RU2470031C2 - Biologically active peptide complexes - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biologically active peptide complexes with immunomodulating and antiviral activity. The disclosed peptide complexes have a three-dimensional structure

wherein X₁ is absent or contains at least 1 amino acid; R1 and R2 are peptide chains which contain amino acid residues, His or Cys, capable of reacting with transition metal ions, wherein R1 contains up to 5 amino acid residues or is absent; R2 contains up to 3 amino acid residues or is absent.

EFFECT: peptide complexes rich in histidine and, primarily alloferon peptides with Zn ions, enable to produce preparations with a directed mechanism of action and enable their design in accordance with the understanding of the structure of the medicinal target.

3 cl, 7 dwg, 2 tbl, 6 ex

Description

The present invention relates to proteins and biologically active peptides with immunomodulating and antiviral activity.

Known compounds of peptide, polypeptide and protein nature, used in medicine as antiviral agents. Among drugs-inducers of interferons (IFN) of the 1st type are known as high molecular weight compounds [Ershov F.I., Kiselev O.I. Interferons and their inducers from molecule to drug. - M .: ed. Geotar - Media, 2005 - p. 356], [Berg K., Bolt G., Andersen H., Owen T.C. Zink potentiates the antiviral action of human IFN-alpha tenfold. J. Interferon Cytokine Res, 2001, Jul; 21 (7): 471-4], and low molecular weight inductors. Of the second, first of all, it should be noted the domestic drug cycloferon and the American drug imiquimod. These funds relate to derivatives of acridones and benzimidazoles, respectively. For imiquimod and related derivatives, the type of Toll-like receptors is known, with which this group of drugs interacts, causing the induction of IFN-α synthesis in various cells [Ershov F.I., Kiselev O.I. Interferons and their inducers (from molecule to drug). M .: "Publishing House. Geotar - Media ”, 2005. - Page 356].

The biological activity of low molecular weight peptides is widely known. This primarily refers to peptides of animal and plant origin with antibacterial activity [Boman H. Peptide antibiotics and their role in innate immunity. Anu. Rev. of Immunol., 1995, Vol.13, p. 61-92]. At the same time, a number of peptides have been described that have a direct antiviral or antitumor effect [Akiyama N., Hijikata M., Kobayashi A., Yamori T., Tsuruo T., Natori S. Anti-tumor effect of N-β-alanyl- 5-S-glutathionyl dihydroxyphenylalananine (5-S-GAD) a novel anti-bacterial substance from an insect. Anticancer Research, 2000, Vol.20, p. 357-362].

Amphibians and insects peptides occupy a special place in this series [Bulet P., Hetru C., Diamarcq J., Hoffmann D. Antimicrobial peptides in insects: structure and function. Devel Comp. Immunol., 1999, Vol.23, p.329-344, Chinchar VG, Wang J., Murti G., Carey C., Rolling-Smith L. Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin -2P, two antimicrobial peptides isolated from frog skin. Virology, 2001, Vol. 288, p. 351-357].

Known immunomodulating peptides - alloferons (RF patent No. 2172322). The main area of application of alloferons is the treatment of viral infections. Alloferons are the closest analogues of the invention in terms of chemical structure and mechanism of action.

It should be noted that the authors of the invention for patent No. 2172322 consider variations of only the primary structure of alloferons and do not betray the key significance of the distribution of histidine residues.

In addition, alloferons should be attributed to rather “weak” interferon inducers, which is obvious from a comparison of their activity with cycloferon.

At the same time, the structure of alloferons attracts attention to the regularity of arrangement of histidine residues and repeating glycine residues. Improving the structure of alloferons is possible in the direction of giving them elements of the tertiary structure, for example, by introducing metal ions.

Also known is the heminpeptide and its pharmaceutically acceptable salts, having virucidal and antiviral effects, containing metal ions, which can be used Zn, Cu, Fe, Mn (RF patent 2296131). However, this compound belongs to another class of peptides and is not an immunomodulator.

Peptide complexes with the Zn ⁺⁺ ion possessing elements of an organized tertiary structure and activity of the first type of interferon inducers are not described in the literature.

The need for modification of histidine-containing peptides by Zn ⁺⁺ ion is due to the following reasons:

1. Biologically active short peptides have an unorganized type of secondary structure, which inevitably reduces their biological activity, the ability to interact with other macromolecules, and metabolic stability.

2. The biological and pharmacological activity of peptides largely depends on the efficiency of transport into cells. Giving peptides a compact structure increases the efficiency of their translocation through membranes and, therefore, pharmacological activity [Leng Q., Mixson J. Modified branched peptides with histidine - rich tail enhance in vitro gene transfection. Nucl. Acids Res., 2005, Vol. 33, e40].

3. The formation of complexes of histidine-containing peptides with the Zn ⁺⁺ ion leads to a fundamental change in the properties of the peptides, making them identical with the domains of transcriptional activators of viruses and cells.

The present invention is the development of complexes of peptides organized in a three-dimensional structure. The developed complexes have a high ability to bind to other groups of molecules and exhibit a wide range of pharmacological effects, including the induction of IFN type I, and act at different levels of cellular functions, which will allow them to create new drugs for the prevention and treatment of viral infections.

A new family of biologically active peptides was developed on the basis of known peptides enriched in histidine residues, alloferons and their homologs using protein domains with known functions as a prototype Zn-finger. Alloferons are used as a matrix of peptides from 6 to 35 amino acid residues in length. The peptides so constructed have the ability to form complexes with the Zn ⁺⁺ ion to form oligomers and aggregates, and in terms of structural and biological properties they satisfy the requirements for immunomodulators.

The proposed peptide complexes have a three-dimensional structure and are described by the following structural formula:

where X _{1 is} absent or contains at least 1 amino acid; R1 and R2 are peptide chains containing amino acid residues capable of interacting with transition metal ions; moreover, R1 contains up to 5 amino acid residues or is absent, R2 contains up to 3 amino acid residues or is absent.

The ability of natural peptides enriched in histidine residues to bind to metal ions has been confirmed by a number of studies [Hua Zhao H., and Waite J.H. Proteins in Load-Bearing Junctions: The Histidine-Rich Metal-Binding Protein of Mussel Byssus, Biochemistry. 2006, 45 (47): 14223-14231].

The invention is illustrated by the data given in the diagrams and figures:

fig. 1. Analysis of consensus sequences of peptides of the alloferon family;

fig. 2. Computer model of A1 polypeptide;

fig. 3. Theoretically possible structural variants of complexes A1 with Zn ⁺⁺ ion;

fig. 4. Kinetics of binding of alloferon A1 to Zn ⁺⁺ by light scattering;

fig. 5. Analysis of the binding of Al peptide to sorbent HiTrap, balanced Ni ⁺⁺ ;

fig. 6. Induction of Type I Interferons;

fig. 7. The protective effect of the studied drugs in fatal influenza infection in mice.

When developing the present invention, the peptide used in Table 2 under the name Alloferon 1 (SEQ ID NO 1) was used as the basic structure, Alloferon 1 was synthesized by solid-phase synthesis and used to study the biological activity of the proposed peptides. Studies, the results of which are given in the examples below, showed that this peptide has the ability to form complexes with transition metals, is an inducer of interferon, and has antiviral activity. Computer analysis of databases on the structure and properties of proteins and peptides has established that this compound belongs to a new previously unknown family of biologically active peptides. The polypeptides enriched in histidine and glycine, with introduced metal ions, have immunomodulating and antiviral activity and, in addition, zinc ions potentiate their biological activity.

The specified sequence peptides were synthesized by the solid-phase method of peptide synthesis using the Boc / Bzl strategy on phenylacetamidomethyl polymer (PAM). The peptides were obtained on the synthesizers of the peptides Coupler-250 and Applied Biosystems 430A.

For temporary protection of the α-amino groups, a tert-butyloxycarbonyl group was used, which was removed with trifluoroacetic acid. To block the side radicals of trifunctional amino acids, benzyl and acyl type protective groups were used: dinitrophenyl for histidine, mesitylene sulfonyl for arginine, 2-chlorobenzyloxycarbonyl for lysine, formyl for tryptophan, 2,6-dichlorobenzyl for tyrosine, O-benzyl esters for tyrosine. Methionine was introduced into the condensation as a sulfoxide derivative.

Removal of the temporary protective groups was carried out with undiluted trifluoroacetic acid, and neutralization was carried out according to the “in situ” procedure by adding N, N'-diisopropylethylamine at the condensation stage directly to the reaction mixture.

The program of addition of one amino acid residue during chain extension of the peptidyl polymer based on the total content of acylamino acid on the polymer 0.2 mmol is shown in the table. The pre-activation of the carboxyl component was carried out for 30 minutes using oxybenzotriazole and diisopropylcarbodiimide. Under these synthesis conditions, in all cases, after the addition of the required amount of amino acid residues corresponding to the sequence of the peptide fragment, a satisfactory gain in the peptidyl polymer was obtained.

The removal of the side protective groups and the cleavage of the peptide from the resin was carried out under the influence of anhydrous hydrogen fluoride in the presence of scavengers, mainly m-cresol. With this treatment, all side protective groups were removed, and the peptide was cleaved from the high molecular weight matrix, the release time ranged from one to one and a half hours.

To prevent undesirable side reactions in the synthesis of peptides containing methionine (in particular, sulfur alkylation with a tert-butyl radical, as well as its partial oxidation during the growth of the peptide chain), the methionine residue is smoothly introduced into the sequence of the peptidyl polymer in the form of a sulfoxide derivative, which at the final stages of peptide deprotection restored to methionine. This reduction reaction took place with a satisfactory result under the action of ammonium iodide, either on a fully unblocked peptide or at a stage when the peptide was still on the resin.

Table 1 The program of joining one amino acid residue N p / p Operation Reagents Repeatability Time min The volume of reagents, ml one. Removing Boc protection (release) Trifluoroacetic acid one 2 5 2. Re-release Trifluoroacetic acid one 2 5 3. Flushing Dimethylformamide 3 one 10 four. Condensation 1.0 mmol of oxybenzotrizole ester of the corresponding amino acid derivative one twenty 5 + Diisopropylethylamine (0.7 mmol in dimethylformamide) 5. Flushing Dimethylformamide 3 one 10 6. Wash Methylene chloride 3 one 10 7. Ninhydrin test * * - With a positive ninhydrin test, re-condensation was carried out.

All synthesized peptide preparations were purified using preparative reverse phase liquid chromatography on a Dynamax 60 A column, 22.5 × 250 mm (Gilson liquid chromatograph, France) and were characterized by amino acid analysis of peptide hydrolysates after hydrolysis with methanesulfonic acid in the presence of tryptamine (Alpha Plus amino acid analyzer, LKB, Sweden).

The ability to achieve the objectives of the invention is confirmed by the following examples.

Example 1. Analysis of the structure and consensus sequences of the families of Alloferon peptides.

To analyze the consensus of the sequences of peptides of the alloferon families, the BioEdit v.7.09 Ibis Biosciences (USA) program was used. The homology of the amino acid sequences of alloferons is presented in table 2.

table 2 Homology of the sequence of alloferons peptide one 2 3 four 5 6 7 8 9 10 eleven 12 13 fourteen SEQ ID NO 1 His Gly Val Ser Gly His Gly Gln His Gly Val His Gly Alloferon 1 SEQ ID NO 2 Cys Val Val Thr Gly His Gly Ser His Gly Val Phe Val Alloferon 10 SEQ ID NO 3 Ile Ser Gly His Gly Gln His Gly Val Pro Alloferon 11 SEQ ID NO 4 Cys Gly His Gly Asn His Gly Val His Alloferon 12 SEQ ID NO 5 Ile Val Ala Arg Ile His Gly Gln Asn His Gly Leu Alloferon 13 SEQ ID NO 6 His Gly Ser Asp Gly His Gly Val Gln His Gly Alloferon 14 SEQ ID NO 7 Phe Gly His Gly His Gly Val Alloferon 15 SEQ ID NO 8 His Gly Asn His Gly Val Leu Ala Alloferon 16 SEQ ID NO 9 His Gly Asp Ser Gly His Gly Gin His Gly Val Asp Alloferon 17 SEQ ID NO 10 His Gly His Gly Val Pro Leu Alloferon 18 SEQ ID NO 11 Ser Gly His Gly Ala Val His Gly Val Met Alloferon 19 SEQ ID NO 12 Gly Val Ser Gly His Gly Gln His Gly Val His Gly Alloferon 2 SEQ ID NO 13 Tyr Ala Met Ser Gly His Gly His Gly Val Phe Ile Alloferon 20 SEQ ID NO 14 Val Ser Gly His Gly Gln His Gly Val His Alloferon 3 SEQ ID NO 15 Ser Gly His Gly Gln His Gly Val Alloferon 4 SEQ ID NO 16 Pro Ser Leu Thr Gly His Gly Phe His Gly Val Tyr Asp Alloferon 5 SEQ ID NO 17 Phe Ile Val Ser Ala His Gly Asp His Gly Val Alloferon 6 SEQ ID NO 18 Thr His Gly Gln His Gly Val Alloferon 7 SEQ ID NO 19 His Gly His Gly Val His Gly Alloferon 8 SEQ ID NO 20 Leu Ala Ser Leu His Gly Gln His Gly Val Alloferon 9 SEQ ID NO 21 His Gly Tyr Thr Ser His Gly Ala His Gly Val Gemagglutin 377-388 Consensus- His Gly His Gly sequencing Structural X1 His Gly X2 R Gly X3 formula

RF patent No. 2172322 provides a sequence of alloferons without presenting a consensus sequence, which does not allow an accurate assessment of the “core” core portion of peptides and to separate significant modifications from non-essential ones.

As a result of the analysis, it seems possible to divide the family of alloferons into 3 families with consensus sequences:

SGHGQ-HGV, VSGHGQ-HGV, SGHGQ-HGV, which is justified by the above computer calculations (Fig. 1) of the sequences of peptides of the alloferon family.

Example 2. Computer simulation of peptide A1 (analysis of the tertiary structure)

To understand the structure of short peptides, computer simulation can be used to evaluate the structure of the peptide as a whole and its individual domains. In particular, it was also necessary to evaluate the possibility of forming complexes of these peptides with the Zn ⁺⁺ ion. For this, a computer simulation of peptide A1 was carried out, having the following structure: His-Val-Ser-His-Gly-Gln-His-Gly-Val-His-Gly (A1). The construction of a simple complex A1 with the Zn ⁺⁺ ion allows one to demonstrate the formation of a peptide loop stabilized by the coordinate bonds of histidine residues with the Zn ⁺⁺ ion.

Computer simulation of peptide A1 (Fig. 2) showed that the short peptide forms a relaxed loop in which the Zn ⁺⁺ ion can interact with the histidine residues available for interaction. Moreover, the general structure of the polypeptide corresponds to the possibility of complex formation of the Zn ⁺⁺ ion with at least three histidine residues at positions 1, 6, and 9.

In the simplified model (Fig. 3) of Zn - A1, it is seen that a significant fraction of glycine residues are in the N-terminal half of the molecule. This corresponds to a secondary structure such as beta layers. The C-terminal part has an alpha-helical structure with imidazole histidine rings exposed inwardly and interacting with the Zn ⁺⁺ ion.

The figure shows an example with Zn ⁺⁺ . Zn ⁺⁺ can be placed in almost any position.

A - intramolecular complex Zn - A1, organized in the form of a loop.

B - intermolecular complex Zn - A1, organized into a dimer. Aggregation can be carried out by the addition of new A1 molecules due to intermolecular incorporation of the Zn ⁺⁺ ion in regions “a” and “b” or in the center of the linear polypeptide upon interaction of the Zn ⁺⁺ ion with histidine residues at positions 6 and 9.

When analyzing the structure of A1, the high content and regular arrangement of histidine residues are noteworthy. Figure 2 shows that A1 polypeptide forms an almost perfect saddle structure. In such a confirmation, Gis residues - 1, 6, and 9 are maximally accessible for interaction with the Zn ⁺⁺ ion.

An important conclusion may be the conclusion that complexation with an excess of peptide with respect to Zn ⁺⁺ can lead to the formation of intermolecular aggregates (Fig. 2 - B). This structural transition fundamentally changes the properties of peptides, giving them the compact structure necessary for biological activity, as demonstrated in a number of studies [Rydengard V., Nordahl E. A., Schmidtchen A. Zinc potentiates the antibacterial effects of histidine-rich peptides against Enterococcus faecalis . FEBS Lett., 2006, Vol. 273, p. 2399-2406].

Example 3. Alloferon and its closest analogues are Zn ⁺⁺ - binding peptides.

The study of the binding of the Zn ⁺⁺ ion with alloferon 1 (A1) and its homologues was carried out according to the method described by [Shi Y., Beger RD, Berg JM Metal binding properties of single amino acid deletion mutants of zinc finger peptides: studies using cobalt (II) as a spectroscopic prob. Biophys. J., 1993, Vol. 64, p. 749-753]. The binding of the Zn ⁺⁺ ion to peptide A1 was studied by light scattering using an ISS, Campaign, IL fluorimeter at 400 nm and exciting light 398 nm.

Figure 4 shows graphs of the interaction of the Zn ⁺⁺ ion with peptide A1.

For analysis conditions, see Shi Y. et al. (1993).

A - (open circles) the interaction of A1 with Zn (NO ₃ ) ₂ . The molar excess of the Zn ⁺⁺ ion with respect to the peptide was 1:10. The solid line is the saturation of the peptide with Zn ⁺⁺ ion. When fully saturated, the bulk of the peptide passed into aggregates. EDTA was added to the aggregates. As a result of the addition of EDTA, the complex quickly dissociated and the peptide (alloferon) passed into the soluble phase.

Figure 4 shows that Zn ⁺⁺ (Zn (NO ₃ ) ₂ ) interacts with peptide A1, which leads to an exponential increase in light scattering and subsequent aggregation of the peptide in the form of polydisperse nanoparticles up to 50-60 nm in diameter with subsequent formation of a suspension of large aggregates. When the chelating agent EDTA is added, aggregates and complexes of peptide A1 are dissolved.

Thus, peptide A1 is able to interact with the Zn ⁺⁺ ion to form soluble complexes in the first phase.

Example 4. Peptides interact with Zn ⁺⁺ , exhibiting high affinity for nickel sorbents.

Chromatography on HiTrap columns showed that A1 behaves like oligohistidine and has a fairly high affinity for this sorbent and is completely eluted with an imidazole solution. Elution was performed with a gradient of phosphate buffer / 0.5M imidazole (Fig. 5).

Example 5. Induction of type 1 interferons

Induction of type 1 interferons was studied according to the method published previously [Ershov F.I., Kiselev O.I. Interferons and their inducers (from molecule to drug). M .: "Publishing House. Geotar-Media ”, 2005 - p. 356, Chemysh et al., 2002]. Figure 6 shows the results of testing drugs for the ability to induce type 1 interferons. As can be seen from the data obtained, the maximum activity in the induction of interferons was possessed by the Zn-A1 peptide. The peptide Zn - A2 was somewhat inferior to it. Unmodified peptide A1 showed a rather high level of ability to induce interferons, but however, it was significantly inferior to derivatives in complex with Zn ⁺⁺ ion and corresponded to cycloferon activity.

In example 6, it will be seen that these data correlate with the protective effect of the drugs in case of fatal influenza infection in mice.

Example 6. Antiviral activity in a model of experimental lethal influenza pneumonia in white mice caused by influenza A.

To test the antiviral activity of the peptide complexes, a model of lethal influenza infection was used on white outbred mice of both sexes weighing 10-12 g, obtained from the Rappolovo nursery. We used the strain of the influenza virus A / Aichi / 2/68 (H3N2), adapted to white mice in the laboratory and showing high pathogenicity, causing infection with the development of pneumonia and death within 5-10 days depending on the dose of the virus.

Peptides and their derivatives were administered to animals once 6 and 12 hours before infection in an amount of 1-2 μg / kg of animal weight intraperitoneally. A placebo in the control group of animals served as saline or phosphate buffer in equal volume.

The virus was pre-titrated in animals and its concentration was determined by the lethality of mice. For the experiment, the virus was administered to animals intranasally under light ether anesthesia at a dose of 0.2 and 5 LD ₅₀ . In each observation group, 10 mice were taken. Observation of animals was carried out up to 15 days, i.e. the period during which experimental flu has a 100% mortality rate in animals.

Daily recorded weight and mortality of animals in the control and experimental groups. Based on the obtained mortality rates, the percentage of mortality in each group was calculated (the ratio of the number of animals killed in 15 days to the total number of infected animals in the group), and the protection index. The data obtained are presented in Fig. 5. An analysis of the results showed that the effect of the studied preparations A1 against the influenza A virus pathogenic for mice was comparable with the effectiveness of the protective effect of the reference drug Remantadin (80-87% at a dose of virus 1 LD ₅₀ ). The high protective effect of Zn-Al complexes indicates that the formation of Zn ⁺⁺ with A1 strongly potentiates the activity of peptides of type A1. The testing method used in this case indicates that the protective effect should be attributed mainly to the induction of interferons. The drug showed maximum activity when used in a prophylactic regimen.

Fig. 7 shows the protective effect of the studied drugs in case of fatal influenza infection in mice. Based on the foregoing, it can be argued that the developed peptide has all the claimed properties.

Complexes of peptides enriched with histidine, and, first of all, peptides of the alloferon family with Zn ⁺⁺ ion, will allow the creation of drugs with a directed mechanism of action and their design in accordance with an understanding of the properties and structure of peptides, as well as the nature of the drug target.

Claims (3)

1. Peptide complexes in which the peptide is organized into a three-dimensional structure, and characterized by the general structural formula:

where X _{1 is} selected from Gln, Ser, Asn, Val, Ala, Phe, Asp or absent;
R ₁ and R ₂ are peptide chains containing His- or Cys- amino acid residues capable of interacting with transition metal ions, while
R _{1 is} selected from: His-Gly-Val-Ser-Gly-, Cys-Val-Val-Thr-Gly-, Cys-Gly-, His-Gly-Ser-Asp-Gly-, Gly-, His-Gly- Asp-Ser-Gly-, Val-Ser-Gly-, His-Gly- or absent;
R ₂ selected from: -Val-His-Gly, -Val-Phe-Val, -Val-His, -Val-Asp or absent. 2. The peptide complexes according to claim 1, having the properties of inducers for the synthesis of interferon. 3. Peptide complexes according to claim 1, having antiviral activity.

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