JP2007254422A - Peptide expressing interferon-like biological activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new low molecular weight peptide expressing interferon-like biological activity at a high level and usable as medicines including low molecular weight viral disease treating agents. <P>SOLUTION: The peptide contains a specific sequence of 21 amino acid residues or an amino acid sequence obtained by the deletion, substitution or addition of one or more amino acids in the above peptide and expresses interferon-like biological activity. These new peptides have high interferon activity compared with conventional peptides such as Syr6 and are expected to be applicable as medicines. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a peptide that expresses an interferon-like biological activity, and particularly relates to a novel peptide that can significantly express an interferon-like biological activity.

  The growth and differentiation of animal cells are carried out through intercellular signal transduction molecules called cytokines such as so-called growth factors, lymphokines, monokines, interferons, chemokines, hematopoietic factors, neurotrophic factors, and so on, through specific receptors on the cell surface. It is controlled by showing biological activity. For example, antiviral activity and antitumor activity of interferon, formation of granulocyte colony of granulocyte colony stimulating factor (G-CSF), production of erythropoietin (EPO) erythrocytes, proliferation of megakaryocytes of thrombopoietin (TPO), etc. Many of them are used to treat actual diseases.

  Of these, interferons are mainly classified into three types, interferon-α, interferon-β, and interferon-γ, due to differences in antigenicity, and the presence of more than 20 subtypes of interferon-α has been confirmed. . On the other hand, interferon is classified into type I and type II according to the binding mode to the receptor, interferon-α and interferon-β are classified as type I interferon, and interferon-γ is classified as type II interferon. Furthermore, both interferon-α and interferon-β are believed to bind to the same receptor. Two subtypes, AR-1 and AR-2, having different affinity for interferons are known as type I interferon receptors. In the former, the primary structure has been clarified, but the higher order structure has not been clarified. The higher order structure of the latter has been clarified by NMR (see Non-Patent Document 4, protein data bank registration number: 1 nu6). In addition, the three-dimensional structure coordinates of human interferon-β have already been clarified (see Non-Patent Document 5, protein data bank registration number: 1au1).

  Cytokine production is performed by culturing a large amount of natural or genetically modified cells and microorganisms having cytokine-producing ability. However, in this case, in order to obtain a cytokine of a single molecular species, a complicated operation of further purification from the culture supernatant or disrupted solution of cells or microorganisms is required. Furthermore, since the cytokine obtained in this way is a high molecular weight protein, it must be administered intravenously, intramuscularly or subcutaneously as a protein preparation, and is generally difficult to do by the patient himself / herself. It was necessary by medical personnel such as doctors. There is a desire for small molecule agonists with possible cytokine activity such as simpler administration methods instead of injections, such as nasal administration, rectal administration, or topical administration.

  The attempt includes, for example, a method of mimicking cytokine activity with a peptide having a small molecular weight. A low molecular weight peptide can be prepared by chemical synthesis and has excellent stability. As such an example, there is a report on preparation of a low molecular peptide that mimics EPO (see Non-Patent Document 1) and TPO (see Non-Patent Documents 2 and 3) using a phage peptide library method.

However, since interferon-β is a high molecular protein consisting of 166 amino acid residues, it is still not easy to design a non-peptide low molecular weight compound directly from this three-dimensional structure. A general understanding of low molecular weight has not been obtained. For this reason, it has been considered impossible to design small molecule agonists and antagonists of interferon. Thereafter, obtaining a peptide that mimics the interferon activity by selecting a sequence that binds to the neutralizing antibody against the cytokine from a random amino acid sequence was examined. As a result, a peptide of 15 amino acid residues Syr6 (sequence of the sequence listing of the present application) 13 peptides (see Patent Document 1) including the number 12 have been acquired. For Syr6, three-dimensional structural coordinates and three-dimensional structural motifs have also been estimated (see Patent Document 2).
However, since Syr6 has insufficient interferon activity, it has been practically difficult to apply it to pharmaceuticals such as viral disease therapeutic agents.

Wrightton, N .; C. Science, 273: 458-463 (1996). Cwirla, S.M. E. Science, 276: 1696-1699 (1997). Kimura, T .; J. et al. Biochem. 122: 1046-1051 (1997). Anglister et al., Structure v11 pp. 791-802 (2003) Karpusas, M .; Et al., Proc. Natl. Acad. Sci. USA, 94: 11813-11818 (1997). International Publication No. WO01 / 048478 Pamphlet JP 2005-29471 A

  An object of this invention is to provide the peptide which expresses interferon-like biological activity notably and can be applied to pharmaceuticals, such as a viral disease therapeutic agent.

  As a result of intensive studies to solve the above problems, the present inventors have created a new peptide that has higher interferon activity than that of a conventional peptide, such as Syr6, and is expected to be applied to pharmaceuticals. The first success. That is, the present invention is as follows.

[1] A peptide shown in the following (A) or (B).
(A) a peptide comprising the amino acid sequence described in SEQ ID NO: 1 (B) an amino acid sequence described in SEQ ID NO: 1, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and interferon-like biology [2] The peptide according to [1], which comprises the amino acid sequence according to any one of SEQ ID NOs: 2 to 11.

  INDUSTRIAL APPLICABILITY According to the present invention, a novel low molecular weight peptide that can highly express interferon-like biological activity can be provided. Moreover, since the said peptide can express interferon-like biological activity higher than the conventionally reported peptide, the application to the therapeutic agent of a low molecular viral disease which was difficult with the prior art can be expected.

  In the present specification, amino acids and peptides are represented by abbreviations adopted by the IUPAC-IUB Biochemical Nomenclature Committee (CBN) shown below. Unless otherwise specified, the sequence of amino acid residues of a peptide is represented such that the N-terminal to C-terminal is from the left end to the right end, and the N-terminal amino acid is number 1.

  A or Ala: alanine residue, D or Asp: aspartic acid residue, E or Glu: glutamic acid residue, F or Phe: phenylalanine residue, G or Gly: glycine residue, H or His: histidine residue, I Or Ile: isoleucine residue, K or Lys: lysine residue, L or Leu: leucine residue, M or Met: methionine residue, N or Asn: asparagine residue, P or Pro: proline residue, Q or Gln : Glutamine residue, R or Arg: Arginine residue, S or Ser: Serine residue, T or Thr: Threonine residue, V or Val: Valine residue, W or Trp: Tryptophan residue, Y or Tyr: Tyrosine Residue, C or Cys: Cysteine residue.

Embodiments of the present invention are described below, but the present invention is not limited thereto.
(1) Peptide The peptide of the present invention is a peptide that expresses an interferon-like biological activity. Specifically, first, (A) a peptide comprising the amino acid sequence described in SEQ ID NO: 1 (hereinafter referred to as peptide (A)) .).
In the amino acid sequence described in SEQ ID NO: 1 (KRRSVQARWX ₁ AAFX ₂ LX ₃ LYRRR), the 10th amino acid residue (X ₁ ) is an amino acid residue excluding glutamic acid (E). Of these, glutamine (Q) or alanine (A) is preferable. The 14th and 16th amino acid residues (X ₂ and X _3, respectively) are amino acid residues excluding the aspartic acid residue (D). Of these, asparagine (N) and alanine (A) are preferable. X ₁ , X ₂ and X ₃ may be the same base or different bases. As such an amino acid sequence described in SEQ ID NO: 1, the amino acid sequences described in SEQ ID NOS: 2, 7, and 8 are most preferable.

  The peptide (A) may be any peptide that includes the amino acid sequence described in SEQ ID NO: 1 as a part thereof, but a peptide consisting of the amino acid sequence described in SEQ ID NO: 1 is most preferable. Specific examples of such a peptide (A) include peptides (KR2QNNNR3, KR2QNNNR3-A11, and KR2QNNNR3-A13 of Example 1) having the amino acid sequences described in SEQ ID NOs: 2, 7, and 8 in the Sequence Listing. it can.

On the other hand, the peptide of the present invention includes cases in which amino acid mutations other than X ₁ , X ₂ and X _{3 in} the amino acid sequence described above are included on the condition that the interferon-like biological activity is expressed. That is, (B) a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 and expressing an interferon-like biological activity (hereinafter referred to as peptide (B ) Is included in the scope of the present invention. The amino acid sequence described in SEQ ID NO: 1 is as described in the description of the peptide (A) above.

  The number of amino acids substituted, deleted, or added in the amino acid sequence described in SEQ ID NO: 1 may be one or more, and it is determined by the position and type of amino acid residues in the three-dimensional structure of the peptide. Can do. Specifically, the interferon-like biological activity can be made within a range that does not significantly impair, preferably 12 or less, more preferably 9 or less, more preferably 5 or less, particularly 3 or less. It is desirable that

  The site of the amino acid to be substituted, deleted or added is not particularly limited. Preferred sites for substitution include the first, second, third, fourth, sixth, eighth and eighteenth amino acids. In the case of deletion, the 19th, 20th and 21st amino acids can be mentioned. In the case of addition, addition of the first amino acid to the N-terminal side is preferable.

  In the case of substitution or addition, the type of amino acid after substitution or addition is not particularly limited. The amino acid after substitution is preferably alanine, histidine and arginine, and in the case of substitution at only one site, alanine is preferred. The amino acid after addition is preferably histidine and arginine.

Peptide (B) is an amino acid sequence in which one type of mutation selected from the above substitutions, deletions and additions, or a combination of two or more types of mutations is inserted at any position of the amino acid sequence described in SEQ ID NO: 2 In particular, those having only substitution or a combination of substitution, deletion and addition are preferred. Examples of the amino acid sequence into which substitution is inserted include the amino acid sequences described in SEQ ID NOs: 3 to 6 and 9 to 11, respectively. Examples of the amino acid sequence having a combination of substitution, deletion and addition include the amino acid sequences described in SEQ ID NOs: 10 and 11.
The peptide (B) may be any peptide that includes at least a part of the amino acid sequence containing the mutation as described above, but a peptide consisting of an amino acid sequence containing the mutation is most preferable. Specific examples of such peptide (A) include peptides having the amino acid sequences described in SEQ ID NOs: 3 to 6 and 9 to 11 in the sequence listing (KR2QNNNR3-A1, KR2QNNNR3-A3, KR2QNNNR3- of Example 1). A5, KR2QNNNR3-A7, KR2QNNNR3-A15, HisQNN, and R5QNN).

The peptide (B) further needs to express an interferon-like biological activity (interferon-like biological activity). Interferon-like biological activity means an activity similar to that of interferon. Here, interferon is meant to include all interferons regardless of antigenicity or receptor binding mode, and includes, for example, all of interferon-α, β, γ, type I interferon, type II interferon, and the like. Such biological activities of interferon include binding activity with interferon receptors (such as AR-1 and AR-2), activity inhibiting interferon binding to interferon receptors, and antiviral activity.
The typical peptide (B) described above is preferably a peptide comprising the amino acid sequence represented by any of SEQ ID NOs: 4 to 11, and particularly preferably the amino acid represented by any of SEQ ID NOs: 4 to 11. A peptide consisting of a peptide consisting of a sequence can be mentioned.
In Example 5 described later, a peptide comprising the sequence shown in SEQ ID NO: 2 to 11 in the sequence listing as a specific example of the peptide (A) and peptide (B) of the present invention described above has interferon-like biological activity. Among them, it was shown to express antiviral activity in particular. In particular, the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 has also been proved to exhibit binding activity with the interferon receptor and activity that inhibits binding of interferon to the interferon receptor in Examples 2 to 4 described later. ing.

(2) Peptide Synthesis The peptides of the present invention can be produced according to methods generally known to those skilled in the art of peptide synthesis. In particular, solid phase synthesis methods in peptide synthesis (for example, RB Merrifield et al., J. Am. Chem. Soc., 85, 2149 (1963) can be mentioned). In the synthesis, an auto peptide synthesizer (Model 433A, manufactured by Applied Biosystems) can be used. Even if the target peptide is a peptide having amino acid substitution / deletion / addition, it can be synthesized by the following method. The synthesis procedure is performed according to a commercial manual. Although the synthesis method is illustrated below, this method is illustrated and is not limited thereto.

  In the synthesis, protecting groups, reagents and resins commonly known to those skilled in the field of peptide synthesis described above can be used. As the protecting group, a tert-butyloxycarbonyl (t-Boc) group or a 9-fluorenylmethyloxycarbonyl (Fmoc) group can be used, and an Fmoc group is preferred. As coupling reagents, O-benzotriazole-N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HBTU), dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), symmetric mixed acid An anhydride method or the like can be used, and HBTU is preferable. As the solid phase resin, HMP resin, amide resin, MAP resin, etc. can be used when Fmoc group is used, and PAM resin, MBHA resin, etc. are used when Boc group is used. can do.

  When an autopeptide synthesizer is used as the apparatus, the target peptide can be produced by sequentially binding from the C-terminal amino acid. The synthesis scale can be 0.1 mmol to 2.0 mmol of each peptide, preferably 1.0 mmol. After synthesis, the peptide is cleaved from the resin. At this time, when the Fmoc group is used as the protecting group, trifluoroacetic acid (TFA) or trimethylsilyl bromide (TMSBr) can be used, whereas when the Boc group is used, hydrofluoric acid. (HF), trifluoromethanesulfonic acid (TFMSA) or trimethylsilyl trifluoromethanesulfonate (TMSOTF) can be used.

By further separating and purifying the obtained peptide, a highly purified one can be used. Separation and purification can be performed using various types of chromatography. For example, the peptide can be lyophilized and then diluted with water to prepare an aqueous solution, which can be performed by high performance liquid chromatography (hereinafter abbreviated as HPLC). The separation and purification by HPLC is preferably performed by reverse phase liquid chromatography. As an eluent, use a mixed solution of water (which may contain an organic acid such as trifluoroacetic acid, for example) and an organic solvent (which is preferably an organic solvent miscible with water, and acetonitrile is particularly preferred). Can do. Next, whether or not a highly pure peptide has been obtained can be confirmed as follows. First, the molecular weight of the peptide contained in the collected peak is measured using a mass spectrometer. Then, fractions containing peptides that match the expected molecular weight estimated from the structural formula are collected. Finally, the purity of the obtained peptide is measured by an HPLC method using a plurality of elution systems, and it is confirmed that the obtained peptide is a single one.
The method for artificially synthesizing a peptide has been described above, but it can also be based on a method of expressing it in various microorganisms, cells, etc. using a gene recombination technique.

(3) Measurement of intermolecular interaction between peptide and interferon receptor The proof that the peptide of the present invention has interferon-like biological activity can be obtained by examining the presence or absence of intermolecular interaction with the interferon receptor. . Various interferon receptors can be used as the interferon receptor, and typical examples include type I interferon receptors such as interferon-α and interferon-β. There are two types of subtypes with different affinity, AR-1 (AR1) and AR-2 (AR2), which can be used for type I interferon receptors. Further, a solubilized receptor in the extracellular region can be used instead of AR-1 and AR-2. Solubilized interferon receptor is prepared as follows.

Total RNA is extracted from 2 × 10 ⁶ human FL cells using Isogen (manufactured by Nippon Gene). The gene in the extracellular region of the interferon receptor AR1 and AR2 chains is isolated by PCR using 1 μg of total RNA and the following primers.

AR1 sense 5'-ggg gaa ttc gta act ggt ggg atc tgc ggc-3 '(SEQ ID NO: 13)
AR1 antisense 5'-ccc gga tcc tta gag gta ttt cct ggt tt-3 '(SEQ ID NO: 14)
AR2 sense 5′-ggg gaa ttc gag aag act cta aaa ata gc-3 ′ (SEQ ID NO: 15)
AR2 antisense 5'-ccc gga tcc ttg gca gat tct gct gat tc-3 '(SEQ ID NO: 16)

The primer pair AR1 is amino acid residues 1 to 436 (Uze, G. et al., Cell, 60, 225-234 (1990)), and the primer pair AR2 is amino acid residues 1 to 243 (Domanski, P. et. al., J. Biol. Chem., 270, 21606-21611 (1995)). Each obtained PCR fragment is ligated to the EcoRI and BamHI sites of the vector HuIgG1 / SRα for expressing a fusion protein with the human IgG1 Fc region. The constructed expression vector is co-transfected into COS-1 cells using the DEAE-Dextran method to transiently express the protein. The soluble interferon receptor produced in the culture solution is purified using a Protein A Sepharose CL-4B column (manufactured by Amersham Pharmacia Biotech). Thus, solubilizing interferon receptor (AR1) ₂ Fc and (AR2) ₂ Fc is obtained.

The intermolecular interaction between the peptide and (AR1) ₂ Fc and (AR2) ₂ Fc can be measured using a BIAcore system using a surface plasmon resonance (hereinafter, SPR) method. Here, the BIAcore system is a measuring device that can detect the degree of interaction between substances, and the change in the refractive index of the solution due to the interaction between the substances in the solution is a change in the intensity of reflected light. As a device to detect as. The surface plasmon resonance (hereinafter referred to as SPR) method is a phenomenon in which the surface plasmon wave, which is an excited state of a metal surface (specifically, a sensor chip) is excited by resonance with light, and the refractive index of the metal surface is changed. In order to respond with high sensitivity, it is a detection method of the BIAcore system that is used to observe minute changes in refractive index caused by specific adsorption of biopolymers such as DNA and proteins to the metal surface. The measurement using the BIAcore system using the SPR method enables reaction between biomolecules, measurement of binding amount, and kinetic analysis in a non-labeled and real-time manner.

  As a specific example of the measurement method, a case where the measurement is performed according to a measurement manual attached to the measurement apparatus will be described below. First, the functional group on the sensor chip is activated. Next, a solubilized interferon receptor is prepared using an appropriate buffer (for example, acetate buffer). The solubilized interferon receptor solution is spotted and immobilized on an activated chip. As the control solution, one that does not immobilize the solubilized interferon receptor solution is used. The active group of the chip after completion of the reaction is blocked. Next, a sample of an appropriate concentration (C) of a synthetic peptide is prepared using an appropriate running buffer (for example, a HEPES buffer containing Tween 20, NaCl). Then, the synthetic peptide sample is added to the chip on which the solubilized interferon receptor is immobilized. The concentration of the sample is increased stepwise, and the signal (Req) (binding amount: RU) at equilibrium at each concentration is followed over time. Finally, a Scatchard plot of Req / C and Req is performed on the obtained concentration-dependent signal, and a dissociation constant (KD) is calculated from the slope of the straight line. From the obtained results, it is confirmed that the synthetic peptide of the present invention binds to the solubilized interferon receptor.

(4) Measurement of inhibitory action of binding between interferon-β and interferon receptor by peptide The fact that the peptide of the present invention has interferon-like biological activity is examined for the presence or absence of inhibitory action on the binding between interferon and interferon receptor. Can be proved by. The interferon receptor is as described in the above item (3). Various interferons can be used as the interferon, but it is necessary to use an interferon corresponding to the receptor.
Such measurement of inhibitory activity can be performed using a BIAcore system using a surface plasmon resonance (hereinafter, SPR) method. As a specific example of the measurement method, a case where the measurement is performed according to a measurement manual attached to the measurement apparatus will be described below. First, the functional group on the sensor chip is activated. Next, interferon such as interferon-β (for example, Feron (manufactured by Toray Industries, Inc.)) is prepared using an appropriate buffer (for example, acetate buffer). The interferon-β solution is spotted and immobilized on an activated chip. A control solution that does not immobilize the interferon-β solution is used. The active group of the chip after completion of the reaction is blocked. Next, a sample in which an appropriate concentration ratio of the peptide and the solubilized interferon receptor (AR2) ₂ Fc or (AR1) ₂ Fc is mixed is used with an appropriate running buffer (eg, TEP20, HEPES buffer containing NaCl). Prepare. Then, the mixed sample is added to a chip on which interferon-β is immobilized. The concentration ratio of peptide / solubilized receptor in the mixed sample is changed stepwise, and the signal (Req) (binding amount: RU) at equilibrium at each concentration ratio is followed over time. The obtained results are expressed as a ratio (control ratio) to the value of Req when only the interferon-solubilized receptor containing no peptide is added to the chip. Here, the decrease in the ratio means that the binding of interferon-β and the solubilized interferon receptor was inhibited by the peptide of the present invention.

(5) Measurement of Antiviral Activity of Peptide The interferon activity of the peptide of the present invention, that is, the antiviral activity, was measured using a bioassay method (Armstrong, JA, A.) using FL cells, which are human amniotic cells, and Sindbis virus. , Methods Enzymology, 78, 381-387 (1981)).

The method for measuring antiviral activity will be described below with an example. First, in a well of a 96-well microtiter plate (Iwaki Glass Co., Ltd.), 4.0 × 10 ⁴ / well of FL cells are seeded and cultured for 24 hours. Thereafter, the peptide is added in an appropriate dilution series and cultured for 24 hours. As a control, interferon such as interferon-β (for example, Feron (manufactured by Toray Industries, Inc.)) or the like is added in an appropriate dilution series and cultured for about 24 hours. Thereafter, Sindbis virus is added to each well and incubated for about 24 hours. The culture solution is discarded, and the plate is immersed in a crystal violet solution containing formalin for about 15 minutes, and the living cells are fixedly stained. Since surviving cells are stained, the concentration at which antiviral activity is expressed can be determined by measuring the absorbance of each well. This examination confirms that the peptide of the present invention has interferon activity.

  The peptides of the present invention described above may be prepared according to methods well known in the pharmaceutical formulation art, for example, conventional solid or liquid vehicles or diluents, as well as types of pharmaceutical additives appropriate for the desired mode of administration (eg, carriers , Excipients, binders, preservatives, stabilizers, fragrances, etc.). Dosage unit formulations containing the sterilized non-toxic and pharmaceutically acceptable vehicle or diluent, etc. may be acceptable.

  The peptides of the invention can be administered by any method appropriate to the disease being treated, which can depend on site-specific treatment or requirements for the amount of drug delivered. Administration can be carried out in the usual manner, for example topically (including in the form of solutions, suspensions, gels, creams or ointments), parenterally (eg subcutaneously, intravenously, Intramuscular, or intrasternal uncoupled injection or infusion methods (including aqueous or non-aqueous solutions or suspensions that are sterile injectable), nasally (including, for example, in the form of inhalation sprays) or rectal (Including in the form of suppositories, for example). The peptides of the present invention can also be administered in a form suitable for immediate release or sustained release. Rapid release or sustained release can be achieved using a suitable pharmaceutical composition, or in particular in the case of sustained release, using a device such as a subcutaneous implant or osmotic pump. Can do.

An effective amount of a peptide of the invention can be determined by one skilled in the art. For example, when administered topically, parenterally, nasally or rectally, the dosage is 0.01-1000 mg / kg, preferably 0.01-100 mg / kg once a day. It can be used in administration or in separate dosage forms. The specific dose level and frequency of administration for a subject can vary depending on various factors and can be determined accordingly. Such factors include, for example, the activity of the specific synthetic peptide used, metabolic stability and duration of action of the peptide; subject species, age, weight, normal health, sex and diet; method of administration and time; The combination of drugs and the severity of the particular disease.
The peptide of the present invention expresses interferon-like biological activity and can be used for treatment of viral diseases and the like.

  EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1
Synthesis of peptides of the present invention Peptides were synthesized by a solid phase method using a peptide synthesizer 433A (Applied Biosystems, Sweden). All reagents were of peptide synthesis grade. For the synthesis, a 9-fluorenylmethyloxycarbonyl (Fmoc) (Applied Biosystems) group was used as a protecting group, and O-benzotriazole-N, N, N ′, N′-tetramethyluronium hexa was used as a coupling reagent. Using fluorophosphate (HBTU) and an amide resin (eg, link amide resin) as the resin, they were coupled by an autopeptide synthesizer in order from the C-terminal amino acid. The synthesis scale used 1.0 mmol of each peptide. After completion of synthesis, the peptide was cleaved from the resin by cleaving the Fmoc group from the resin using TFA, dissolved in distilled water after lyophilization. This was separated and purified using high performance liquid chromatography (HPLC) (Nihon Waters, Tokyo). The HPLC was performed by reverse phase chromatography using an ODS column and eluted with distilled water (A) containing 0.1% TFA and 5-65% gradient acetonitrile (B) over 30 minutes. The detection wavelength was 220 nm. Subsequently, the molecular weight of the peptide contained in the collected peak is measured with a mass spectrometer (Voyager (registered trademark) Elite, Perspective Biosystems, USA), and the fraction that matches the molecular weight estimated from the structural formula Were collected and the purity was confirmed by HPLC. Purity was examined with distilled water (A) containing 0.1% TFA and acetonitrile (B) with a gradient of 5 to 65%, and the obtained peptide was confirmed to be a single peak. Peptides were synthesized as 10 peptides of SEQ ID NO: 2 and 3-11 in the sequence listing. The peptides consisting of each sequence were named KR2QNNR3, KR2QNNR3-A1, KR2QNNR3-A3, KR2QNNR3-A5, KR2QNNR3-A7, KR2QNNR3-A11, KR2QNNR3-A13, KR2QNNR3-A15, HisQNN, and R5QN, respectively.

Example 2
Measurement of intermolecular interaction between peptide having amino acid sequence described in SEQ ID NO: 2 and solubilized interferon receptor ((AR1) ₂ Fc and (AR2) ₂ Fc) Binding of peptide of the present invention and solubilized interferon receptor Was measured using BIAcore 3000 (Biacore AB, Sweden), which is a BIAcore system using the surface plasmon resonance (SPR) method. First, a carboxyl group which is a functional group on the sensor chip CM5 is detected using a mixed solution of N-methyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The chip CM5 was activated by washing.

The (AR1) ₂ Fc solution and the (AR2) ₂ Fc solution were prepared by using (AR1) ₂ Fc and (AR2) ₂ Fc, respectively, using acetate buffer (pH 4.5) to a concentration of 30 μg / ml. Next, according to the manual attached to the BIAcore 3000, each solution is adjusted so that (AR1) ₂ Fc is about 2000 binding amount (RU: resonance unit) in the flow cell 2 and (AR2) ₂ Fc is in the flow cell 3 respectively. It was immobilized on the sensor chip CM5. Flow cell 1 was the control and nothing was immobilized on the control.

Next, the excess carboxyl group on the chip was blocked by reacting with a 1M ethanolamine solution (pH 8.5). The peptide consisting of the amino acid sequence described in SEQ ID NO: 2 was prepared to an appropriate concentration using a running buffer (0.01 M HEPES buffer (pH 7.4) containing 0.005% Tween 20, 0.15 M NaCl). . For the calculation of the concentration, a 6990 value was used as an extinction coefficient at 280 nm (Gasteiger E., et. Al., The Proteomics Protocols Handbook, Humana Press (2005)). The intermolecular interaction between the peptide and (AR1) ₂ Fc and (AR2) ₂ Fc was measured according to the manual at a flow rate of 20 μl / min and a sample addition time of 300 seconds. That is, the concentration of the peptide in each solution was increased stepwise to obtain a signal at equilibrium at each concentration (0 nM, 25 nM, 500 nM and 1000 NM, see FIGS. 1 and 3).

Based on the obtained measurement results, the time course of binding was plotted using analysis software (BIA evaluation) attached to BIAcore 3000. As a result, on the flow cell ₂ (AR1) 2 Fc, flow cell 3 on the (AR2) depending on the concentration in both cases of ₂ Fc, increased binding of the immobilized solubilized receptor was observed (FIGS. 1 and 3). It dissociated rapidly after the end of peptide addition. Further, (AR2) ₂ Fc was subjected to Scathard Plot analysis based on the data obtained by BIACore (FIG. 2). As a result, a negative linearity was observed between the binding amount and the bound / free ratio with a correlation coefficient of 0.9 or more, and the peptide was specifically bound to (AR2) ₂ Fc. It was suggested. Furthermore, from this result, it was calculated that the dissociation constant (K _D ) of the peptide for (AR2) ₂ Fc was about 1.1 μM.
For (AR1) ₂ Fc, Steady state affinity analysis was performed using BIA evaluation (FIG. 4). As a result, it was found that the dissociation constant (K _D ) of the peptide against (AR1) ₂ Fc was 1.9 mM. The χ ² value was 7.6 × 10 ⁻⁴ , and the above analysis was found to be statistically reliable. The control signal was used to calibrate the signal when measuring the sample.

Example 3
Measurement of inhibitory action of binding between interferon-β and solubilized interferon receptor (AR2) ₂ Fc by peptide having amino acid sequence described in SEQ ID NO: 2 Interferon-β by peptide having amino acid sequence described in SEQ ID NO: 2 of the present invention Measurement of the inhibitory effect on the binding of glycine to solubilized interferon receptor (AR2) ₂ Fc was performed using BIAcore 3000 (Biacore AB, Sweden) which is a BIAcore system using the surface plasmon resonance (SPR) method. First, the sensor chip CM5 is washed with a mixed solution of N-methyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The carboxyl group which is the functional group of was activated.

  As the interferon-β solution, a solution prepared by interferon-β (Feron, manufactured by Toray Industries, Inc.) using an acetate buffer (pH 4.5) to a concentration of 30 μg / ml was used. Next, the solution was immobilized on the sensor chip CM5 so that the binding amount (RU) was about 2000 according to the manual attached to the BIAcore 3000. On the other hand, flow cell 1 was used as a control, and nothing was immobilized on the control. Next, the excess carboxyl group on the chip was blocked by reacting with a 1M ethanolamine solution (pH 8.5).

As a mixed solution of peptide and (AR2) ₂ Fc, using a running buffer containing 100 nM (AR2) ₂ Fc (0.01 M HEPES buffer (pH 7.4) containing 0.005% Tween 20, 0.15 M NaCl) The one prepared to an appropriate concentration was used. The concentration was calculated by using 6990 values as an extinction coefficient at 280 nm (see John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005). Pp. 571-607). Measurement of the inhibitory action of the peptide binding of interferon-β and (AR2) ₂ Fc was performed according to the manual with a flow rate of 20 μl / min and a sample addition time of 300 seconds. That is, the peptide concentration ratio in the mixed solution of peptide and (AR2) ₂ Fc was increased stepwise (0 nM, 50 nM, 100 nM, 200 nM, 500 nM) to obtain signals at equilibrium at each concentration. On the other hand, as a control, the same measurement was performed on the same running buffer except that (AR2) ₂ Fc was not added and the peptide 500 nM was added to the same.

Based on the obtained measurement results, the time course of binding was plotted using analysis software (BIA evaluation) attached to BIAcore 3000. As a result, inhibition of binding of (AR2) ₂ Fc to immobilized interferon-β was observed with an increase in the peptide concentration ratio (FIG. 5). The control signal was used to calibrate the signal when measuring the sample. From this result, it was found that the peptide consisting of the amino acid sequence described in SEQ ID NO: 2 has a common interaction site in (AR2) ₂ Fc with interferon-β.

Example 4
[Interferon -β and solubilized interferon receptors by peptide SEQ ID NO: 2 (AR1) ₂ Measurement of inhibition of binding of the Fc]
The measurement of the inhibitory effect on the binding of interferon-β and solubilized interferon receptor (AR1) ₂ Fc by the peptide having the amino acid sequence described in SEQ ID NO: 2 was performed using the BIAcore 3000 BIAcore system using surface plasmon resonance (SPR) (Biacore AB, Sweden). First, the sensor chip CM5 is washed with a mixed solution of N-methyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), so that the sensor chip CM5 is functionalized. The carboxyl group which is a group was activated.

  As the interferon-β solution, an interferon-β (manufactured by Toray Industries, Inc.) prepared using an acetate buffer (pH 4.5) to a concentration of 30 μg / ml was used. Next, the solution was immobilized on the sensor chip CM5 so that the binding amount (RU) was about 2000 according to the manual attached to the BIAcore 3000. On the other hand, flow cell 1 was used as a control, and nothing was immobilized on the control. Next, the excess carboxyl group on the chip was blocked by reacting with a 1M ethanolamine solution (pH 8.5).

As a mixed solution of peptide and (AR1) ₂ Fc, using a running buffer containing 100 nM (AR2) ₂ Fc (0.01 M HEPES buffer (pH 7.4) containing 0.005% Tween 20, 0.15 M NaCl) The one prepared to an appropriate concentration was used. For the calculation of the concentration, 6990 values were used as an extinction coefficient at 280 nm (see John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press (2005) pp. 571-607). Measurement of inhibitory effect of the binding of interferon -β and (AR1) ₂ Fc by peptide SEQ ID NO: 2, flow rate 20 [mu] l / min, sample addition time was performed according to the manual 300 seconds. That is, the peptide concentration ratio in the mixed solution of peptide and (AR1) ₂ Fc was increased stepwise (0 nM, 100 nM, 500 nM), and signals at equilibrium at each concentration were obtained. On the other hand, as a control, the same measurement was performed on the same running buffer except that (AR1) ₂ Fc was not added and the peptide 500 nM was added to the same one.

Based on the obtained measurement results, the time course of binding was plotted using analysis software (BIA evaluation) attached to BIAcore 3000. As a result, inhibition of binding of (AR1) ₂ Fc to immobilized interferon-β was observed with an increase in the peptide concentration ratio (FIG. 6). The control signal was used to calibrate the signal when measuring the sample. From this result, it was found that the peptide consisting of the amino acid sequence described in SEQ ID NO: 2 has a common interaction site in (AR1) ₂ Fc with interferon-β.

Example 5
[Measurement of antiviral activity of peptide]
Interferon activity of the peptide (peptide consisting of each amino acid sequence of SEQ ID NO: 2 to 11 in the Sequence Listing) and peptide SY6 (see amino acid sequence described in SEQ ID NO: 12), that is, antiviral activity, is the FL cell that is a human amniotic cell. It was measured by a bioassay method using Sindbis virus (Armstrong, JA, Methods in Enzymology, 78, 381-387 (1981).

In a well of a 96-well microtiter plate (Iwaki Glass Co., Ltd.), FL cells were seeded with 4.0 × 10 ⁴ / well and cultured for 24 hours. Thereafter, each peptide was added in a dilution series of 0.65 μM to 83.3 μM and cultured for 24 hours. As a control, interferon-β (Feron, Toray Industries, Inc.) was added in a dilution series of 0.52 IU / ml to 66.7 IU / ml and cultured for about 24 hours. Thereafter, Sindbis virus was added to each well and incubated for about 24 hours. The culture solution was discarded, and the plate was immersed in a crystal violet solution containing formalin for about 15 minutes, and the living cells were fixedly stained. Since surviving cells are stained, the concentration at which antiviral activity is expressed can be determined by measuring the absorbance of each well.

The titer was calculated by setting the OD value (C _OD ) of the cell control (interferon non-treated and virus non-treated) to 100%, and the OD value (V _OD ) of the virus control (interferon non-treated and virus treated) to 0%. The OD value was converted to% value to obtain OD%. Further, (C _OD + V _OD ) / 2 was set to _OD 50%. A linear equation (y = A × LogX + B) was calculated from the dilution factor at two points across 50% from the dilution curve with OD% as the Y axis and the common logarithm of the dilution factor (LogX) as the X axis. In addition, A and B in the formula were calculated by the following formulas.

A = {(OD% of L) − (OD% of H)} / {(LogL) − (LogH)}
B = (OD% of H) −A × (LogH)
H: Dilution factor on the low dilution side between two points that sandwich OD50% L: Dilution factor on the high dilution side between two points that sandwich OD50%

LogX at OD 50% and further dilution factor X at OD 50% (EXP.TITER) were calculated from the created primary equation.
LogX at OD 50% = (50−B) / A
EXP. TITER = 10 ⁽ LogX at ^{OD50% )}
For the plate standard, the plate standard titer (400 IU / ml) was calculated using EXP. Factor was calculated by dividing by TITER.
Factor = (400) / (EXP.TITER)
EXP. For each lane of the measurement sample. The antiviral activity value was obtained by multiplying TITER by Factor and the predilution factor (FIG. 7).

It is drawing which shows the measurement result of the intermolecular interaction of the peptide represented by sequence number 2, and (AR2) ₂ Fc. It is drawing which shows Scatchard Plot computed from the measurement result of the intermolecular interaction of the peptide represented by sequence number 2, and (AR2) ₂ Fc. It is a figure which shows the measurement result of the intermolecular interaction of the peptide represented by sequence number 2, and (AR1) ₂ Fc. It is drawing which shows the Steady state Affinity analysis result computed from the measurement result of the intermolecular interaction of the peptide represented by sequence number 2, and (AR1) ₂ Fc. It is drawing which shows the measurement result of the inhibitory action of the coupling | bonding of the peptide represented by sequence number 2, and solubilized interferon receptor (AR2) ₂ Fc. It is drawing which shows the measurement result of the inhibitory action of the coupling | bonding of the peptide represented by sequence number 2, and solubilized interferon receptor (AR1) ₂ Fc. It is drawing which shows the measurement result of the antiviral activity of the peptide represented by sequence number 2-12.

Claims (2)

The peptide shown in the following (A) or (B).
(A) a peptide comprising the amino acid sequence described in SEQ ID NO: 1 (B) an amino acid sequence described in SEQ ID NO: 1, comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and interferon-like biology That expresses sexual activity   The peptide according to claim 1, comprising the amino acid sequence of any one of SEQ ID NOs: 2 to 11.

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