WO2003066089A1

WO2003066089A1 - Megsin ligands

Info

Publication number: WO2003066089A1
Application number: PCT/JP2003/001316
Authority: WO
Inventors: Kiyoshi Kurokawa; Toshio Miyata
Original assignee: Kiyoshi Kurokawa; Toshio Miyata
Priority date: 2002-02-08
Filing date: 2003-02-07
Publication date: 2003-08-14
Also published as: AU2003244385A1; JPWO2003066089A1

Abstract

It is found out that megsin ligands are serine proteases. Megsin binds to plasmin and trypsin, which are serine proteases, and thus inhibits the activities of these enzymes. Hyperexpression of megsin is causative of renal failure. Therefore, renal failure can be treated by controlling the function of megsin with the use of these ligands.

Description

Technical field

The present invention relates to ligands for proteins expressed in kidney cells. The ligands of the present invention can be applied to the treatment of mesangial proliferative glomerulonephritis, and to Z or prophylaxis. Background art

As many as 60 trillion different cells in the body have essentially identical genomic DNA. For normal physiological function, the expression of these genes is tightly controlled by cell lineages and the signals that cells receive. Therefore, it is extremely important to elucidate genes specifically expressed in individual cell types.

Mesangium is located at the center of the leaflet of the capillary loop of the glomerulus and is the core tissue that connects the leaflets. The mesangium is covered by the glomerular basement membrane, and the capillary lumen is connected to cells (mesangial cells) separated by endothelial cells and the inner transparent layer in the glomerular basement membrane, which consists of three layers. It is composed of intangible substances (mesangial matrix). It is known that mesangial cells play a central role in maintaining the structure and function of renal glomeruli, and in the development of glomerular diseases such as glomerulonephritis and glomerulosclerosis. It is considered to be a major factor. And mesangial cells also have central pathophysiological significance in various types of nephritis. For example, proliferation of mesangial cells and accumulation of extracellular glomerular matrix are important pathological findings of glomerulosclerosis in patients with various glomerular diseases such as chronic nephritis and diabetic nephropathy [D. Schlondorff, Kidney Int., 49, 1583-1585 (1996); RBSterzel et al "Glomerular mesangial cells. Immunologic Renal Diseases, pp595-626 (199 By the way, the present inventor isolated a gene that is particularly strongly expressed in mesangial cells by large-scale DNA sequencing and database analysis, and determined the entire nucleotide sequence thereof. Then, the sequence of a novel protein (megsin) consisting of 380 amino acids encoded by the full-length cDNA clone of megsin was determined (WO 99/15652). Furthermore, amino acid homology search by FASTA program was performed using the Swiss Prot database. 1985); R. Carrrell et al "Cold Spring Harbor Symp. Quant. Biol., 52, 527 (1987); EKOKruithof et al" Blood, 86, 4007 (1995); J. Potempa et al "J. Biol. Chem., 269, 15957 (1994); E. Remold-O'Donnell, FEBS Let "315, 105 (1993)] [T. Miyata et al., J. Clin. Invest., 102, 828-83 6 (1998)].

Analysis of various tissues and cells by Northern blot and reverse transcription polymerase chain reaction revealed that megsin was weakly expressed in human fibroblasts, smooth muscle cells, endothelial cells, and keratinocytes, and was particularly strongly expressed in mesangial cells. (Ie, the expression of the megsin gene has a specificity of 4 in mesangial cells). These findings are further described in in situ hybridization [T. Miyata, et al., J. ClinJnvest., 102, 828-836 (1998); D. Suzuki, et al "J. Am. Soc. Nephrol., 10, 26 06-2613 (1999)] and immunohistochemistry using a megsin-specific antibody [R. Inagi, Biochem. Biophys. Res. Commun., 286, 1098-1106 (2001) Comparison of the expression level of megsin in the kidney tissue between patients with IgA nephropathy or diabetic nephropathy and healthy subjects showed that megsin in patients with IgA nephropathy and diabetic nephropathy was compared. Expression is significantly higher [T. Miyata et al, J. Clin. Invest., 102, 828-836 (1998), D. Suzuki et al, J. Am. Soc. Nephrol., 10, 2606-2613. (1999)] In an experimental mesangial cell proliferative glomerulonephritis model using rats (Thy-1 nephritis model), A similar increase in the expression of megsin was observed [M. Nangaku et al., Kidney Int., 6641-652 (2001)].

To better understand the role of megsin in mesangial function, we overexpressed human megsin cDNA in the mouse genome. Two strains of megsyn transgenic mice were obtained, which showed progressive expansion of the mesangial substrate, mesangial cell proliferation, and increased immune complex deposits (WO 01/24628). These findings indicate that megsin has a biologically significant effect on mesangial function. Interestingly, single genetic manipulation of megsin can generate experimental and early mesangial lesions present in human glomerulonephritis. Disclosure of the invention

Thus, the transgenic mouse of megsin, which has a structural characteristic of serine protease inhibitor (SERPIN) and may be deeply involved in renal disease, progressively expands mesangial substrates and proliferates mesangial cells. , And increased immune complex deposits, indicating that nephritis may have been induced by its SERPIN activity. Therefore, finding and specifying a ligand (protease) for megsin is necessary to elucidate the biological properties of mesangial cells, to investigate the causes of diseases associated with mesangial cells, and to treat and diagnose mesangial cell-related diseases. It is effective for

Screening a compound that specifically inhibits the complex between megsin and a ligand can be a useful tool for the development of a therapeutic agent for nephritis.

An object of the present invention is to provide a megsin ligand that is effective for treatment, diagnosis, and the like of a disease associated with mesangial cells. Another object of the present invention is to provide a method for evaluating the activity of a test compound that interferes with the binding between megsin and a megsin ligand. Further, the present invention has an activity of interfering with the binding between megsin and a megsin ligand. It is an object of the present invention to provide a method for screening compounds.

The present inventors have investigated the enzymes that form a complex with megsin and the enzymes whose activities are inhibited by megsin using various serine proteases and recombinant megsin. They found that plasmin trypsin, a species, had these effects, and completed the present invention. It was known that megsin had a characteristic structure in the SERPIN superfamily, but it was not confirmed that it actually had a serine protease inhibitory action. In addition, no information has been obtained on which proteases are inhibited in many proteases. That is, the present invention relates to the following megsin ligand, and a method for treating and preventing or preventing mesangial proliferative glomerulonephritis using a megsin ligand, and a screening method.

[2] A pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis, comprising as an active ingredient serine protease or a protein functionally equivalent to serine protease.

[3] A method for evaluating the activity of a test compound that interferes with the binding between megsin and a megsin ligand, comprising the following steps, wherein the megsin ligand comprises a serine protein

—Ze, or a method that is a fragment thereof.

1) a step of contacting megsin, a megsin ligand, and a test compound in any order described in a) to d) below under conditions in which megsin and the megsin ligand form a complex;

a) after contacting the test compound with megsin, and further contacting with a megsin ligand;

b) contacting the test compound with megsin in the presence of megsin ligand;

'After contacting with syn, further contact with test compound Let or

d) After contacting megsin ligand and test compound, further contact with megsin

2) Step of evaluating the effect of the test compound on the formation of a complex between megsin and a megsin ligand

[4] the method of [3], wherein the serine protease is plasmin or trypsin;

[5] The effect of the test compound on the formation of a complex between megsin and a megsin ligand was evaluated by the method described in [3], and the formation of the complex was compared to that in the absence of the test compound. A method for screening a compound having an activity of inhibiting the formation of a complex between megsin and a megsin ligand, comprising a step of selecting a compound to be inhibited.

[6] A method for inhibiting the activity of megsin, comprising the step of bringing a compound obtainable by the screening method according to [5] into contact with megsin.

[7] An inhibitor of megsin activity, comprising a compound obtainable by the screening method of [5].

[8] A pharmaceutical composition for treating mesangial proliferative glomerulonephritis and for preventing or preventing Z, comprising a compound obtainable by the screening method according to [5] as an active ingredient.

[9] An inhibitor of plasmin and / or trypsin activity, comprising megsin or a protein functionally equivalent to megsin as an active ingredient.

[10] Megsin or a protein functionally equivalent to megsin, plasmin

A method for inhibiting the activity of plasmin and Z or trypsin, comprising the step of contacting Z or trypsin.

As used herein, “treatment” refers to slowing or reversing the progress of any disease or condition to which the “treatment” applies, or such disease or condition. Means to relax either. In the present specification, "prevention" means

"Treatment" means maintaining a situation that does not result in either the disease or condition to which it applies.

As used herein, mesangial proliferative glomerulonephritis refers to a renal disease involving proliferation of renal mesangial cells and an increase in mesangial matrix. Known examples of such kidney diseases include IgA nephropathy, membranous proliferative glomerulonephritis, SLE (systemic lupus erythematosus) nephropathy, diabetic nephropathy, and cryoglobulin nephropathy. The present invention relates to megsin ligand. A megsin ligand is a substance that binds to megsin and interferes with its function. The present inventor has found that megsin binds to a specific protease and inhibits its enzymatic activity.

The megsin ligands discovered by the present inventors are the serine proteases plasmin and trypsin. Therefore, a megsin ligand can be defined as a substance having an activity of interfering with the inhibitory action of megsin on serine protease. Megsin ligand is useful for modulating the action of megsin. As described above, megsin has been shown to cause renal dysfunction caused by the proliferation of mesangial cells. Therefore, substances that interfere with the action of megsin are useful for treating renal dysfunction caused by overexpression of megsin.

The serine protease refers to a protein hydrolase having serine at the active center. The amino acid sequence constituting the active center is generally Asp-Ser-Gly. Megsin force It was revealed that SEKPIN has a characteristic structure, but it is not known that it actually binds to serine protease and inhibits its enzymatic action. Plasmin and trypsin can be mentioned as desirable serine proteases in the present invention.

Further, the serine protease fragment in the present invention refers to a fragment containing an amino acid sequence constituting a region necessary for binding to megsin in an amino acid sequence constituting serine protease. The fragment of the serine protease in the present invention may be any This can be clarified by preparing a library of serine proteases, collecting fragments capable of binding to megsin, and analyzing the amino acid sequence.

A fragment of serine protease can be obtained, for example, by digestion with a protease. Alternatively, a fragment encoding a serine protease can be obtained by isolating the cDNA encoding the serine protease and expressing the fragment. The gene fragment is prepared, for example, by amplifying a target region by PCR.

Plasmin, which is a megsin ligand of the present invention, is known as a fibrinolytic enzyme that hydrolyzes L-arginine and L-lysine peptides and esters, and particularly solubilizes fibrin. However, plasmin was not known to have ligand activity for megsin. Trypsin is a serine protease that hydrolyzes L-arginyl and L-lysyl carboxyl-linked peptides, amides and esters. Trypsin was also not known to have ligand activity for megsin.

In addition, the present invention relates to a pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis, comprising as an active ingredient a serine protease or a protein functionally equivalent to a serine protease. The present invention also relates to a method for treating mesangial proliferative glomerulonephritis, and a method for preventing or preventing Z-proliferative glomerulonephritis, comprising the step of administering serine protease or a protein functionally equivalent to serine protease. Alternatively, the present invention relates to the use of a serine protease or a protein that is functionally equivalent to a serine protease in the manufacture of a pharmaceutical composition for the treatment and prevention of mesangial proliferative glomerulonephritis. The serine protease used in the pharmaceutical composition of the present invention can be, for example, plasmin or trypsin.

In the present invention, the protein functionally equivalent to the serine protease is megsin. Refers to a protein having an activity of regulating the inhibitory effect of megsin on serine protease by binding to. Such a protein may be, for example, a fragment of a serine protease containing a binding domain to megsin. Fragments of serine proteases can be obtained by cleaving proteins. Methods for cleaving specific amino acid sequences by enzymatic or chemical reactions are known.

Furthermore, it consists of a DNA containing a nucleotide sequence encoding serine protease and an amino acid sequence encoded by DNA that hybridizes under stringent conditions, and regulates the inhibitory effect of megsin on serine protease. Proteins having activity are included in the functionally equivalent proteins of the present invention. It is considered that many of the sequences that can hybridize to a specific sequence under stringent conditions have an activity similar to that of the protein encoded by the specific sequence. The stringent conditions generally indicate the following conditions. That is, hybridization is performed at 4 × SSC at 65 ° C., and the plate is washed with 0.1 × SSC at 65 ° C. for 1 hour. The temperature conditions for high predication and washing, which greatly affect the stringency, can be adjusted according to the melting temperature (Tm). Tm varies depending on the ratio of the constituent bases to the hybridized base pairs and the composition of the hybridization solution (salt concentration, sodium formamide-dodecyl sulfate concentration). Therefore, those skilled in the art can empirically set conditions that give equivalent stringency in consideration of these conditions.

Alternatively, a functionally equivalent protein in the present invention preferably has an amino acid sequence constituting serine proteinase of at least 85%, more preferably at least 95%, homologous search by FASTA or BLAST homology search. Includes proteins consisting of amino acid sequences with liposomes.

In the present invention, a functionally equivalent protein comprises an amino acid sequence comprising one or more amino acids in the amino acid sequence constituting the protein, wherein the amino acid sequence has substitutions, deletions, insertions, and additions of Z or amino acids. Binds to megsin and binds megsin serine protein And proteins having an activity of regulating an inhibitory effect on the enzyme.

The amino acid sequence of plasminogen, which is a precursor of plasmin, and the nucleotide sequence of cDNA encoding it are known (J. Biol. Chem. 1990 Apr. 15; 265 / ll: 6104-ll; GenBank Accession No.NM — 000301). Similarly, for trypsin, the amino acid sequence of trypsinogen, its precursor, and the nucleotide sequence encoding it have been clarified (Gene 1986; 41 / 2-3: 305-10; GenBank Accession No. M22612). The number and location of amino acid mutations in proteins are not limited as long as their functions are maintained. The number of mutations is generally within 30%, or within 20%, for example, within 10%. A more preferred number of mutations is within 5% or 3% of all amino acids. A particularly preferred number of mutations can be within 2% of all amino acids or within 1% of all amino acids. The functionally equivalent protein of the present invention includes a case in which a mutation of several amino acids is substituted as a plurality of amino acids. Several are, for example, 5, or even 4 or 3, or 2, even 1 amino acids.

In order to maintain the function of the protein, the amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution. For example, amino acids belonging to each group as shown below are amino acids having properties similar to each other within the group. Substitution of these amino acids for other amino acids in the group often does not impair the essential function of the protein. Such amino acid substitution is called a conservative substitution and is known as a technique for converting an amino acid sequence while maintaining the function of a protein.

Non-polar amino acids: Ala, Val, Leu, lie, Pro, Met, Phe, and Trp Non-charged amino acids: Gly, Ser, Thr, Cys, Tyr, Asn, and Gin Acidic amino acids: Asp, and Glu

Basic amino acids: Lys, Arg, and His

Such proteins are obtained by transforming other prokaryotic or eukaryotic host cells by incorporating the gene encoding the protein into the appropriate expression vector DNA. Can be expressed. Examples of expression vectors include pET-3 [Studier & Moffatt, J. Mol. Biol. 189, 113 (1986)] for Escherichia coli and pEF-BOS [Nucleic Acids Research 18, 5322 for COS cells]. (1990)], pSV2-gpt [Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78, 2072 (1981)], and in the case of CHO cells, pVYl [WO 89/03874]. Respectively. Further, by linking a gene encoding another polypeptide to the gene of interest and expressing it as a fusion protein, purification can be facilitated, and then the protein of interest can be cut out. Known proteins to be fused include a histidine tag, a c-myc tag, an MBP-tag, and a GST-tag. Vectors capable of expressing inserts with these tags fused are commercially available.

In addition, the present invention relates to a method for evaluating the activity of a test compound in interfering with the binding between megsin and a megsin ligand, comprising the following steps. In the evaluation method of the present invention, the megsin ligand refers to a serine protease or a fragment thereof. Examples of serine mouth proteases include plasmin-trypsin.

1) contacting megsin, a megsin ligand, and a test compound in any order described in a) to d) below under conditions in which megsin and the megsin ligand form a complex;

b) contacting the test compound with megsin in the presence of megsin ligand; c) contacting the megsin ligand with megsin followed by further contacting the test compound; or

d) After contacting the test compound with megsin ligand, further contact with megsin

The activity of the test compound of the present invention to interfere with the binding between megsin and a megsin ligand. The method of evaluation is useful for screening for compounds that interfere with the binding of megsin to the megsin ligand. That is, the present invention evaluates the effect of a test compound on the formation of a complex of megsin and a megsin ligand by the above-described evaluation method, and evaluates the compound that inhibits the formation of a complex as compared to the formation of a complex in the absence of the test compound. And a method for screening a compound having an activity of inhibiting the formation of a complex between megsin and a megsin ligand.

By the screening method of the present invention, compounds that interfere with the binding between megsin and a megsin ligand are selected. Such compounds can be used to control the activity of megsin. More specifically, such a compound can be used as an activity inhibitor for megsin or a method for inhibiting megsin activity. That is, the present invention relates to a megsin activity inhibitor comprising the compound selected by the screening method. In addition, the present invention includes the step of administering a compound capable you to select by the screening method, the treatment of Mesangiumu proliferative glomerulonephritis, and / ⁷ or a method for the prevention. Alternatively, the present invention relates to the use of a compound obtainable by the above-mentioned screening method in the manufacture of a pharmaceutical composition for treating and preventing Z or prophylaxis of mesangial proliferative glomerulonephritis. The present invention also relates to a method for inhibiting the activity of megsin, comprising the step of bringing the compound selected by the screening method into contact with megsin. The present invention has revealed that megsin acts as an inhibitor on serine protease. In the present invention, the activity of megsin refers to an activity of binding to a serine protease and inhibiting its enzymatic action.

The morphological or physiological abnormalities of renal glomeruli caused by overexpression of megsin are considered to be due to the function of megsin, that is, its inhibitory effect on protease. Therefore, compounds that inhibit the activity of megsin are useful as therapeutic or prophylactic agents for suppressing glomerular abnormalities caused by the action of megsin. In the evaluation method or screening method of the present invention, as the megsin ligand, any protein selected from serine protease or a fragment thereof can be used. For example, the serine proteases human plasmin and trypsin are known proteins. Methods for preparing these proteins are known. Purified proteins of plasmin and trypsin are commercially available.

On the other hand, for proteins functionally equivalent to these serine proteases, for example, fragments thereof can be used. A protein fragment can be obtained by cleaving a full-length protein by enzymatic, chemical, or physical action, and purifying a fragment having a desired activity. Alternatively, an oligopeptide having the required amino acid sequence as a fragment can be obtained by peptide synthesis. As a functionally equivalent protein, specifically, for example, a protein containing an amino acid sequence constituting a binding domain to megsin can be shown. For example, the following peptides containing an amino acid sequence constituting the active center of plasmin and trypsin can be used as a protein functionally equivalent to serine protease.

Plasmin:

MHFCGGTLISPEWVLTAAHCLEKSPRCAGHLAGGTDSCQGDSGGPLV

Trypsin:

YHFCGGSLINSQWWSAAHCYKSGIQCAGYLEGGKDSCQGDSGGPW

The screening method of the present invention can be more easily carried out by labeling one of megsin and a megsin ligand on a solid phase and labeling the other. As the solid phase, magnetic beads, the inner wall of a reaction vessel, or the like is used. For the label, a known label component such as a fluorescent substance, a luminescent substance, or an enzyme active substance may be used. The label can be directly bound to megsin (or a megsin ligand) or indirectly by utilizing an affinity reaction such as avidin-biotin binding. In the present invention, megsin, megsin ligand, and a test compound are contacted under conditions under which megsin and the megsin ligand form a complex. In the present invention The conditions under which megsin and a megsin ligand can form a complex are specifically defined by, for example, physiological pH and salt concentration, and a temperature suitable for forming the complex. More specifically, if the salt concentration is about the same as that of physiological saline and the temperature is 20 to 40 ° C in a buffer giving pH 6-8, megsin and the megsin ligand can be complexed. Coalescence can be produced.

Megsin, a megsin ligand, and a test compound can be contacted in the order described in any of a) to c) above.

a) When the test compound and megsin are brought into contact with each other and then contacted with a megsin ligand, the inhibition of the binding between megsin / megsin ligand by the test compound can be evaluated. B) When the test compound is brought into contact with megsin in the presence of the megsin ligand, the competitive inhibition by the test compound can be evaluated. For competitive inhibition, the ratio of the reaction components is adjusted so that the binding partner is in excess with respect to the component to be inhibited.

Further, c) when the megsin ligand and megsin are brought into contact with each other and then with the test compound, the activity of displacement of the binding between megsin / megsin ligand by the test compound can be evaluated. Alternatively, d) when the test compound is brought into contact with the megsin ligand and then with the megsin, and then the test compound can be evaluated for its inhibitory effect on the binding of megsin. The test compound selected at this time has an effect of preventing the binding of megsin to the megsin ligand by modifying the megsin binding site of megsin ligand.

For example, the oligopeptide P1-14 consisting of the amino acid sequence TEATAATGSNIVEK corresponding to positions 334-347 from the N-terminus in the amino acid sequence of megsin inhibits the inhibitory effect of megsin on enzyme activity by binding to plasmin. I have confirmed. The activity of such a peptide is detected by contacting the three components in the order as described in a), b) or d).

With any of these contact sequences, Can be found. These binding inhibitory activity, competitive inhibitory activity, and displacement activity are all included in the activity of interfering with the binding between megsin / megsin ligand.

Next, the effect of the test compound on the formation of a complex of megsin and a megsin ligand is evaluated using the level of the following complex formed as a result of such a reaction as an index.

A: Complex consisting of megsin and megsin ligand

B: Complex of megsin and test compound

C: Complex of megsin ligand and test compound

The level of formation of these complexes can be easily measured by labeling any of the components constituting the complex. For example, if megsin is immobilized on a solid phase and a megsin ligand is labeled and used, it is possible to know the level of complex formation of A and B using the activity of the solid phase and the labeling of the Z or liquid phase as an index. it can. Alternatively, a tag can be attached to megsin or a megsin ligand, and a substance having an affinity for the tag can be bound to perform a pull-down assay. As the tag, a histidine tag or an HA tag is used.

The elements necessary for the screening of the present invention can be supplied as a pre-combined kit. The screening kit according to the present invention includes megsin and a megsin ligand as essential components, and further includes a reaction vessel, a diluent, a negative control, a positive control, or a method for performing screening using each element. An operation manual describing the order can be included. Further, if necessary, a substrate compound and the like necessary for detecting the labeling component used in the screening can be combined.

In the evaluation method or screening method of the present invention, examples of the test compound include natural or synthetic compounds, various organic compounds, natural or synthetic saccharides, proteins, peptides, expression products of gene libraries, cell extracts, or Cell components and the like can be mentioned.

Further, a compound that can satisfy the substrate specificity of megsin by maintaining or mimicking the three-dimensional structure of the active center site of serine protease can be a candidate for a compound having a binding activity to megsin. As an active center of serine protease, for example, oxy union hole can be shown.

The present invention also relates to an inhibitor of plasmin and z or trypsin activity, which comprises megsin or a protein functionally equivalent to megsin as an active ingredient. According to the present invention, it has been revealed that the ligands of megsin are plasmin and trypsin. Megsin binds to these serine proteases and inhibits their enzymatic activity. Therefore, megsin is useful as a serine protease activity inhibitor. Furthermore, a protein functionally equivalent to megsin can also be used as a serine protease inhibitor.

Human 'megsin is a protein encoded by DNA having the nucleotide sequence shown in SEQ ID NO: 1. The amino acid sequence of human megsin is shown in SEQ ID NO: 2. The megsin of the present invention can target not only human megsin but also proteins functionally equivalent to human megsin. Such a protein may be, for example, a homologue of megsin in another species. In the megsin homolog, for example, the structure of rat megsin and mouse megsin has been elucidated by the present inventors (WO 99/15652). The nucleotide sequence of rat megsin and the amino acid sequence are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the base sequence of mouse megsin and the amino acid sequence are shown in SEQ ID NO: 5 and SEQ ID NO: 6.

In general, eukaryotic genes often show polymorphism, as is known for the human interferon gene. Due to this polymorphism, the protein activity is usually maintained even if one or more amino acid substitutions occur in the amino acid sequence. In general, it is known that protein activity is often maintained by modifying one or several amino acids. Therefore, SEQ ID NO: 2, SEQ ID NO: 4, and The gene encoding a protein consisting of an amino acid sequence obtained by artificially modifying the amino acid sequence shown in any of SEQ ID NO: 6 can be used as long as the protein binds to a protease and inhibits its activity. Can be used for

Hereinafter, megsin derived from humans, rats, or mice and proteins having biologically equivalent functions are collectively referred to as megsins. However, in order to use the megsin ligand according to the present invention for controlling the activity of human proteases or to screen compounds useful as therapeutic agents in humans, it is advantageous to use human megsin DNA. This is because the effect on human megsin can be more faithfully reflected.

Since plasmin is a fibrinolytic enzyme, megsin, which inhibits its activity, can be expected to promote blood coagulation. In addition, trypsin inhibitors have been used for the treatment of knee inflammation. Therefore, megsin having a trypsin inhibitory effect can be expected to be useful as a therapeutic agent for phitis.

According to the present invention, there is provided a pharmaceutical composition for treating and / or preventing mesangial-proliferating glomerulonephritis, comprising any one of the following active ingredients. Serine protease, or a protein functionally equivalent to serine protease, a compound that interferes with the formation of a complex between megsin and a megsin ligand (this compound is selected by the screening method)

Alternatively, the present invention provides a serine protease inhibitor comprising megsin or a protein functionally equivalent to megsin as an active ingredient. The inhibitor of the present invention can be used for a pharmaceutical composition for controlling the activity of serine protease.

These pharmaceutical compositions can be used as they are or after being subjected to various treatments such as dilution with water, and can be used by being blended with pharmaceuticals, quasi-drugs and the like. In this case, the compounding amount is appropriately selected according to the disease state and the product. However, in the case of a systemically administered preparation, the amount is usually 0.001 to 50% by weight, particularly 0.01 to: L 0% by weight. Can do If the content is less than 0.001% by weight, satisfactory preventive or therapeutic effects may not be observed, and if it exceeds 5% by weight, the stability and flavor properties of the product itself may be impaired. Is not preferred.

The megsin ligand of the present invention may be contained in a preparation as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, for example, salts with bases such as inorganic bases and organic bases, and acid addition salts such as inorganic acids, organic acids, basic and acidic amino acids, and the like.

Examples of the inorganic base include alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, aluminum, and ammonium. Examples of the organic base include primary amines such as ethanolamine, etc., secondary amines such as dimethylamine, diethanolamine, hexylamine, and Ν, Ν′-dibenzylethylenediamine, and trimethylamine. And tertiary amines such as triethylamine, pyridine, picoline and triethanolamine. Examples of the inorganic acid include hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Examples of organic acids include formic acid, acetic acid, lactic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, benzoic acid, cunic acid, succinic acid, malic acid, methanesnolefonic acid, ethanesnolefonic acid, and benzenesnolefonic acid And Ρ-tonolenesulfonic acid. Examples of the basic amino acid include arginine, lysine, orditin and the like. Examples of the acidic amino acid include aspartic acid and glutamic acid.

As the administration method of the pharmaceutical composition of the present invention, in addition to oral administration and intravenous administration, transmucosal administration, transdermal administration, intramuscular administration, subcutaneous administration, rectal administration, and the like can be appropriately selected, and depending on the administration method. Thus, it can be used as various preparations. Hereinafter, each preparation will be described, but the dosage form used in the present invention is not limited thereto, and can be used as various preparations usually used in the field of pharmaceutical preparations. Further, the active ingredient constituting the pharmaceutical composition can be encoded by SDNA. Then, the DNA can be incorporated into a vector for gene therapy and used for gene therapy. Administration can be performed by, for example, intraarterial injection, intravenous injection, intranasal administration, intrabronchial administration, intramuscular administration, subcutaneous administration, oral administration, direct administration to the affected area, and the like. The dose varies depending on conditions such as the patient's body weight, age, health degree, and administration method, but those skilled in the art can appropriately select an appropriate dose.

When used for treatment and / or prevention of mesangial proliferative glomerulonephritis, the oral dose of megsin ligand is preferably in the range of 0.03 mg / kg to 30 mg / kg, more preferably. Is between 0.1 mg / kg and 10 mg / kg. In the case of systemic administration, for example, in the case of intravenous administration, the effective blood concentration is 0.2 ii _g / mL to 20 / ig / mL, more preferably 0.5 / 1111 ^ to 10 g / mL. The dosage should be adjusted so that The dose is adjusted according to age, sex, weight, and the like.

Dosage forms for oral administration include powders, granules, capsules, pills, tablets, elixirs, suspensions, emulsions and syrups, and can be appropriately selected. In addition, these preparations can be modified such as sustained release, stabilization, easy disintegration, difficult disintegration, enteric coating, and easy absorption. In addition, the dosage form for topical administration in the oral cavity includes mastication agents, sublingual agents, puccal agents, troches, ointments, patches, liquids, and the like, which can be appropriately selected. In addition, these preparations can be modified such as sustained release, stabilization, easy disintegration, difficult disintegration, enteric coating, and easy absorption. For each of the above dosage forms, known drug delivery system (DDS) technology can be employed. The DDS preparations referred to in the present specification include controlled release preparations, topically applicable preparations (troches, puccal tablets, sublingual tablets, etc.), controlled drug release preparations, enteric coating preparations, gastric coating preparations, etc. This refers to a formulation in the optimal formulation form, taking into account availability and side effects.

The components of a DDS basically consist of a drug, a drug release module, and an encapsulation treatment program. Each component, especially when the release is stopped, A drug with a short half-life that reduces the blood concentration is preferred, an envelope that does not react with the living tissue at the administration site is preferred, and a treatment program that maintains the best drug concentration for a set period of time is preferred. Is preferred. The drug release module basically has a drug reservoir, a release controller, an energy source and a release hole or release surface. It is not necessary to have all of these basic components, and the best mode can be selected by adding or deleting as appropriate.

Materials that can be used for DDS include polymers, cyclodextrin derivatives, and lecithin. Polymers include insoluble polymers (silicone, ethylene butyl acetate copolymer, ethylene butyl urea / reco / le copolymer, etinolace / rerose, ce / rerose acetate, etc.), water-soluble polymers Xyl gel-forming polymers (polyacrylamide, cross-linked polyhydroxyethyl methacrylate, cross-linked polyacryl, polybutyl alcohol, polyethylene oxide, water-soluble cellulose derivatives, cross-linked poloxamers, chitin, chitosan, etc.) Butyl cellulose, methyl butyl ether, partial ester of maleic anhydride copolymer, etc., gastric-soluble polymers (hydroxypropylmethylcellulose, hydroxypropylcellulose, potassium lmellose, macrogol, polybutylpyrrolidone) Dimethacrylate Tylaminoethyl / methyl methacrylate copolymer, etc., enteric polymers (hydroxypropyl methylcellulose phthalate, phthalcellulose acetate, hydroxypropinolemethinolecinolose acetate succinate, force / repoxymethylinoethyl cellulose, acrylic acid Polymers), biodegradable polymers (thermocoagulated or crosslinked anorepmin, cross-linked gelatin, collagen, fibrin, polycyanoacrylate, polydalicholate, polylactic acid, poly) 3-hydroxyacetic acid, polycaprolactone, etc.) Yes, it can be appropriately selected depending on the dosage form.

In particular, silicon, ethylene 'vinylinoleate copolymer, ethylene-butyl alcohol copolymer, and partial esters of methylbutyl ether / maleic anhydride copolymer can be used to control drug release, and cellulose acetate can be used for osmotic pumps. As material It can be used, ethinoresenorelose, hydroxypropinolemethylcellulose, hydroxypropylcellulose, and methylcellulose can be used as a membrane material of a sustained-release preparation, and a crosslinked polyacryl can be used as an adhesive for oral mucosa or ocular mucosa.

Depending on the dosage form (known dosage forms such as oral dosage forms, injections, suppositories, etc.), solvents, excipients, coatings, bases, binders, lubricants, Additives such as disintegrants, dissolution aids, suspending agents, thickeners, emulsifiers, stabilizers, buffers, isotonic agents, soothing agents, preservatives, flavoring agents, fragrances, coloring agents, etc. Can be added.

Each of these additives will be exemplified with specific examples, but is not particularly limited thereto. Examples of the solvent include purified water, water for injection, physiological saline, seaweed oil, ethanol, glycerin and the like.

Examples of the excipient include starches, lactose, glucose, sucrose, crystalline cellulose, calcium sulfate, calcium carbonate, talc, titanium oxide, trehalose, xylitol, and the like. Examples of the coating agent include sucrose, gelatin, cellulose acetate phthalate, and the polymers described above.

Examples of the base include petrolatum, vegetable oil, macrogol, oil-in-water emulsion base, water-in-oil emulsion base, and the like. Examples of the binder include starch and its derivatives, cellulose and its derivatives, gelatin, sodium alginate, tragacanth, natural polymer compounds such as gum arabic, synthetic high molecular compounds such as polybulpyrrolidone, dextrin, hydroxypropyl starch and the like. Can be mentioned.

Examples of the lubricant include stearic acid and salts thereof, talc, waxes, wheat starch, macrogol, hydrogenated vegetable oil, sucrose fatty acid ester, polyethylene glycol and the like.

Disintegrators include starch and its derivatives, agar, gelatin powder, sodium hydrogencarbonate, cellulose and its derivatives, carmellose calcium, hydroxypropyl starch, carboxymethylcellulose and its salts and Bridges and low-substituted hydroxypropylcellulose can be exemplified.

Examples of the solubilizer include cyclodextrin, ethanol, propylene glycol, and polyethylene glycol. Examples of suspending agents include arabia gum, tragacanth, sodium alginate, aluminum monostearate, citric acid, and various surfactants.

Examples of the thickener include carmellose sodium, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, polyvinylinole alcohol, tragacanth, acacia, sodium alginate and the like. Examples of the emulsifier include gum arabic, cholesterol, tragacanth, methinolecellulose, various surfactants, lecithin and the like. Examples of the stabilizer include sodium bisulfite, ascorbic acid, tocopherol, a chelating agent, an inert gas, a reducing substance, and the like. Examples of the buffer include sodium hydrogen phosphate, sodium acetate, boric acid and the like.

Examples of the tonicity agent include sodium chloride, glucose and the like. Examples of the soothing agent include proforce hydrochloride, lidocaine, and benzyl alcohol. Examples of the preservative include benzoic acid and salts thereof, para-hydroxybenzoic acid esters, chloroptanol, inverted soap, benzyl alcohol, phenol, and tyromesal.

Examples of the flavoring agent include sucrose, saccharin, canzo extract, sorbitol, xylitol, glycerin and the like. As the fragrance, spruce tincture, rose oil and the like can be mentioned. Examples of the coloring agent include a water-soluble edible dye, a lake pigment, and the like.

As mentioned above, the formulation of DDS preparations, such as sustained-release preparations, enteric-coated preparations, or controlled-release drug preparations, is expected to have the effect of maintaining the effective blood concentration of the drug and improving bioavailability. it can. However, megsin ligand may be inactivated or degraded in vivo, resulting in a diminished or diminished effect. You. Therefore, by combining a substance that inhibits a substance that inactivates or degrades megsin ligand with a pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis, the effects of the ingredients can be further sustained. Can be converted. These may be incorporated into the preparation or may be administered separately. Those skilled in the art can appropriately identify substances that inactivate or degrade megsin ligand, select substances that inhibit this, and combine or use them in combination.

In the drug product, other ingredients used in the usual composition can be used as additives other than the above, and the addition amount of these ingredients should be a normal amount within a range not to impair the effects of the present invention. Can be.

All prior art documents cited in the present specification are incorporated herein by reference. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a photograph showing SDS-PAGE under non-reducing conditions followed by CBB staining (middle panel on the left) or immunoblotting with anti-t-PA antibody (right panel). Megsin was shown to have functional binding to plasmin but not to other serine proteases such as t-PA ゃ thrombin. * Indicates the band of the complex.

Lane 1: Plasmin, Lane 9: Megsin,

Lane 2: o; 2-AP, 10 Lane 10: Thrombin + megsin, Lane 3: Plasmin + α2-ΑΡ, Lane 11: t-PA,

Lane 4: Megsin, Lane 1 2: PAI-1,

Lane 5: plasmin + megsin, lane 13: t-PA + PAI-l, lane 6: thrombin, lane 14: megsin,

Lane 7: ΑΤΠΙ, 15 Lane 15: t-PA + Megsin Lane 8: Thrombin + ΑΤΙΠ, FIG. 2 is a diagram showing the results of the MCA attestation. ◊: Substrate only, Mouth: Plasmin, Jap: Plasmin + antiplasmin (1: 2), 〇: Plasmin + megsin (1:10), ·: Plasmin + inactivated megsin (1:10), △: Ovalbumin, ▲: anolepmin.

FIG. 3 is a diagram showing the results of SELDI-TOF mass spectrometry. The results of analysis of only plasmin, only megsin, and a mixture of megsin and plasmin from the top are shown. BEST MODE FOR CARRYING OUT THE INVENTION

(Example 1)

(Recombinant megsin)

Recombinant human megsin was purified by a known method [R. Inagi, et al., Biochem. Biophys. Res. Commun., 286, 1098-1106 (2001)]. That is, megsin was obtained from the culture supernatant of Chinese hamster ovary (CHO) cells transfected with megsin transfected. C-myc tag and histidine tag were ligated to the N-terminal side of the full length coding region of megsin cDNA by mutagenesis using PCR. Tag-tagged megsin cDNA was cloned into the mammalian expression vector pREP9 (Invitrogen, Belgium, The Netherlands). In 5% C0 ₂ incubator CHO cells cultured in the beta one 3 7 ° C plated on 6 © El plates were transformed with the Lipofecta mine reagent (GIBCO) and pREP9 containing Hitomegushin cDNA. Stable transformants were selected using 0.5 mg / mL geneticin disulfate (Wako Pure Chemical Industries). Then, a C-myc-histidine tag-conjugated megsin recombinant was purified from the culture supernatant using a (His) 6 abundity column.

(Experiment 1. Megsin-serine protease complex formation test)

The P17-P8 sequence of megsin (EGTEATAAT) is conserved as a consensus sequence for inhibitory SERPIN [T. Miyata, J. CUn. Invest., 102, 828-836 (1998)]. Therefore, the activity of megsin on serine proteases by in vitro complex formation was evaluated. Plasmin, kallikrein, elastase, trypsin (Sigma, St Louis, MO), tissue plasminogen activator (t-PA: Biopool, Ventu) are used as serine proteases to test complex formation with megsin. ra, CA) and thrombin (Calbiochem, La Jolla, CA) were used. Further, as a serine protease inhibitor that comparison, antithrombin ΙΠ (ΑΤΠΙ), od- antitrypsin _{(α 1-ΑΤ), a} 2- antiplasmin (ο! 2-ΑΡ), and positive Minogenakuchi base one coater inhibitor l (PAI -l) was used.

The purified megsin obtained above was combined with a 2: 1 molar ratio of serine protease in phosphate buffered saline (pH 7.4), respectively. Incubate with C for 30 minutes, perform SDS-PAGE analysis under non-reducing conditions, stain with Coomassie brilliant blue (CBB), or Imno-plot analysis using anti-human t-PA antibody (Cedarlane, Ontario, Canada) (For t-PA).

The reaction mixture was also applied to a reverse phase chip (H4 Protein Chip ™: Ciphergen Bio systems, Fremont, CA) and air dried. After the chip was reacted with sinapinic acid, mass spectrometry was performed using a surface-modified / time-of-flight mass spectrometer (SELDI-TOF-MASS) (Ciphergen SELDI Protein Biology System II).

(Experiment 2. MCA Atsushi)

To assess the inhibitory activity of megsin on plasmin activity, purified megsin was incubated with various molar ratios of plasmin in 0.2 M Tris-HCl (pH 8.0) at 37 ° C for 30 minutes, and ImM was incubated with ImM. Reaction with a synthetic fluorescent plasmin substrate, Boc (t-butyloxycarbonyl) -Glu-Lys-Lys-MCA (4-methyl-coumaryl-7-amid) (Peptide Institute, Osaka, Omoto), then lex = 380nm and At em = 460 mn, fluorescence measurements of peptide-MCA to AMC cleavage were performed. Antiplasmin (Sigma) was used as a control.

For the preparation of inactivated megsin, 0.25M trisodium citrate and 10m The purified megsin was incubated in M Tris-HCl (pH 7.4) at 60 ° C for 15 hours.

(Experimental results 1. Complex formation test)

In SDS-PAGE, when plasmin or trypsin was mixed with the recombinant megsin, a new band appeared, indicating the formation of a complex. However, no other band was found for other serine proteases (Figure 1, Table 1). In the table,

◎ 〇 X indicates the depth of the band in the order of X, and X indicates that no band was observed.

The binding of megsin to plasmin was also confirmed by SELDI-TOF mass spectrometry. That is, in the reaction mixture of megsin and plasmin, the peak was identified at the molecular weight of 120,163.7 Da, which coincided with the sum of megsin (47,061.4 Da) and plasmin (73,264.7 Da), and was estimated to be a megsin-plasmin complex. (Figure 3). Complex formation was not observed when recombinant megsin was inactivated by heat (not shown).

(Experimental results 2. MCA Atsushi)

In addition, FIG. 2 and Table 2 show the inhibitory effect of megsin on the enzyme activity of plasmin using MCA Atsee. In the table, {Δ} indicates the strength of the inhibition in order, and X indicates that no inhibition was observed. Ovalbumin, especially as evident from Figure 2.

(△) and albumin (▲) did not inhibit plasmin activity, whereas megsin (〇) showed specific plasmin inhibitory activity. In addition, inactivated megsin (Oka) did not show plasmin inhibitory activity. -These results suggest that megsin and plasmin form a complex (

Table 2

Industrial applicability

The present invention provides a pharmaceutical composition for treating and ^/ or preventing mesangial proliferative glomerulonephritis. The present invention also provides a method for screening the ability of a compound to inhibit the binding of megsin to megsin ligand, wherein the test compound and the megsin ligand form a complex with the test compound and the megsin ligand. The above method is provided by the present invention, comprising the step of contacting under conditions.

Claims

The scope of the claims

A pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis, comprising a serine protease or a protein functionally equivalent to serine protease as an active ingredient.

A method for evaluating the activity of a test compound that interferes with the binding of megsin to a megsin ligand, comprising the following steps, wherein the megsin ligand is serine protease or a fragment thereof.

1) a step of contacting megsin, a megsin ligand, and a testide conjugate in any order described in a) -d) below under conditions in which megsin and the megsin ligand form a complex. ,

b) Contacting test compound with megsin in the presence of megsin ligand

c) after contacting the megsin ligand and megsin, further contacting the test conjugate; or

4. The method according to claim 3, wherein the serine protease is plasmin or trypsin.

The effect of a test compound on the formation of a complex between megsin and a megsin ligand is evaluated by the method according to claim 3, and the effect of the test compound on the formation of a complex in the absence of the test compound is evaluated. A Starry Jung method for a compound having an activity of inhibiting the formation of a complex between megsin and a megsin ligand, comprising the step of comparing and selecting a compound that inhibits the formation of a complex.

A method for inhibiting megsin activity, comprising a step of bringing a compound obtainable by the screening method according to claim 5 into contact with megsin.

A megsin activity inhibitor comprising a compound obtainable by the screening method according to claim 5.

A pharmaceutical composition for treating and / or preventing mesangial proliferative glomerulonephritis, comprising a compound obtainable by the screening method according to claim 5 as an active ingredient.

An inhibitor of plasmin and Z or trypsin activity, comprising megsin or a protein functionally equivalent to megsin as an active ingredient.

A method for inhibiting plasmin and / or trypsin activity, comprising the step of contacting megsin or a protein functionally equivalent to megsin with plasmin or trypsin.