Asp Asn Arg Arg Arg Pro) was synthesized using a peptide synthesizer model 430A (Applied Biosystems). The Cys residue was protected by acetoamide methylation. After purification by reverse-phase HPLC, each peptide preparation was shown to be homogeneous by reverse-phase HPLC analysis. After dissolving in Tris-HCl buffer, the pH was adjusted to 7.0 to 8.0 before use in the experiment.

Example 6

Materials and reagents

(1) Reagent

All the following chemical reagents used were of the highest analytical quality and are commercially available. DFP: Diisopropylfluorophosphate, Tris-HCl, Bovine Serum Albumin (BSA), Tween 80 and Forball 13-Myristate 13-Acetic Acid (Sigma), Sodium Phosphate (Seikagaku), I ODO- B EAD S (PI ERCES), calf serum and cell culture reagent (GI BUCO), plastic container for cell culture (CORNING). UK and single-chain perokinase (scuPA)

(Mochida Pharmaceutical), small molecule perokinase (LMWUK) (PR0T0GEN).

(2) Protein

The N-terminal amino acid sequence was sequenced on a Sequencer Model 900A (Applied Biosystem).

PAI-1 was isolated from the conditioned medium of cultured human HT1080 cells. Sakata, Y., Okada, M., Nororo, A. & Matsuda, M. (1988) J. Biol. Chem. 263, 1960-1969; Sakata, Y., Murakami, T., Noro, A., Mori, K. & Matuda, M. Blood 77, 1949-1957). PA I-1 is based on I ODO-B as described by Kaneko et al. (Kaneko, M., Sakata, Y., Matsuda, M. & Mimuro, J. (1992) J. Biochem. Ill, 244-248). It was labeled with Na [125I] (2575 Ci / mmol, Dupont-New England Nuclear Company) using EAD S. The labeled PAI-1 had a specific radioactivity of 1.84 × 109 cpm / mg.

(3) Monoclonal antibody against perokinase

BAL B / c mice are immunized with purified human UK, a mouse monoclonal antibody against human UK is selected, and Sakata, Y., Okada, M., Noro, A. & Matsuda, M., et al. (1988) J. Biol. Chem. 263, 1960-1969; Sakata, Y., Murakami, T., Noro, A., Mori, K. & Matuda, M. Blood 77, 1949-1957) And cloned according to Kollerand Mils in method. Monoclonal anti The body (MoAbs) was obtained from mouse ascites by Mimuro, J., Koike, Y., Sumi, Υ. & Aoki, N .: Blood 69, 446-453 (1987), Wakabayashi et al. , Sakata, Y., Aoki, N. (1986) J. Biol. Chem. 261, 11097-11105) and separated using protein A-coupled sepharose. Mo Abs were analyzed by Western blotting for binding to UK functional domains. UK was separated by SDS-PAGE under reducing and non-reducing conditions. After electrophoresis, the sample was transferred to a nitrocellulose membrane and incubated with an appropriate Mo Ab. Conjugated antibodies were obtained from Mimuro, J., Koike, Y., Sumi, Υ & Aoki, N. Blood 69, 446-453 (1987), Wakabayashi et al. (Wakabayashi, K., Sakata, Y., Aoki, N. (1986) J. Biol. Chem. 261, 11097-11105) detected with an enzyme-linked anti-mouse IgG antibody .. By Western blotting, MoAb J TU was converted to UK B containing a catalytic domain. And bound MoAbJTU-A3 to the UKA chain containing the growth factor domain and the kringle domain.

(4) Separation of N-terminal UK fragment containing kringle domain

UK76 (^ g in the presence of plasmin (70 g / ml)

Incubated in 800 1 at 37 ° C for 20 minutes. Restriction proteolysis by plasmin was determined by SDS-PAGE, confirming LMWUK, N-terminal fragment of growth factor and UK containing kringle domain. The reaction solution was applied to a lysine-linked spharose column in the presence of aprotinin (100 U / ml) to remove plasmin. The unbound fraction was applied to a monoclonal antibody JTU-C3 conjugated Sepharose column to remove LMWUK and uncleaved UK (JTU-C3 binds to the catalytic domain of UK). Unbound fractions were collected and analyzed by SDS-PAGE and MoEb J TU-A3 by pentane blotting.

Example 7 Binding site of PAI-1 to synthetic peptides

Flat bottom microtiter plates (96-well, polystyrene) at 4 ° C in 100 1 phosphate buffer (20 mM sodium phosphate, 0.14 M NaCU pH 7.4) containing UK N-terminal fragment (1 / zg / ml) Coated with C for 16 hours. Block with BSA (3%), Tween 80 (0.01%), BSA (lmg / ml) and polyethylene glycol 6000 (4%,

After washing three times with a phosphate buffer (binding buffer) containing (W / V), the plates were incubated in the presence of various concentrations of [1251] -PAI-1 in the binding buffer. After washing three times in the binding buffer, the radioactivity in each well was measured using a gamma counter (Arrow force, Tokyo, Japan). The dissociation constant was calculated after the scatcher analysis by the method of Kaneko, M, J., Matsuda, M. & Sakata, Y. (1991) Biochem. Biophys. Res. Commun. 178, 1160-1166 (Fig. 1). ). That is, FIG. 1 shows the results of confirming the establishment of a screening system for PAI-1 inhibitors using the N-terminal fragment of UK instead of UK. Using the screening system thus obtained, the PAI-11 binding to the N-terminal fragment of UK was studied in the presence of a synthetic peptide (FIG. 2).

In this experiment, it was found that U107 showed strong binding inhibition, and U103 also showed weak inhibition. However, U373 and U99 did not show inhibition. This indicates that U107 and U103 inhibit the inhibition of UK by PAI11, and the inhibition sites are on PAI11 and in the N-terminal fragment of UK.

Example 8

Binding of P A I-1 to synthetic peptides

Analysis of binding of PAI-1 to UK N-terminal fragment and synthetic peptide. Flat bottom microtiter plate (96-well, polystyrene) was used to remove 10 µg / ml of peptide. The plate was coated in 100 phosphate buffer solutions (20 mM sodium phosphate, 0.14 M NaCK ρΗ7.4) at 4 ° C. for 16 hours. Block with B SA (3%) and Tween 80 (0.

After washing three times with a phosphate buffer (binding buffer) containing BSA (1 mg / ml) and polyethylene glycol 6000 (4%, W / V), the plates were treated with various concentrations of [ 1251] I was incubated in the presence of PA I-1. After washing three times in the binding buffer, the radioactivity in each well was measured using a gamma counter (Arrow force, Tokyo, Japan). The dissociation constant is determined by the method of Kaneko et al. (Kaneko, M., J., Matsuda, M. & Sakata, Y. (1991) Biochem. Biophys. Res. Commun. 178, 1160-1166) after scatter analysis. Calculated.

As shown in FIGS. 3 and 4, the peptides U103, U107 and U373 bound to PAI-1 in a concentration-dependent manner. On the other hand, U99 did not bind to PA I-1. These results indicate that the peptide U 103 and U 107 groups and the peptide U 373 have different binding sites on PAI-1 but all bind to PAI-1 and are peptides that inhibit PA I-1. You can see that there is. On the other hand, it is shown that U99, which has no effect in Example 7 and Example 8, does not inhibit PAI-11.

As shown in Table 1, the dissociation constants of the above experiments were as follows: U 103, U 107, and U 373 were 3.25 ± 2.92, 0.487 ± 0.10, and 78.1 ± 4.6 Kd of PAI-1, respectively. Met. table 1

Dissociation constant (K d) K peptide K d (nM) for binding of synthetic peptide to PAI-11 U 99 not detected

U 1 03 3.25 ± 2.92

U 1 07 0.487 ± 0.10

U 37 3 78.1 ± 0.460

Example 9

Effect of synthetic peptides on UK / P A I-1 complex formation

UK (lnM) and PAI-11 (lnM) were incubated at 37 degrees in the presence or absence of various concentrations of the synthetic peptide in TBS / Tween. The reaction was stopped by adding 10 mM p-APMS F. The concentration of the UK / PA I-1 complex is

Enzyme-linked immunosorbent assay using monoclonal antibodies against PAI-1 and Sakata et al.

Y., Okada, Μ ·, Noro, A. & Matsuda, M. (1988) J. Biol. Chem. 263, 1960-

1969, Sakata, Υ ·, Murakami, T., Noro, A., Mori, K. & Matuda, M .: Blood

77, 1949-1957) as described by the affinity for the purified polyclonal antibody against UK. The second-order rate constants are calculated by Hekman, K. M. & Losku-toff, DJ. (1988) Arch. Biochem. Biophys. 262, 199-210), Kaneko, M.,

Sakata, Y., Matsuda, M. & Mimuro, J. (1992) J. Biochem. Ill, 244-248; Kaneko, M., Mimuro, J., Matsuda, M. & Sakata, Y. (1991) Biochem Biophys. Res. Commun. 178, 1160-1166) was calculated under the pseudo-first-order rate constant. After incubating equimolar concentrations of UK and PAI-11 at 37 ° C in the presence or absence of 100 M peptide for various periods of time, add lOmMp-APMS F to stop the reaction, and then use UK / PAI- 1 The concentration of the complex was determined as described above. To confirm that synthetic substrate S-2444 (Riki, Stockform, Sweden) has an inhibitory effect on the peptide, the presence or absence of the peptide It was used to record the inhibition of UK hydrolytic activity by PA I-1 in the absence. UK (5 nM) in the presence or absence of various concentrations of PAI-1 (2.

5nM) for 10 minutes at 37 ° C. The remaining activity of UK is S—

Quantified by measuring the 2444 water dissolution rate.

Figure 5 shows that the inhibition of UK by PAI-1 is U107, U373 and

It is recognized by U103 and its effects are shown to be strong in U107 and U373, while weak in U103.

Example 10

Effect of synthetic peptides on LMWUK / P A I-1 complex formation

LMWUK (InM) and PA I-1 (InM) were incubated at 37 degrees in the presence or absence of various concentrations of synthetic peptides in TBS / Tween. The reaction was stopped by adding lOmMp-APMSF. Concentration of LMWUK / PA I-11 complex was determined by enzyme immunoassay using monoclonal antibody against PA I-11 and Sakata et al.

(Sakata, Y., Okada, Μ ·, Noro, Α. & Matsuda, M. (1988) J. Biol. Chem. 263, 1960—1969; Sakata, Y., Murakami, T., Noro, A., Mori, K. & Matuda,

M .: Blood 77, 1949-1957) as described by the affinity for the purified polyclonal antibody for LMWUK. Hekman et al. (Hekman, KM & Loskutoff, DJ (1988) Arch. Biochem. Biophys. 262, 199-210), Pitt et al. (Wittwer, AJ & Sanzo, MA (1990) Thromb. Hemostas. 64 , 270-275), Kaneko, M., Mimuro, J., Matsuda, M. & Sakata,

Y. (1991) Biochem. Biophys. Res. Commun. 178, 1160-1166) Calculated under stated pseudo-first-order rate constants.

After equimolar concentrations of LMWUK and PAI-11 were incubated at 37: C for various periods in the presence or absence of ΙΟΟ ^ 各種 peptide, lOmMp-APMS F was added. After stopping the reaction, the concentration of the LMWUK / PA I-1 complex was determined as described above. To confirm that the synthetic substrate S-2444 (Riki, Stockform, Sweden) has an inhibitory effect on peptides, PAI-1 inhibits the hydrolytic activity of LMWUK in the presence or absence of peptides. Used for recording. LMWUK (5 nM) was incubated with PA 分間 -1 (2.5 nM) in the presence or absence of various concentrations of the peptide for 10 minutes at 37 ° C. Residual activity of LMWUK was quantified by measuring the rate of S-2444 water hydrolysis.

The results showed that, as shown in FIG. 6, only U373 inhibited the inhibition of LMWUK by PAI-1. However, LMWUK is a degradation product of UK and physiologically the inhibition of UK by PAI-1 is important. This result shows that the action sites of the peptide U373 and ぺ peptide U103 and U107 groups It means different. Example 1 1

Second order reaction rate constants as a result of conducting experiments according to the methods of Examples 9 and 10.

100 MU 107, lOO ^ MU 373 or both U 107 and U 373 in the presence or absence of 100 ί と each with UK and P A I — 1 or LMWUK

The second-order rate constant in the inactivation of PAI-1 was determined.

As shown in Table 2, in the case of UK and PA I-1, it was observed that the rate constant was reduced about 1/10 compared to the case where both U107 and U373 were absent. However, the coexistence of U107 and U373 synergistically reduced the rate constant to about 1/100. On the other hand, in the case of LMWUK and PAI-11, the rate constant did not decrease even when U107 was added, and only when U373 was added, the rate constant decreased. Even when U107 and U373 coexisted, only the effect of U373 alone was shown.

As mentioned above, since LMWUK is a degradation product of UK, inhibition of the reaction between UK and PAI-1 is more important when considering the therapeutic effect as a drug, The inhibition of the reaction between LMWUK and PAI-11 has significance in the investigation of the reason for the synergistic effect in the former. In other words, it shows that the action sites of U373 and U107 and U103 are different.

Table 2

Second-order rate constants for inactivation of LMWUK with PAI-11 or of PAI-11 with UK in the presence or absence of peptide

Kassoc (M-IS-IX 10-6)

Peptide

UK / P A I-1 LMWUK / P A I 1 1 None 86.0 ± 3.90 5.70 ± 0.80

U 1 07 8.60 ± 0.60 5.50 ± 0.90

U 373 8.33 ± 0.57 0.40 ± 0.05

U 1 07 + U 373 0.41 ± 0.02 0.40 ± 0.02 Industrial applicability

The peptide compound of the present invention is a novel compound which, when used in combination with uPA, can sufficiently serve to protect uPA from PAI-11 inhibition. Prevention and treatment of thrombosis such as cerebral infarction by using the peptide compound of the present invention, prevention and treatment of reocclusion after reopening with a thrombolytic agent, disseminated intravascular coagulation syndrome (DIC), etc. It can be used as a medicament for the prevention and treatment of organ damage associated with the disease, a diagnostic agent for uPA and PAI-1 in blood, or a reagent for the above-mentioned diseases and PAI-11 research.

Claims

The scope of the claims

1 A peptide or a derivative or salt thereof, comprising an amino acid sequence having at least four amino acids containing a guanidine group or an amino group as a functional group.

2. The peptide according to claim 1, wherein the number of amino acids is from 4 to 28, or a derivative or salt thereof.

2. The peptide according to claim 1, wherein the peptide has a tertiary amino acid sequence.

Asn Arg Arg Arg Pro Trp Cys Tyr

4. The peptide according to claim 1, wherein the peptide has a quaternary amino acid sequence.

lie Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp

5. The peptide or a derivative or salt thereof according to claim 1, wherein the inhibitor inhibits the inhibition of urokinase type plasminogen activator by a plasminogen activator inhibitor.

(6) After the peptide of claim 1 or a derivative or salt thereof is attached to the substrate to prevent non-specific adsorption, the binding frequency of radiolabeled plasminogen activator-inhibitor is observed. A screening method comprising screening a plasminogen activator inhibitor.

7. The screening method according to claim 5, wherein the substrate is a microtiter plate.

8 Thrombosis such as cerebral infarction and reocclusion and severe 2. A peptide having an amino acid sequence according to claim 1, which is used for the prevention and treatment of organ disorders associated with disseminated intravascular coagulation (DIC) in patients with infectious diseases. Pharmaceuticals containing derivatives or salts.

9 Drugs for thrombosis such as cerebral infarction, reocclusion after reopening with thrombolytic agents, diseases such as disseminated intravascular coagulation (DIC) and plasminogen activator inhibitors 2. The peptide having an amino acid sequence according to claim 1 or a derivative or salt thereof, which is used for a physical reagent.

10. Diagnosis for perokinase-type plasminogen activator and plasminogen activator inhibitor in blood containing the peptide having the amino acid sequence of claim 1 or a derivative or salt thereof. medicine.