JP2002531465A - Antiviral peptide - Google Patents

Abstract

  (57) [Summary] The present invention comprises an amino acid chain comprising 10 to 25 amino acid domains, wherein the majority of the half amino acids in the domain are positively charged amino acids, and the majority of the remaining half amino acids in the domain are uncharged amino acids. And a peptide used as an antiviral agent. The present invention further provides for the use of peptides and / or oligomers for the manufacture of a medicament for the treatment of viral infection, as well as the use of said peptide consisting of at least two peptides linked together via an optional spacer used as an antiviral agent. For oligomers.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel peptide derived from a natural salivary peptide having antiviral activity.

[0002] Viruses can only be sought with chemotherapeutic agents with great difficulty. One reason for this is the fact that virus growth is closely related to host cell function. When trying to remove the virus, the host cell will also suffer at least some irreparable damage. In addition, classical antibiotics used to combat bacteria and other microorganisms have little or no effect on viruses. Vaccination is effective for many viral infections, whereas antiviral therapy (treatment) is only available for some viral infections (herpes simplex virus (HSV) and human immunodeficiency virus (HIV)). The antiviral agent has virus specificity. In many viral infections, no effective antiviral agent is known.

[0003] It is an object of the present invention to provide a wide range of activities, namely the viral env.
It is an object of the present invention to provide a novel antiviral agent having activity against a plurality of viruses including DNA and RNA viruses irrespective of whether or not they have elope.

[0004] The objective is to consist of an amino acid chain comprising 10 to 25 amino acid domains, the majority of the amino acids of the domain being positively charged amino acids and the majority of the other half amino acids of the domain being uncharged amino acids. This is achieved by the present invention by using a peptide which is as an antiviral agent.

[0005] The structure of these peptides has several variants. First, the active domains can form α-helices, of which 1-, 2-, 5-, 6-
, 9- (12-, 13-, 16-, 19-, 20-, 23- and 24-) at least a majority contains a positively charged amino acid, and the 8-position contains a positively charged or uncharged amino acid. , Peptide and 3-, 4-, 7-, 10- (11-, 14-
, 15-, 17-, 18-, 21-, 22-, 25-) at least a majority contain uncharged amino acids. These peptides have amphiphilicity in the side chain direction, ie a maximum hydrophobic moment of 100 °. Briefly, these peptides are hydrophobic on the left and hydrophilic on the right or vice versa. These peptides are referred to herein as "Type I" (peptides).

The domains can further form α-helices, of which 1-
, 2-, 5-, 6-, 9- (12-, 13-, 16-, 19-, 20-, 23-
And at least a majority of the 24-)-positions contain uncharged amino acids, the 8-position contains positively or uncharged amino acids, and 3-, 4-, 7-, 10- (11-
, 14-, 15-, 17-, 18-, 21-, 22-, 25-) at least a majority contain positively charged amino acids. These peptides have amphiphilicity in the side chain direction, ie a maximum hydrophobic moment of 100 °. Briefly, these peptides are hydrophobic on the right and hydrophilic on the left or vice versa. These peptides are referred to herein as "Type II" (peptides).

Further, the domains can form an α-helix, one of which
At least a majority of the-to 6- (or 7- or 8- or 9- or 10- or 11- or 12-) positions comprise an uncharged amino acid, and the 7- (or 8- or 9- or 10- or Positively charged amino acids have been found at positions 11- or 12- or 13-) to 25-. These peptides are amphiphilic in the side chain direction, ie, 100
It has a maximum hydrophobic moment of °. These peptides are hydrophobic at the top,
It is hydrophilic at the bottom or vice versa. Such a peptide is referred to as "Type I
II "(peptide).

Further, the domains can form an α-helix, one of which
At least a majority of the-to 6- (or 7- or 8- or 9- or 10- or 11- or 12-) positions contain a positively charged amino acid, and the 7- (or 8- or 9-)
Alternatively, uncharged amino acids are found at positions 10- or 11- or 12- or 13-) to 25-. Since these peptides also have amphiphilicity in the side chain direction,
It has a maximum hydrophobic moment of 100 °. These peptides are hydrophobic at the bottom and hydrophilic at the top or vice versa. Such a peptide is called "Type IV" (peptide).

Finally, the domains can form so-called β-strands, of which 1-, 3-, 5-, 7-, 9- (1-, 13-, 15-, 17-, 19-
, 21-, 23- and 25-) contain at least a majority of the positively charged amino acids and are 2-, 4-, 6-, 8-, 10- (12-, 14-, 16-, 18).
At least a majority of the positions-, 20-, 22-, 24--) contain uncharged amino acids. Such β-strands are amphiphilic in the side chain direction and have a maximum hydrophobic moment of 180 °. The β-strand structure is more planar than the α-helix, in short, is hydrophobic on the left and hydrophilic on the right, or vice versa. These are "Type V" peptides.

[0010] The positively charged amino acid is preferably selected from the group consisting of ornithine (O), lysine (K), arginine (R) and histidine (H), while the uncharged amino acid is preferably a fatty acid. Group amino acids glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I), methionine (M) which is an amino acid having a bipolar side chain, asparagine (N), glutamine (
Q), serine (S) and threonine (T), and phenylalanine (F), tyrosine (Y) and tryptophan (W), which are amino acids having an aromatic side chain
Selected from the group consisting of The amino acids at the boundary between hydrophilic and hydrophobic may be selected from both groups or from the remaining amino acids.

When one of the positively charged amino acids and / or one of the uncharged amino acids is replaced with a random amino acid, in principle no difference in activity can be detected. The majority of the positively charged amino acids is therefore preferably a value obtained by subtracting 1 from the total number of the positively charged amino acids, and the majority of the uncharged amino acids is the total number of the uncharged amino acids. It is preferably a value obtained by subtracting 1 from.

The domain can be part of a larger peptide, but can itself constitute an entire peptide. When the domain forms part of a larger peptide, the accompanying C-terminal and / or N-terminal amino acids may be random amino acids.

[0013] Further, these domains include peptide oligomers, (other peptides, lipids,
Can form part of more complex structures such as oligosaccharides, (radioactive) isotope tracers (labeling isotopes), hybrid peptides and peptide conjugates (along with organic acceptor ligands) It is. The peptide drug may be encapsulated in Willion liposomes, for example, to better ensure the cell-cell activity of the peptide.

The following type I is particularly recommended. KRLFKELKFSLRKY (Peptide 3) KRLFKELLFSLRKY (Peptide 4) KRLFKELKKSLRKY (Peptide 5) KRLFKELLKSLRRKY (Peptide 6) OOLFOELOSLOOY (Peptide 7) OOLFOELLYKLKLKLKLKLKLKLKLKLKLKLKLKLKKLKP

A preferred type II peptide has the following amino acid sequence: LLLFLKKRKKKRY (peptide 11)

The peptide according to the present invention may further comprise a modified form. These modifications are, for example, selective cleavage of a synthetic resin that modifies the N-terminal amide ring having acetic anhydride or the like or the C-terminal. For the latter, substitution of the C-terminal carboxyl group with an amide group, ester group, ketone group, aldehyde group or alcohol (hydroxyl) group can be assumed. Examples of the peptide having such a modification include the following. KRLFKELKFSLRKY-amide (peptide 12) KRLFKELLFSLRKY-amide (peptide 13)

In addition to single peptides, oligomers can be produced. These are preferably linear oligomers of the peptides of the invention. Coupling may be head-to-tail and tail-to-tail, as well as head-to-tail, either by direct or post-synthetic enzymatic coupling. Advantages of peptide oligomers (see below)
As described in the examples, there is good efficacy and a broad spectrum of activity. Usually a spacer must be inserted. In direct synthesis of head-to-tail linked oligomers, the spacer is of a non-natural amino acid of the correct length, such as β-alanine, γ-aminobutyric acid, ε-
It is inserted using a chain such as aminocaproic acid. Commercially available hetero-bifunctional coupling agent (reagent) for binding a peptide antigen to a protein carrier (eg, 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide (EDC), m-maleimidobenzoyl)- N-hydroxysuccinimide ester (MBS), N
-Succinimidyl 3- [pyridyldithio] propionate (SPDD), etc.)
Is used to make linear oligomers with insertion spacers. For head-to-head and tail-to-tail bonds, aspartic acid (D), glutamic acid (E), ornithine (O), lysine (K), serine (S) and cysteine are used.

Oligomers very suitably used in the present invention are oligomers of peptide 10 and peptide 11 having the following amino acid sequence: α, ε- (KRLFKKLLFSLRKY) ₂ -K-amide (peptide 10-dimer) α, ε- (LLLFLLKKRKKKRKKY) ₂ -K-amide (peptide 11-dimer)

The peptides described herein have no or little hemolytic activity.

In vitro potency assays have demonstrated that the peptides described herein have no toxic effect on human erythrocytes and monkey renal cells (vero-cells).

The peptide and / or oligomer thereof according to the present invention can be used in or as an antiviral agent. Their antiviral activity is further described in the examples below.

[0022] Part of the present invention also includes the use of said peptides and / or oligomers for the manufacture of a therapeutic agent for a viral infection.

The peptide of the present invention and an agent derived therefrom (construct)
ts) can be used in different drug dosage forms for the treatment of various viral disorders. Examples include (oral) sprays, ointments, gels, and sweetened tablets (candy) for the treatment of herpes, after ulcers and viral bronchial infections.
lozenges).

The peptides according to the invention and the oligomers derived therefrom can be used in different drug dosage forms for the treatment of herpes, after ulcers and viral bronchitis infections. Particularly recommended forms are (mouth) spray,
There are ointments, gels and sweetened tablets (lozenges).

The present invention will be further described by way of examples, which are provided for illustrative purposes only and do not limit the present invention in any way.

<Examples> Example 1 (Peptide synthesis) The peptide of the present invention was prepared by the method of Van't Hof et al.
91) and Helmerhorst et al. (1997). The peptide is 9-
Synthesized using the T-bag method adapted to fluorenylmethoxycarbonyl ((Fmoc) chemistry). The p-benzyloxybenzyl alcohol resin to which the first N-Fmoc protected amino acid is already attached is
-Placed in the bag. The coupling reaction was performed in N, N-dimethylformamide. After the amino acid chain is completed, the amino acid chain is cleaved from the resin and the side chain protecting groups are 5% thioanisole, 5% phenol, 5% water and 8%.
Removed with a mixture of 5% trifluoroacetic acid. Purity analysis is by reversed-phase HPLC
(Reverse phase high performance liquid chromatography) and showed a single peak with only a few peptide contaminants (less than 5%).

[0027] All peptides were 2 mg / l in 10 mM sodium phosphate buffer pH 7.4.
Dissolved to a concentration of ml and stored at -20 ° C. The exact peptide concentration used in the antiviral potency assay was determined by amino acid analysis.

Table 1 shows an overview of the peptides 2 to 13 thus prepared. In the table, peptides 1 and 2 indicate histidine 5 and its C-terminal, respectively.

[0029]

[Table 1]

Example 2 (Antiviral activity against herpes simplex (herpes) virus (HSV)) In a test vial, 10 μl of HSV (Department of Virology, Leiden University Hospital (
Phylum), experimental (room) variant 96-6700P (TCID ₅₀ (50% tissue culture infectivity)
The peptide and NaPB were supplemented to 10 ^{5 to} 10 ⁶ )) to a volume of 200 μl. As a positively charged control (sample), the peptide was replaced with human neutrophil defensin pool (HNP _1-3 ). The test bottle was then incubated at 37 ° C. for 3 hours. afterwards,
The 10-fold dilutions were added to Dulbecco's with 2% fetal calf serum (FSC _i ).
Made in Modified Eagles Medium (DMEM).
Various peptides synthesized in the same manner were used as negatively charged controls (samples) in this experiment.

The monkey kidney cells (Vero-cells) were added to a detachment buffer (0.2% in PBS).
(5% trypsin and 0.03% EDTA mixture), washed and brought to a cell concentration of 2 × 10 ⁵ cells per ml DMEM + 2% FCS _i . 96
Wells-plates (Nunclo)
n)), 100 μl of cell suspension (2 × 10 ⁴ cells) were added per well. Acyclovir (ACV) was administered about 2 hours before infection (
At t = -2 hours), several reservoirs were added as control agents. At time t = 0, 50 μl of diluent was added to each reservoir.

After culturing for 3 days in an individual CO ₂ -drying room at 37 ° C., the cytopathological effect (cpe
) Was evaluated by counting under a microscope, and TCID ₅₀ (50%
Tissue culture infectious titer was determined by the Reed & Muench method.
ethod) (Dulbecco and Ginsberg, "Virology", J. B. Lippincott, Philadelphia, 2nd edition (1988) (Dulbecc
o & Ginsberg, "Virology", JB Lippincot
t Co. , Philadelphia, 2nd edition, 1988)
It measured using.

FIG. 1 shows that at sufficiently high concentrations (50 μg / ml), peptide 10 is at least as effective as acyclobar or HNP.

Thereafter, the effectiveness of peptide 10 was measured at appropriate times. The concentration of the control sample was HNP _1-3 50 μg / ml for Acyclovir (ACV) and 5 μg / ml for Acyclovir (ACV).
ml. The same formulation was used for the first part of the test for this purpose,
Samples were collected after 5 minutes, 30 minutes, 1 hour, 2 hours, and 3 hours.
. The results are shown in FIG.

Thereafter, as described above, a series of HNPs and a peptide 10 according to the present invention were compared for dilution activity in the same manner. Acyclobar was again used as a positively charged control (sample). FIG. 3 shows that peptide 10 is more effective than HNP at high concentrations.

Example 3 Removal of Measles Virus Different concentrations of peptide 11 and peptide 10 dimer in PBS (pH 7.4) were mixed with 5-10 μl of measles virus stock in a reagent bottle and 200 μl of After the final volume, the cells were cultured at 37 ° C. for 3 hours. After the elapse of the culture time, the test bottle was placed on ice, and immediately, 2% FCS, 100 U / ml penicillin G, 100 μg / m 2
Serial dilutions were made in DMEM using 1 streptomycin and 20 mM HEPES buffer, pH 7.4. A series of dilutions are placed on a tissue culture monolayer,
TCID ₅₀ was measured using the above-mentioned Read and Munch method.

FIG. 5 shows the result. Measles virus T as a result of peptide 11
The difference in the effect of pre-incubation time between CID ₅₀ and the TCID ₅₀ of the adenovirus as a result of the dimerization of peptide 10 suggests that different mechanisms of activity co-exist.

Example 4 (Human immunodeficiency virus type 1 (HIV-1) with a peptide according to the present invention)
Neutralization) Virus Journal Vol. 69 523-527
(1995) (Groenlink M et al., J. Virol 6).
9, 523-527 (1995)).
The neutralization efficacy test was performed for b. In essence, this potency assay is tested using an inoculum of 457 TCID ₅₀ HIV / ml for neutralization incubated for 2 hours at 37 ° C. with a series of two-fold dilutions of test peptide (final concentration up to 800 μg / ml). Resulting in the ability of different peptides to neutralize the virus. The culture was performed in a 5 mM phosphate buffer. Evaluation was based on the cytopathic effect (syncytium formation) detected on MT-2 cells on days 7, 14 and 21. Table 2 shows the results on day 21.

[0039]

[Table 2]

Table 2 shows that peptide 11 has the strongest neutralizing activity. Furthermore, only peptide 11 was found to be cytotoxic (damaging) at the highest concentration (800 μg / ml).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/12 C07K 14/00 // C07K 7/08 ZNA 14/47 14/00 A61K 37/02 14 / 47 43/00 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ , TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , A, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Fant Hof Willem The Netherlands NL- 2313 ZZ Leiden Serpencarde 61 (72) Inventor Nivering Peter Hendrikus The Netherlands NL-2215 SL Fall Hout Chopin Lahn 5 F term (reference) 4C076 AA06 AA09 AA24 CC35 4C084 AA02 AA07 AA12 BA01 BA23 BA23 BA23 BA23 BA23 4 BA36 BA41 CA59 NA14 ZB332 4H045 AA10 BA16 BA18 CA40 EA29 FA33

Claims (31)

[Claims] 1. An amino acid chain comprising 10 to 25 amino acid domains, wherein the majority of half amino acids in the domain are positively charged amino acids and the majority of amino acids in the other half of the domain are uncharged amino acids. A peptide used as an antiviral agent. 2. The domain forms an α-helix and comprises 1-, 2-, 5-
, 6-, 9- (12-, 13-, 16-, 19-, 20-, 23- and 24-
) Position contains a positively charged amino acid, the 8-position contains a positively charged or uncharged amino acid, and 3-, 4-, 7-, 10- (11-, 14-, 1
The peptide according to claim 1, wherein at least a majority of the 5-, 17-, 18-, 21-, 22- and 25-) positions contain an uncharged amino acid. 3. The domain forms an α-helix and at least a majority of the 1- to 6- (or 7- or 8- or 9- or 10- or 11- or 12-) positions are uncharged amino acids. And 7- (or 8- or 9- or 10- or 1
The peptide according to claim 1, wherein the peptide contains a positively charged amino acid at the 1- or 12- or 13-) to 25-position. 4. An amino acid wherein the domain forms an α-helix and at least a majority of the 1- to 6- (or 7- or 8- or 9- or 10- or 11- or 12-) positions are positively charged. The peptide according to claim 1, wherein the peptide comprises an uncharged amino acid at positions 7- (or 8- or 9- or 10- or 11- or 12- or 13-) to 25-. 5. The method according to claim 1, wherein said domains form a so-called β-strand,
-, 5-, 7-, 9- (11-, 13-, 15-, 17-, 19-, 21-, 2
At least a majority of the 3- and 25-) positions contain positively charged amino acids and are 2-, 4-, 6-, 8-, 10- (12-, 14-, 16-, 18-, 20-,
The peptide of claim 1, wherein at least a majority of the 22- and 24-) positions contain uncharged amino acids. 6. The positively charged amino acid is ornithine (O), lysine (K
The peptide according to any one of claims 1 to 5, wherein the peptide is selected from the group consisting of), arginine (R) and histidine (H). 7. The uncharged amino acid is glycine (aliphatic amino acid).
G), alanine (A), valine (V), leucine (L) and isoleucine (I
), Methionine (M) which is an amino acid having a bipolar side chain, asparagine (N)
Glutamine (Q), serine (S) and threonine (T), and phenylalanine (F) which is an amino acid having an aromatic side chain, tyrosine (Y) and tryptophan (W). The peptide according to any one of claims 1 to 6, wherein 8. The peptide according to claim 1, wherein the majority of the positively charged amino acids is a value obtained by subtracting 1 from the total number of the positively charged amino acids. 9. The peptide according to claim 1, wherein the majority of the uncharged amino acids is a value obtained by subtracting 1 from the total number of the uncharged amino acids. 10. The peptide according to claim 1, wherein the domain constitutes the whole peptide. 11. The peptide according to claim 1, wherein the N-terminus is amidated. 12. The method according to claim 12, wherein the C-terminal carboxyl group is an amide, ester, ketone,
The peptide according to any one of claims 1 to 11, wherein the peptide is substituted with an aldehyde or alcohol group. 13. The peptide according to claim 2, wherein said domain has the following amino acid sequence: KRLFKELKFSLRKY (peptide 3) 14. The peptide according to claim 2, wherein said domain has the following amino acid sequence: KRLFKELLFSLRKY (peptide 4) 15. The peptide according to claim 2, wherein said domain has the following amino acid sequence: KRLFKELKKSLRKY (peptide 5) 16. The peptide according to claim 2, wherein said domain has the following amino acid sequence: KRLFKELLKSLRKY (peptide 6) 17. The peptide according to claim 2, wherein said domain has the following amino acid sequence: OOOLFOELOSLOOY (peptide 7) 18. The peptide according to claim 2, wherein said domain has the following amino acid sequence: OOOLFOELLOSLOOY (Peptide 8) 19. The peptide according to claim 2, wherein said domain has the following amino acid sequence: KRLFKKLKFSLRKY (peptide 9) 20. The peptide according to claim 2, wherein said domain has the following amino acid sequence: KRLFKKLLFSLRKY (peptide 10) 21. The peptide according to claim 2, wherein said domain has the following amino acid sequence: LLLFLKKRKKKRY (peptide 11) 22. The peptide oligomer according to any one of claims 1 to 21, which comprises at least two mutually linked peptides which may be used as an antiviral agent and may be via a spacer if necessary. 23. The bond of the monomeric peptide is a head-to-head bond, ie, N-
23. The oligomer according to claim 22, wherein the ends are facing each other. 24. The bond of the monomeric peptide is a tail-to-tail bond, ie, C-
23. The oligomer according to claim 22, wherein the ends are facing each other. 25. The bond of a monomeric peptide is a head-to-tail bond or a tail-to-head bond, that is, the bond between the C-terminus of one monomer and the N-terminus of a second monomer or vice versa. 23. The oligomer according to claim 22, wherein 26. The amino acid sequence α, ε- (KRLFKKLLFSLKKY) ₂
25. The oligomer according to claim 24 having -K-amide (peptide 10-dimer). 27. The amino acid sequence α, ε- (LLLFLLKKRKKRKY) _Two 25. The oligomer according to claim 24, having -K-amide (peptide 11-dimer).
- 28. Use of a peptide according to any one of claims 1 to 21 and / or an oligomer according to any one of claims 22 to 25 for the manufacture of a therapeutic agent for a viral infection. 29. A pharmaceutical composition for the treatment of viral infection, comprising one or more peptides according to claims 1 to 21 and / or oligomers according to claims 22 to 25 and one or more suitable excipients. . 30. The pharmaceutical composition according to claim 29, which is in the form of a spray, ointment, gel or candy. 31. A peptide according to any of claims 1 to 21 which is part of a hybrid peptide (along with other peptides, lipids, oligosaccharides, (radioactive) isotope tracers, organic receptor ligands, etc.). And a peptide polymer conjugate.