JP5803267B2 - Antibacterial agent containing S-nitrosated α1-acid glycoprotein - Google Patents

Description

The present invention relates to an antibacterial agent containing S-nitrosated α ₁ -acid glycoprotein as an active ingredient.

α ₁ -acid glycoprotein is a serum protein also called orosomucoid having a molecular weight of about 44 kDa, consisting of 183 amino acids and 5 sugar chains. The sugar chain content of α ₁ -acid glycoprotein is 45% of the molecular weight, which is extremely high among other proteins, and since it has a sialic acid residue at the end of the sugar chain, it is most negatively charged in serum. (PI value: 2.7). The serum concentration of α ₁ -acid glycoprotein is 0.5 to 1.0 g / L in normal, but increases about 2 to 4 times in blood and tissues during infection, inflammation, etc. It is thought to function as an anti-inflammatory and immunomodulatory molecule. However, the detailed mechanism of action still remains unclear. In addition, in this α ₁ -acid glycoprotein, two variants of “F1 ^* S form” and “A form” are present in the living body at a ratio of about 2 to 3: 1. One of the differences in the amino acid sequence between the “F1 ^* S form” and the “A form” is the 149th free cysteine (Cys-149) residue that only the A form has. However, reports on the functional differences are limited to ligand binding ability.

Nitric oxide (NO) becomes peroxynitrite (ONOO ⁻ ) and exhibits a bactericidal action. On the other hand, when one electron is oxidized to NO ⁺ , residual molecules such as glutathione (GSH) and protein cysteine remain. It reacts with the group and is converted to chemically stable S-nitrosothiol (RS-NO). So far, the present inventors have reported that S-nitrosated form (SNO-HSA) such as human serum albumin (HSA) is NO itself or S-nitrosoglutathione (GS-NO) which is a low molecular RS-NO. It has already been clarified that it exhibits antibacterial activity several hundred times stronger than that (Patent Document 1).

Japanese Patent Laid-Open No. 2005-206577

  An object of the present invention is to provide a safe antibacterial agent having high antibacterial activity and having an effect on various bacteria.

The present invention provides an antibacterial agent containing S-nitrosated α ₁ -acid glycoprotein or a variant thereof as an active ingredient.

In one embodiment, the S-nitrosated α ₁ -acid glycoprotein is S-nitrosated α ₁ -acid glycoprotein variant A.

In one embodiment, the S- nitrosated alpha ₁ - acid glycoprotein variant A body, 149 cysteine residue is nitrosated S- nitrosation alpha ₁ - is an acidic glycoprotein variant A body.

In one embodiment, the S- nitrosated alpha ₁ - acid glycoprotein, at least one sialic acid has been removed S- nitrosated alpha ₁ sugar chain terminal - is an acidic glycoprotein.

In one embodiment, the S-nitrosated α ₁ -acid glycoprotein is S-nitrosated asialo α ₁ -acid glycoprotein.

The present invention is also an S-nitrosated α ₁ -acid glycoprotein variant A or a variant thereof from which at least one sialic acid at the sugar chain end has been removed.

In one embodiment, at least one sialic acid at the end of the sugar chain obtained by removing sialic acid with neuraminidase from S-nitrosated α ₁ -acid glycoprotein variant A or a variant thereof is removed. S-nitrosated α ₁ -acid glycoprotein variant A or a variant thereof.

In one embodiment, the S-nitrosated α ₁ -acid glycoprotein variant A from which at least one sialic acid at the sugar chain end has been removed has a cysteine residue at position 149 S-nitrosated. Yes.

The antibacterial agent of the present invention has strong antibacterial activity due to the efficient transport of nitric oxide (NO) into bacteria by S-nitrosated α ₁ -acid glycoprotein which is an active ingredient. It is possible to provide a safer drug than being an in vivo substance. The S-nitrosated α ₁ -acid glycoprotein of the present invention can be stored by lyophilization because the S-nitroso group is stably present after lyophilization. The S-nitrosated α ₁ -acid glycoprotein from which sialic acid of the present invention has been removed has particularly strong antibacterial activity.

It is process drawing which shows the manufacturing method of S-nitrosation alpha ₁ -acid glycoprotein variant A body. It is explanatory drawing of the NO addition rate determination method of S-nitrosated protein by HPLC. It is a figure which shows the structure of the N-linked sugar chain of α ₁ -acid glycoprotein. It is a graph which shows the antibacterial activity of S-nitrosated alpha ₁ -acid glycoprotein variant A body. It is a graph which shows the antibacterial activity of S-nitrosated alpha ₁ -acid glycoprotein variant A body. It is a graph which shows the antibacterial activity of S-nitrosated asialo α ₁ -acid glycoprotein variant A. It is a graph which shows NO transport efficiency from various S-nitrosated protein into bacteria. It is a graph which shows the relationship between the fluorescence intensity of DAF-FM which is a NO detection probe, and the growth rate of Escherichia coli. It is a figure which shows the modification method of the bacterial cell surface of Escherichia coli, and the measuring method of NO transport amount to the bacterial body by modification. It is a graph which shows the change of the NO transport amount into the microbial cell by modification of the microbial cell surface of Escherichia coli.

The antibacterial agent of the present invention contains S-nitrosated α ₁ -acid glycoprotein or a variant thereof as an active ingredient.

The α ₁ -acid glycoprotein (hereinafter also referred to as AGP) is not particularly limited in its origin. For example, AGP derived from human, monkey, cow, dog, pig, mouse can be mentioned. Preferably, it is AGP derived from human. Human-derived AGP is a serum protein with a molecular weight of 44100, consisting of 183 amino acid residues and 5 sugar chains. There are two variants of human-derived AGP (A-form and F1 ^* S-form). The amino acid sequence of AGP variant A-form (hereinafter also referred to as AGP / A-form) is represented by SEQ ID NO: 1, and variant F1 ^* S The amino acid sequence of the body is shown in SEQ ID NO: 2. In the present invention, the AGP • A form is preferably used. AGP can be obtained from plasma by a known method, or can be obtained by a conventional method using a gene recombination technique.
Sequence number 1: QIPLCANLVPVPITNATLDRITGKWFYIASAFRNEEYNKSVQEIQATFFYFTPNKTEDTIFLREYQTRQNQCFYNSSYLNVQRENGTVSRYEGGREHVAHLLFLRDTKTLMFGSYLDDEKNWGLSFYADKPETTKEQLGEFYEALDCLKPR
SEQ ID NO: 2: QIPLCANLVPVPITNATLDQITGKWFYIASAFRNEEYNKSVQEIQATFFYFTPNKTEDTIFLREYQTRQDQCIYNTTYLNVQRENGTISRYVGGQEHFAHLLILRDTKTYMLAFDVNDEKNWGLSVYADKPETTKEQLGEFKEPLEDCGE

  An S-nitrosated AGP variant is an S-nitrosated AGP variant. The AGP variant is not particularly limited as long as it is an AGP in which an amino acid residue is substituted, deleted, or inserted and has the same activity as AGP. Preferred are variants in which at least one amino acid residue of AGP is replaced with cysteine, or variants in which at least one cysteine has been inserted. Mutants in which 1 to 3 cysteines are substituted or inserted are preferred. The mutant can be obtained by a conventional method using a gene recombination technique.

  The S-nitrosated AGP of the present invention or a variant thereof is an AGP in which an S-nitroso group (—NO) is added to a thiol group of at least one cysteine residue of AGP or a variant thereof to become —SNO or a derivative thereof It is a mutant. The cysteine residue may be a naturally occurring cysteine residue or an inserted or substituted cysteine residue. Preferable examples include S-nitrosated AGP · A form in which the cysteine residue at position 149 of the AGP · A form is nitrosated.

  S-nitrosation can be achieved by a known method such as reaction with nitrite. However, although there are examples in which S-nitrosation of proteins has been successful, such as hemoglobin, in general, S-nitrosation is a reaction under severe conditions for proteins. Therefore, it is preferable to use a method capable of introducing NO into a thiol group under milder conditions, and a method using isoamyl nitrite (De Master EG et al., Biochemistry, 34, p.11494-11499, 1995) and a method of reacting with n-butyl nitrite (Meyer DJ et al., FEBS Letters, 345, p. 177-180, 1994) can be preferably used.

Multiple sugar chains are added to AGP or a variant thereof, and by removing at least one sialic acid at the end of the sugar chain, an AGP or AGP variant from which sialic acid has been removed is obtained. . By removing sialic acid, a new galactose residue appears at the non-reducing end of the sugar chain. By removing a plurality of sialic acids, the isoelectric point of α ₁ -acid glycoprotein increases and the molecular weight decreases.

The asialo α ₁ -acid glycoprotein is one in which sialic acid has been removed by 95% or more from the α ₁ -acid glycoprotein, and preferably all sialic acid has been removed. For example, α ₁ -acid glycoprotein from which 14 sialic acids and 15 sialic acids have been removed from α ₁ -acid glycoprotein can be mentioned. When sialic acid is removed, the same number of galactose residues appear instead at the non-reducing end of the sugar chain.

  Five sugar chains are added to the AGP • A body. Specifically, sugar chains are added to the 15th, 38th, 54th, 75th, and 85th asparagines. These sugar chains are branched and sialic acid is present at the end. By removing at least one sialic acid, an AGP • A body from which sialic acid has been removed can be obtained. 5 or more, more preferably 10 or more, and still more preferably AGP • A form or a variant thereof from which all sialic acid present at the sugar chain terminal has been removed. For example, AGP • A form from which 14 or 15 sialic acids have been removed may be mentioned. The AGP • A form from which 95% or more of the sialic acid present at the sugar chain end has been removed is the asialo AGP · A form.

  The method for removing the sialic acid at the end of the sugar chain is not limited. For example, the removal by an enzyme etc. are mentioned. Preferably it is removed by an enzyme. Examples of the enzyme include neuraminidase.

  The antibacterial agent of the present invention can efficiently transport nitric oxide into bacteria. The nitric oxide transport mechanism is thought to involve an S-nitroso transfer reaction from S-nitrosated AGP to a thiol group on the cell surface. Therefore, the antibacterial agent of the present invention has excellent antibacterial properties against various bacteria, and is extremely useful as a therapeutic agent for various bacterial infections such as Gram-positive bacterial infections and Gram-negative bacterial infections. is there. Examples of the infectious diseases include streptococci (Group A β-streptococci, pneumococci, etc.), Staphylococcus aureus (MSSA, MRSA), Staphylococcus epidermidis, enterococci, Listeria, meningococcus, bacillus, pathogenic E. coli (0157). : H7), Klebsiella (Klebsiella pneumoniae), Proteus, Bordetella pertussis, Pseudomonas aeruginosa, Serratia, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Chlamydia, Clostridium, etc., tuberculosis, cholera, diphtheria, dysentery , Scarlet fever, anthrax, trachoma, syphilis, tetanus, leprosy, legionella, leptospira, Lyme disease, wild goose disease, Q fever and the like.

  The antibacterial agent of the present invention can be made into preparations such as injections, external preparations, and oral preparations. It can also be used in combination with other antibacterial agents. In the case of an injection, it can be made into a liquid or a lyophilized preparation by a conventional method, and can contain a stabilizer such as sucrose.

  The amount of the active ingredient contained in the antibacterial agent of the present invention, that is, the dose having antibacterial activity should be sufficient to exert the desired therapeutic effect on the prevention or disease process and condition. The dose to a patient varies depending on conditions such as the age of the individual patient to be treated and the degree of infection, but generally the dose of 1 mg to 10 g of active ingredient per day is 1 day. 1 to 4 times for prophylaxis or treatment.

The present invention is also referred to as S-nitrosated α ₁ -acid glycoprotein variant A form from which at least one sialic acid at the sugar chain end has been removed (hereinafter also referred to as sialic acid-removed S-nitrosated AGP · A form). ) Or a variant thereof.

  A sialic acid-removed S-nitrosated AGP · A form or a variant thereof means that at least one sialic acid at the end of the sugar chain of AGP · A form or a variant thereof is removed and at least one cysteine residue It is an AGP • A isomer in which the thiol group is S-nitrosated or a variant thereof.

  As described above, the AGP · A isomer is a serum protein having a molecular weight of 44,100 consisting of 183 amino acid residues and 5 sugar chains. The AGP • A body has the amino acid sequence represented by SEQ ID NO: 1.

  That at least one sialic acid at the end of the sugar chain has been removed means that it is added to each of the asparagine residues at positions 15, 38, 54, 75, and 85 of the AGP • A form or a variant thereof. At least one sialic acid at the end of the sugar chain is removed, preferably 5 or more, more preferably 10 or more are removed, and more preferably, the sialic acid present at the end of the sugar chain is removed. More than 95% is removed. For example, 14 or 15 sialic acids have been removed.

  S-nitrosation means that an S-nitroso group (—NO) is added to a thiol group of at least one cysteine residue of the AGP • A isomer or a variant thereof to make -SNO. S-nitrosation may be performed before or after removal of sialic acid. Preferably, the thiol group of cysteine at position 149 is S-nitrosated.

  The AGP / A mutant is an AGP / A mutant in which an amino acid residue is substituted, deleted, or inserted. As for the substitution, deletion, insertion position, number of amino acid residues, etc. of the amino acid residues, the sialic acid-removed S-nitrosated AGP • A mutant is the same as the sialic acid-removed S-nitrosated AGP • A As long as it has strong antibacterial activity, it is not particularly limited.

  The same method as described above can be used for the method for obtaining the AGP • A form, the method for removing sialic acid at the sugar chain end, and the method for S-nitrosation.

  In one embodiment, the sialic acid-removed S-nitrosated AGP • A or a variant thereof is obtained by removing sialic acid from an S-nitrosated AGP • A or a variant thereof by neuraminidase. In another embodiment, the sialic acid-depleted S-nitrosated AGP • A or a variant thereof is obtained by S-nitrosating an AGP • A or a variant thereof from which at least one sialic acid has been removed by neuraminidase. can get. It is obtained by mixing 1 to 100 mg, preferably 20 mg of AGP · A isomer with neuraminidase 0.05 to 0.5 unit, preferably 0.2 unit, and reacting at 37 ° C. for 2 to 48 hours, preferably 24 hours. Sialic acid-removed S-nitrosated AGP · A is preferred.

  A preferred sialic acid-removed S-nitrosated AGP · A form of the present invention is a sialic acid-removed S-nitrosated AGP * A form in which the cysteine residue at position 149 is S-nitrosated. In addition, it is an S-nitrosated asialo AGF · A form in which sialic acid is removed by 95% or more. A more preferable sialic acid-removed S-nitrosated AGP · A form is an S-nitrosated asialo AGF · A form in which the cysteine residue at position 149 is S-nitrosated, and its molecular weight is about 40,000. .

  The sialic acid-removed S-nitrosated AGP • A body or a mutant thereof of the present invention exhibits a strong antibacterial action. This substance can efficiently transport nitric oxide into bacteria. It is considered that the nitric oxide transport mechanism involves an S-nitroso transfer reaction from a sialic acid-removed S-nitrosated AGP • A body to a thiol group on the cell surface. Therefore, the sialic acid-removed S-nitrosated AGP · A body of the present invention has excellent antibacterial properties against various bacteria, and various bacterial infectious diseases such as Gram-positive bacterial infections and Gram-negative bacterial infections. It is extremely useful as a therapeutic agent.

  EXAMPLES Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

(Example 1)
As the AGP fraction separated and purified from human serum, the one provided by General Chemistry and Serum Therapy Research Institute or purchased human serum-derived AGP (G9885, Sigma-Aldrich) was used. Separation and purification of each AGP variant was performed according to the method of Herve et al. That is, after washing 50 mL of iminodiacetate-Sepharose gel (18-8769-01, Chelating Sepharose Fast Flow, GE Healthcare Bioscience) with ion-exchanged water, 0.2 M CuCl ₂ aqueous solution was flowed to chelate Cu ²⁺ ions. Next, excess Cu ²⁺ ions were removed by flowing 0.1 M sodium acetate buffer (pH 3.8). Thereafter, a mobile phase of 20 mM sodium phosphate buffer (pH 7.0) +0.5 M NaCl was stabilized by flowing at a flow rate of 0.4 mL / min, and a sample in which 20 mg of AGP was dissolved in 1 mL of mobile phase was added to the column. . After the addition, F1 ^* S body that eluted without being held for about 1.5 hours was collected. Next, a mobile phase to which imidazole (final concentration 20 mM) was added was passed, and Form A eluted after about 5 hours was collected. Each of the separated variants was dialyzed with ion exchange water and then lyophilized. Furthermore, the AGP variant ratio was calculated from the recovery rates of F1 ^* S and A forms.

  A 3-fold molar amount of 1,4-dithiothreitol (hereinafter referred to as DTT) (100 mM potassium phosphate buffer (pH 7.4) +5 mM EDTA) was added to and mixed with the obtained AGP · A body. Reduction of the SH group of the free Cys residue present was performed. This was subjected to gel filtration, and 10 times the amount of S-nitrosoglutathione (GS-NO) (100 mM potassium phosphate buffer (pH 8.0) +1 mM DTPA) was added to the filtrate, mixed, and S- Nitrosation reaction was performed. The mixture was subjected to gel filtration, and S-nitrosated AGP · A form (hereinafter referred to as SNO-AGP) was obtained in the filtrate (FIG. 1).

The S-nitrosation site of SNO-AGP was analyzed by HPLC flow reactor system. First, AGP variant F1 ^* S body purified from plasma was S-nitrosated as described above. Further, C149R in which cysteine at position 149 of AGP variant A was mutated to arginine was obtained by the method of Nishi et al. (Nishi K, et al., Drug Metab Pharmacokinet, 25, 200-207, 2010). The obtained C149R was S-nitrosated as described above. Each NO addition rate was quantified by the method of Akaike et al. (T. Akaike, et al., J. Biochemistry, 122, 459-466, 1997). That is, in the HPLC flow reactor system shown in FIG. 2, the azo dye, which is a reaction product of NO ₂ ⁻ and Griess reagent, produced by reacting Hg ²⁺ with the above SNO-AGP, F1 ^* S form and C149R at 540 nm. By measuring, various S-nitrosated proteins were separated and quantified. Table 1 shows the NO addition rate of each protein. From the results in Table 1, it was confirmed that the cysteine at position 149 of SNO-AGP was S-nitrosated.

(Example 2)
Asialo AGP · A in which 0.2 unit of neuraminidase was added to 20 mg of AGP · A obtained in Example 1, mixed and reacted at 37 ° C. for 24 hours to remove 95% or more of sialic acid at the sugar chain end. Got the body. The obtained asialo AGP · A form was S-nitrosated according to Example 1 to obtain an S-nitrosated asialo AGP · A form (hereinafter referred to as SNO-asialo-AGP) (FIG. 3). In the obtained SNO-asialo-AGP, 95% or more of sialic acid was removed, and the molecular weight was about 40,000.

Example 3
The SNO-AGP solution obtained by gel filtration in Example 1 was freeze-dried by a conventional method to obtain a freeze-dried preparation. Moreover, when the stability of SNO-AGP in liquid form at pH 7 and pH 5 was evaluated, it was confirmed that pH 7 had a half-life of 13 days and pH 5 had a half-life of 24 days, which was very stable. .

Example 4
The SNO-asialo-AGP solution obtained in Example 2 was freeze-dried by a conventional method to obtain a freeze-dried preparation.

(Comparative Example 1)
S-nitrosated human serum albumin (hereinafter referred to as SNO-HSA) was obtained by the method described in JP-A-2005-206657.

(Test Example 1)
Gram-negative bacteria, E. coli, S. typhimurium LT2, P. aeruginosa, and Gram-positive bacteria, B. subtilis, pyogenic hemolytic The antibacterial activity of SNO-AGP was examined using S. pyogenes.

Each strain was cultured overnight in M9 medium (64 g Na ₂ HPO ₄ , 15 g KH ₂ PO ₄ , NaCl 2.5 g, NH ₄ Cl 5 g / L) supplemented with 20 wt% glucose and three times with M9. After washing, the number of bacteria was adjusted to OD ₆₃₀ = 0.05 ± 0.01 in M9 medium. To this medium, SNO-AGP obtained in Example 1, SNO-HSA obtained in Comparative Example, and phosphate buffer (PBS) as a control were added and allowed to stand at 37 ° C. for 9 hours, and then OD ₆₃₀ was added. It was measured. The growth rate was calculated by comparing this value with the control group.

The growth inhibition rate of SNO-AGP and SNO-HSA for E. coli is shown in FIG. Further, 50% inhibitory activity concentration (IC ₅₀ ) against various bacteria is shown in Table 2 and FIG. As is clear from FIG. 5, SNO-AGP inhibited the growth of bacteria at an extremely low concentration. The antibacterial activity of SNO-AGP was the strongest among the known S-nitrosated proteins.

(Test Example 2)
The test was conducted in the same manner as in Test Example 1 except that SNO-asialo-AGP obtained in Example 2 was used instead of SNO-AGP. The results are shown in FIG. The 50% inhibitory activity concentration (IC ₅₀ ) was 65 nM with SNO-AGP and 800 pM with SNO-asialo-AGP. It was revealed that SNO-asialo-AGP has an antibacterial activity 80 times that of SNO-AGP, inhibits the growth of bacteria at a very low concentration, and has a very high antibacterial activity.

(Test Example 3)
The NO transport efficiency from SNO-AGP, SNO-asialo-AGP and SNO-HSA into bacteria was examined.

  SNO-AGP, SNO-asialo-AGP, and SNO-HSA were added to the E. coli culture solution, and E. coli was cultured at 37 ° C. for 7 hours. After addition of intracellular fluorescein-FM diacetate (DAF-FM DA), which is an intracellular NO detection probe, the cells are further cultured for 1 hour, and then fluorescence that is an indicator of NO in the E. coli body is measured at excitation wavelength: 485 nm and measurement wavelength: 535 nm. did. The results are shown in FIG. 7 and FIG. The antibacterial activity of SNO-AGP, SNO-asialo-AGP and SNO-HSA was dependent on the ability to transport NO into bacteria.

(Test Example 4)
The mechanism of NO transport from SNO-AGP to bacteria was examined. The thiol group on the cell surface of Escherichia coli was modified with N-ethylmaleimide (NEM) to examine whether a change in the amount of NO taken into the cell occurred. The method is shown in FIG. The results are shown in FIG. As is clear from FIG. 10, the intracellular transport of NO was inhibited in E. coli in which the thiol group on the cell surface was modified with NEM. Therefore, it was found that the S-nitroso transfer reaction from SNO-AGP to the thiol group on the cell surface is involved in the NO transport mechanism of SNO-AGP.

  The antibacterial agent of the present invention and the S-nitrosated AGP A-form from which the sialic acid of the present invention has been removed or a variant thereof has a very high antibacterial activity and has a wide antibacterial spectrum, thereby treating various infectious diseases. It can contribute to the medical field as a medicine.

Claims (4)

A antimicrobial agent containing an acidic glycoprotein as an active ingredient, the S- nitrosated alpha ₁ - - S- nitrosated alpha ₁ acid glycoprotein, at least one sialic acid sugar chain termini are removed An antibacterial agent which is S-nitrosated α ₁ -acid glycoprotein . The antibacterial agent according to claim 1, wherein the S-nitrosated α ₁ -acid glycoprotein is S-nitrosated α ₁ -acid glycoprotein variant A. The S- nitrosated alpha ₁ - acid glycoprotein variant A body, 149 cysteine residue is nitrosated S- nitrosation alpha ₁ - antimicrobial according to claim 2 is an acidic glycoprotein variant A body Agent. The antibacterial agent according to any one of claims 1 to 3, wherein the S-nitrosated α ₁ -acid glycoprotein is S-nitrosated asialo α ₁ -acid glycoprotein.

Images