JP2003088397A - Plasminogen fragment having neovascularization- inhibiting activity and method for searching compound which induce production of the plasminogen fragment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently searching a compound which accelerates the conversion of plasminogen into an angiotensin-like activity-having fragment, and to provide the new plasminogen fragment. SOLUTION: This method for efficiently searching the compound which accelerates the conversion of the plasminogen into the angiotensin-like activity- having fragment. A point which utilizes the acceleration of the activation of the plasminogen as an index is an important point. The plasminogen fragment produced by the action of the compound identified by the method. The plasminogen fragment is a new molecule which has a molecular weight of 50 to 72 kDa and contains K1-K5 and whose protease domain is partially linked with a disulfide bond, and inhibits the proliferation, migration and lumen formation of cultured vascular endothelial cells similarly to angiostatin.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for searching a compound that promotes the production of a plasminogen fragment having an angiogenesis-inhibiting action, and the use of the compound having the activity.

[0002]

BACKGROUND OF THE INVENTION Angiostatin is a protein having a molecular weight of about 40,000, which is produced by limited degradation of plasminogen (an inactive precursor of fibrinolytic enzyme plasmin) (a single polysylline from the first kringle region to the fourth kringle region of plasminogen) It is a peptide) and inhibits the growth and metastasis of cancer by inhibiting angiogenesis. Angiostatin has shown dramatic effects in animal studies and is currently in clinical trials in the United States.
Similar activities have also been reported for plasminogen partially decomposed products such as kringle 1-3, kringle 1-5, and kringle 5.

Cancer treatment with angiostatin is carried out by intravenous administration of purified angiostatin (recombinant protein using Escherichia coli or highly purified elastase digestion fragment of plasma plasminogen). Both purification methods require a large cost, and in the case of angiostatin production by elastase digestion, angiostatin cannot be selectively produced due to the low substrate specificity of elastase. Of the angiostatin), and the activity of the produced angiostatin does not appear reproducibly. Also, angiostatin is a protein and needs to be administered directly into the blood. Furthermore, systemic administration of angiostatin is difficult to inhibit angiogenesis in a cancer tissue-specific manner. In contrast, compounds that promote the production of angiostatin or plasminogen fragments with similar activity in vivo, which mainly occur in cancer tissues, are considered to exert their effects depending on the regulatory ability of the body. , And is expected to show more effective angiogenesis-inhibiting activity (anti-cancer effect) according to various pathological conditions such as cancer progression.

No examples of compounds having such activity have been known so far. Further, in order to search, discover, and identify this, for example, a reaction in which angiostatin is produced from plasminogen (see the section of means for solving the following problems) is reconstructed and produced in vitro. Plasminogen fragments must be analyzed by methods such as SDS polyacrylamide gel electrophoresis, which is very labor, time and cost consuming to perform.

[0005]

The object of the present invention is to provide an effective and convenient method for searching for a substance which promotes the conversion of plasminogen into a fragment having angiostatin-like activity, which is identified by this method. The utilization of the compound.

[0006]

[Means for Solving the Problems] Plasminogen is about 92
It is a single-chain glycoprotein of kDa and has N-terminal peptides (NTP) and five kringle regions (K
1, K2, K3, K4, K5), and a serine protease region. The plasminogen activator hydrolyzes Arg561-Val562 to form the active enzyme plasmin, which expresses many physiological functions such as thrombolysis, tissue reconstruction, wound healing, and ovulation. On the other hand, a plasminogen fragment having a kringle region (angiostatin consisting of K1-K5, K1-K3, K1-K4.5,
K5 and the like) inhibit the proliferation, migration and lumen formation of vascular endothelial cells to inhibit angiogenesis. Due to this action, angiostatin is currently under clinical development as an anticancer agent. Angiostatin production from plasminogen proceeds by the following pathway. Plasminogen is activated to plasmin by the urokinase-type plasminogen activator (uPA), which is abundant in tumor tissues, and a part of it is reduced by the action of phosphoglycerate kinase in tumor tissues and the disulfide bond in K5 is cleaved. This molecule undergoes limited degradation by plasmin and the like to become K1-K4.5, which is further subjected to limited degradation by matrix metalloprotease (MMP) in tumor tissue to K1-K4.
(Angiostatin). Since plasminogen in the circulating blood has an intramolecular bond between NTP and K5 and has a tight conformation, it is resistant to activation by a plasminogen activator. By relaxing the conformation of plasminogen, the inventor can promote the conversion of plasminogen to plasmin and the fragmentation of plasminogen by proteases such as plasmin, thus producing a plasminogen fragment having an activity similar to that of angiostatin or angiostatin. I hypothesized that it would be possible. That is, it was considered that a compound that promotes the production of a plasminogen fragment having angiostatin-like activity can be discovered by using the promotion of plasminogen activation as an index. In order to confirm this, an in vitro reaction system for measuring the activation of plasminogen was used to search for compounds that promote this activity from the culture extract of about 5000 strains of microorganisms. As a result, Stachybotrysmic
rospora) IFO30018 strain produced staplabin (compound 1 according to claim 2) and SMTP-4 to 8 (compounds 2 to 6 according to claim 2), and plactin produced by the asporus deficient bacterium F164 strain. (Plactin) B and D (compounds 7 and 8 according to claim 2), Streptomyces genus (S
treptomyces sp. ) Thioplabin A, B and C produced by strain R1401 (compounds 21 to 23 according to claim 2), Streptomyces sp.
Complestatin (complement produced by the 1631 strain
statin) and chloropeptin (chlorop)
eptin) I (compounds 24 and 2 according to claim 2)
5) was identified as the active substance. In addition, as a result of examining the activity of the chemically synthesized plactin derivative, it was revealed that the compounds 9 to 20 described in claim 2 have the same activity.

These compounds convert plasminogen into uPA
When activated with, it transiently dramatically promoted plasmin production, but resulted in the subsequent fragmentation of plasmin. The generated plasminogen fragment was a novel molecule having a molecular weight of 50 kDa to 72 kDa, containing K1-K5, and further having a part of the protease region linked by a disulfide bond. It was revealed that the plasminogen fragments obtained by the action of various compounds inhibit the proliferation, migration and lumen formation of cultured vascular endothelial cells in the same manner as angiostatin, and are useful as angiogenesis inhibitors.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail.

A. The first of the present invention is the development of a method for efficiently searching for a compound that promotes the conversion of plasminogen into a fragment having angiostatin-like activity. As described in the section of the means for solving the above-mentioned problems, it is an important point of the present invention to search for this with the promotion of plasminogen activation as an index. As a result, 96 holes or more (38
Automatic measurement using an assay plate of 4 wells, 1536 wells, etc. has become possible, resulting in a dramatic increase in search efficiency.

B. The second aspect of the present invention is a compound identified by the above method (compounds 1 to 25 according to claim 2).
It is a plasminogen fragment produced by the action of. It spans a molecular weight of 50 kDa to 72 kDa,
A novel molecule containing -K5 and having a part of the protease region linked by a disulfide bond, it inhibited proliferation, migration, and tube formation of cultured vascular endothelial cells in the same manner as angiostatin.

EXAMPLES Examples of the present invention will be specifically described below.

Example 1 A method for efficiently searching for a compound which promotes conversion of plasminogen into a fragment having angiostatin-like activity.

The method for measuring activity using a 96-well assay plate will be described below. 100 nM plasminogen, 5
0 IU / ml uPA, 100 μM Val-Leu-
Lys-p-nitroanilide (substrate for plasmin), 1
00 mM NaCl and 0.01% Tween80
50 μl of Tris-HCl buffer (pH 7.4) containing 9
Incubation was performed at 37 ° C. in a 6-well assay plate, and the absorption at 405 nm was measured with a microplate reader (every 1 to 3 minutes). The initial reaction rate (plasmin production rate) can be determined by plotting the obtained absorbance on the vertical axis and the square of time on the horizontal axis. A fixed amount of the microbial culture extract was added to this reaction solution after drying under reduced pressure, and the one that gave an increase in the initial reaction rate was used as the active sample. The numerical values in the examples show one example, and the concentration of each component, the components and pH of the buffer solution, the amount of the reaction solution, the reaction temperature, and the reaction time in this measurement system can be appropriately changed. Is not limited to these numbers. Also, instead of plasminogen, N-terminal lysine-type plasminogen (Lys-p
lasminogen) or plasma (1% final concentration)
From about 50%) to other plasminogen activators (such as tissue-type plasminogen activator) instead of uPA.

As a result of searching for compounds that promote this activity from the culture extracts of about 5,000 strains of microorganism, Streptomyces sp. R1401 strain and Streptomyces sp.
Strong activity was observed in the culture solutions of the 1 strain, the spore-free incomplete bacterium F164 strain, and the Stachybotris microspora IFO30018 strain. The active substance was purified and isolated, and the structure was determined. As a result, compounds 1 to 8 and compounds 21 to 25 according to claim 2 were obtained.
Was identified. Further, as a result of examining the activity of the chemically synthesized plactin derivative, it was shown that the compounds 9 to 20 described in claim 2 had the same activity (Table 1). Further, FIG. 1 shows an example of the result of measuring the main activity.

[0014]

[Table 1]

Example 2 Fragmentation of plasminogen by the action of compounds 4, 8, 22 and 24 (FIG. 2).

In a control experiment the results of which are shown in FIGS. 2A and B, plasminogen (100 nM), uPA (50 IU /
ml) with 100 mM NaCl and 0.01% T
50 μl Tris-HCl buffer (pH 80) containing ween 80
Incubated at 37 ° C. in 7.4), and a part (45 μl) of the reaction solution was taken from 0 to 60 minutes after the reaction. To this, 5 µl of 1 mM Val-Leu-Lys-p-nitroanilide was added, and the mixture was incubated at 37 ° C and optionally (1
Absorbance at 405 nm was measured with a microplate reader every 3 minutes. The plasmin concentration was calculated from the slope of the obtained straight line (FIG. 2A). At the same time, a part of the reaction solution (210 μl) was mixed with 70 μl of 40% trichloroacetic acid and the protein was recovered by centrifugation. After washing this with acetone, 11 μl of SDS sample buffer (2%
SDS, 5% 2-mercaptoethanol, 10% sucrose, 0.02% bromophenol blue (bromop
(62.5 mM Tris-HCl, pH 6.8) containing henol blue), and then plasminogen fragmentation was observed by SDS gel electrophoresis using 10% polyacrylamide gel (FIG. 2B). Compound 4 (250 μM), Compound 8 (60 μM),
Compound 22 (30 μM), and compound 24 (0.3 μM
The results obtained with M) are shown in Figures 2A, C, D, E and F, respectively.

As shown in FIG. 2A, the plasmin concentration produced by the activation of plasmin was increased with time.
Consistent with this, an increase in A and B chains of plasmin was confirmed by SDS gel electrophoresis (Fig. 2B). On the other hand, in the presence of SMTP-6, the plasmin activity was transiently increased, but then decreased (FIG. 2C). SDS gel electrophoresis showed a decrease in the amount of plasmin B chain with a decrease in the activity, but the amount of A chain increased with time (FIG. 2D). The results show that in the presence of compound 4, the plasmin A chain is relatively stable, but the B chain is fragmented. Similarly, in the presence of compound 8, compound 22, and compound 24, an increase in plasmin concentration was not observed after 10 to 30 minutes after the reaction (FIG. 2A), which was consistent with the ratio of B chain to plasmin A chain. Decrease (Fig. 2
D, 2E, 2F) and B chain fragmentation was shown to be significantly induced.

Example 3 Structural analysis of plasminogen fragments (FIG. 3, FIG. 4, Table 2, Table 3).

The plasminogen fragment obtained by the method described in Experimental Example 2 was fractionated by SDS gel electrophoresis (FIG. 3) using a 20% polyacrylamide gel under reducing conditions, and the fraction was subjected to PVDF (polyvinylidene dif).
Coomassie Brilliant Blue (Coomassie Brilliant Bl)
ue) Stained with R-250. The plasminogen fragment band (arrows 1 to 5 in FIG. 3) was cut out, and the N-terminal amino acid sequence was analyzed by a protein sequencer, and the results shown in Table 2 were obtained. In addition, the plasminogen fragment obtained by the method described in Experimental Example 2 was lyophilized, reductively carboxymethylated, and then desalted using ZipTip (Millipore), followed by mass spectrometry (matrix-associa).
tedlaser destruction ioniz
ation time off mass
(MALDI-TOF-MS) spectrometer
y). The results obtained are shown in Table 3.

[0020]

[Table 2]

[0021]

[Table 3]

Based on the obtained information, the structure of the plasminogen fragment was determined as shown in FIG. This result is
The plasminogen fragment produced in the presence of the active compound is
It is shown to be a heterogeneous molecule consisting of various fragments of partially fragmented plasmin A chain and B chain linked by disulfide bonds. No such substance has been reported to date.

Example 4 Purification of plasminogen fragments (Figure 5).

The reaction solution containing the plasminogen fragment obtained by the method described in Experimental Example 2 was dialyzed against water at 4 ° C. overnight and freeze-dried. The obtained dried product was dissolved in a 0.1% trifluoroacetic acid solution, and μBONDASPHER was dissolved.
E 5μ C8-300Å column (19 x 150
mm; Waters) for fractionation by high performance liquid chromatography. At this time, a mixed solvent of 2-propanol and a 0.1% trifluoroacetic acid solution was used as a developing solvent, and the 2-propanol concentration was 2 at 0% at a flow rate of 5 ml / min.
After feeding for 0 minutes, the rate was increased at a rate of 0.8% per minute. Detection was absorption at 210 nm and excitation at 287 nm 34
It was performed by measuring the fluorescence at 8 nm. A peak with an elution time of about 60 to 62.5 minutes was collected and freeze-dried.
This was sterilized by filtration with a filter having a 0.45 μM pore size, and then analyzed by SDS gel electrophoresis using 7.5% polyacrylamide gel under non-reducing conditions (FIG. 5).

As a result of electrophoresis, it was found that the purified plasminogen fragment had a molecular weight of 66 to 72 kDa (FIG. 5).

Example 5 Proliferation and migration of vascular endothelial cells by plasminogen fragment
Inhibition of tube formation (Figure 6).

In the following experiment (1), the plasminogen fragment purified by the method of Example 4 was used. Also (2),
The plasminogen fragment used in the experiments (3) and (4) was prepared by the following method. Plasminogen (100n
M) to compound 4 (250 μM) or compound 8 (60
μM) with uPA (50 IU / ml), 100 m
After incubation in a Tris-HCl buffer solution (pH 7.4) containing M NaCl and 0.01% Tween 80 at 37 ° C. for 1 hour, the reaction solution was adjusted to 1 mM with phenylmethanesulfonyl sieve powder (phenyl).
Metanesulfonylfluoride) was added and dialyzed against purified water overnight at 4 ° C. The obtained dialysate was freeze-dried and then sterilized by filtration with a filter having a 0.45 μM pore size.

(1) Bovine capillary endothelial cells (BCEC)
Growth inhibition. BCEC (12,500 cells) was added to Dulbecco's modified Eagle medium (Dulbecco ') containing 0.5 ml of 10% fetal bovine serum in a 24-well tissue culture plate.
s modified Eagle medium;
Seed using DMEM). After culturing the cells for 24 hours, the medium was removed, and the plasminogen fragment (0.4 to 40 μg / ml) purified by the method of Example 4 and 0.2
Add 5 ml of DMEM containing 5% fetal bovine serum,
Incubated for minutes. After that, further 3 ng / ml
0.25 ml of DMEM containing basic fibroblast growth factor (bFGF) and 5% fetal bovine serum was added and the culture was continued for 3 days. Thereafter, the medium was removed and 100 μl of 0.
Phosphate buffered saline containing 05% trypsin was added,
After standing at 37 ° C. for 5 minutes, the cells were suspended and the number of cells was measured with a hemocytometer.

As shown in FIG. 6A, the plasminogen fragment purified by the method of Example 4 inhibited the growth of cultured BCEC by 17-53% at 0.2-20 μg / ml.

(2) Human umbilical vein endothelial cells (HUVE
Growth inhibition of C). HUVEC is an endothelial cell growth medium (EG
M; 2% fetal bovine serum, 20 ng / ml epidermal growth factor (EGF), 1 μg / ml hydrocortisone (hyd
rocortisone) and 12 μg / ml bovine brain extract (endothelial cell)
l basal medium) (Clonetic
s, San Diego, CA, USA). HUVECs (2,5
00 cells) in a 96-well tissue culture plate with 0.1 ml of E
Seeding was done with EGM without GF. After culturing the cells for 24 hours, the medium was removed, and plasminogen fragment (0.6 to 6 μg / ml) or angiostatin (K1-K3) (20 μg / ml) and 0.1 ml of EG were added.
F-free EGM was added and incubated for 20 minutes. Then, EGM further containing 20 ng / ml epidermal growth factor
0.1 ml was added and the culture was continued for 3 days. Then, the medium was removed and 0.1 ml of EGM and 10 μl WST-
One reagent (cell proliferation measurement kit based on MTT method) (Dojindo) was added, and the mixture was incubated at 37 ° C for 4 hours. Then, the absorbance at 405 nm was measured and used as an index of the cell number.

As shown in FIG. 6B, the plasminogen fragment produced by the action of compounds 4 and 8 inhibited the growth of cultured HUVECs by 68-86% at 0.3-3 μg / ml.
Angiostatin (K1-K3) used for comparison
Showed 74% growth inhibition at 10 μg / ml.

In order to confirm the specificity of this growth inhibitory effect, the effects of plasminogen fragment and angiostatin (K1-K3) on the growth of cells other than vascular endothelial cells were examined. The cells used were HT1080 human fibrosarcoma, CHO.
HT1 in K1 cells and primary rat dermal fibroblasts
080 cells and rat fibroblasts were grown in DMEM medium containing 10% fetal calf serum, and CHO-K1 cells were grown in RPMI1640 medium containing 10% fetal calf serum in the same manner as in the experiment using vascular endothelial cells. The activity was measured. As a result, as shown in FIG. 6C, it was shown that the plasminogen fragment generated by the action of compounds 4 and 8 did not affect the proliferation of these cells at 10 μg / ml, similar to angiostatin (K1-K3).

(3) HUVEC migration inhibition. HUVEC
Was cultured overnight in EGM containing 0.1% bovine serum albumin without serum and EGF, and then suspended in the same medium supplemented with 20 μg / ml plasminogen fragment or 20 μg / ml angiostatin (K1-K3). Incubated for 20 minutes. An 8 μm pore polycarbonate membrane insert coated with 15 μg fibronectin was added to 0.1% bovine serum albumin and 10 ng / ml b.
It was inserted into a 24-well tissue culture plate containing 0.5 ml of EGM containing FGF, and 0.5 ml of the above cell suspension (containing 110,000 cells) was seeded on a polycarbonate membrane. This was cultured for 5 hours and the polycarbonate membrane insert was taken out. After removing the cells on the upper surface of the membrane with a cotton swab, the cells that migrated through the membrane were stained with Giemsa,
It was observed under a microscope (100 times).

As shown in FIG. 6D, the plasminogen fragment produced by the action of compounds 4 and 8 inhibited the migration of cultured HUVEC by 23-31% at 10 μg / ml. In addition,
Angiostatin (K1-K3) used for comparison showed 37% inhibition.

(4) Inhibition of HUVEC lumen formation. In a 24-well tissue culture plate, 2.6% fetal bovine serum and 0.24
0.2 ml MCDB13 containing% type I collagen
1 medium (Clonetics) was added to form a gel,
HUVEC (40,000 cells) was inoculated into this using MCDB131 medium containing 0.1 ml of 2% fetal bovine serum. After culturing the cells for 2 hours, remove the medium,
10 μg / ml plasminogen fragment or 10 μg /
0.1 ml of angiostatin (K1-K3), 2.6% fetal calf serum and 0.24% type I collagen.
2 ml of MCDB131 medium was overlaid. After 30 minutes, 0.2 ml MCDB131 medium containing 10 μg / ml plasminogen fragment and 2% fetal bovine serum was further added.
Incubated for hours. Then, the cells were observed with an inverted microscope (40 times).

As shown in FIG. 6E, the plasminogen fragment produced by the action of compounds 4 and 8 strongly inhibited HUVEC lumen formation at 10 μg / ml. A similar inhibitory action was also observed for angiostatin (K1-K3) used for comparison.

[0037]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to effectively and efficiently search for a compound which promotes the conversion of plasminogen into a fragment having angiostatin-like activity. Moreover, the plasminogen fragment generated by the action of the compound identified by this method is a novel angiogenesis inhibitor, and it was possible to provide this.

[Brief description of drawings]

FIG. 1 shows an example of activity measurement results.

FIG. 2. Acceleration of plasminogen activation by the action of active compounds and the resulting fragmentation of plasmin. In A, compound 4 (SMTP-6), 8 (practin D),
22 (thioplabin B) and 24 (com
Plestatin) shows the time course of plasminogen activation, and B to F show the results of SDS gel electrophoresis of the reaction in the presence of control (no compound added) and compounds 4, 8, 22, and 24, respectively. . Arrow A in the figure
Indicates the position of the A chain of plasmin, and the arrow B indicates the position of the B chain.

FIG. 3: Analysis of plasminogen fragments generated by the action of active compounds. Compound 4 (SMTP-6), 8 (plac
The plasminogen fragment generated by the action of tin (D), 22 (thiolabin B) and 24 (completestatin) was fractionated by SDS gel electrophoresis using a 20% polyacrylamide gel under reducing conditions.
Arrow B in the figure indicates the B chain of plasmin.

FIG. 4: Several examples of the structure of plasminogen fragments produced by the action of active compounds. The thick solid line shows the peptide chain and the thin solid line shows the disulfide bond between cysteine residues. Each amino acid is represented by a one-letter code.

FIG. 5 Purification of plasminogen fragments generated by the action of active compounds. Compound 4 (SMTP-6), 8 (plac
tin D), 22 (thiolabin B) and 24 (completestatin), the resulting plasminogen fragment was bound to μBONDASPHERE 5μ.
After purification by high performance liquid chromatography using a C8-300Å column, it was analyzed by SDS gel electrophoresis using 7.5% polyacrylamide gel under non-reducing conditions. M
Indicates a molecular weight marker, and Plg indicates plasminogen. Lane 1 is a plasminogen fragment generated in the presence of compound 4 (before purification), lane 2 is a sample after the purification, lane 3 is a plasminogen fragment generated in the presence of compound 24 (before purification), lane 4 is a sample after the purification, Lane 5 is a plasminogen fragment generated in the presence of compound 22 (before purification), lane 6 is a sample after the purification, lane 7 is a plasminogen fragment generated in the presence of compound 8 (before purification),
Lane 8 is a sample after the purification.

FIG. 6: Inhibition of vascular endothelial cell proliferation, migration and tube formation by plasminogen fragments produced by the action of active compounds. A for BCEC, B for HUVEC, C for HT1
The effect on proliferation of 080 cells, rat fibroblasts and CHO-K1 cells is shown. D is HUVEC's migration,
E shows the effect of HUVEC on lumen formation. AS
(K1-K3) represents angiostatin (K1-K3).

Continued front page    (72) Inventor Tomotaka Harada             2-12-36 Honmachi, Shiki City, Saitama Prefecture             Ruins Hill Shiki Ichibankan No. 106 (72) Inventor Hu Weimin             5-11 Miyanishi-cho, Fuchu-shi, Tokyo Lou             Eheim Fuchu No. 201 F-term (reference) 4B050 CC06 DD11 LL01                 4B063 QA01 QA18 QQ20 QQ36 QR16

Claims (2)

[Claims] 1. A method for searching for a compound which leads to the formation of a plasminogen fragment having angiogenesis inhibitory activity. 2. Compounds 1 to 2 identified by the method of claim 1.
A plasminogen fragment produced by the action of 5.