RU2302425C2 - Peptides exhibiting antimicrobial activity - Google Patents

Abstract

FIELD: biotechnological methods.

SUBSTANCE: two forms of peptide H₂N-Ser¹-Met²-Trp³-Ser⁴-Gly⁵-Met⁶-Trp⁷-Arg⁸-Arg⁹-Lys¹⁰-Leu¹¹-Lys¹²-Lys¹³-Leu¹⁴-Arg¹⁵-Asn¹⁶-Ala¹⁷-Leu¹⁸-Lys¹⁹-Lys²⁰-Lys²¹-Leu²²-Lys²³-Gly²⁴-Glu²⁵-X, where X is free carboxylic group or Lys26 are isolated from Central Asian spider Lachesana tarabaevi, the both exhibiting antimicrobial; activity.

EFFECT: enlarged assortment of antimicrobial agents.

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Description

The invention relates to biochemistry, specifically to biologically active peptides with antimicrobial activity, which may find application in biotechnology and medicine.

The number of known antibiotics is growing rapidly, bacteria, fungi, plants and animals serve as natural sources, many are de novo chemical synthesis products. Nevertheless, only about 200 antibiotics are actually used in medical practice, while most are not used for a number of reasons (toxicity, inactivation in the body, etc.). A number of antibiotics are indispensable medications used for infections previously considered incurable. However, due to the increase in the number of microorganisms causing infectious diseases, as well as due to changes in the etiological structure of infectious diseases, the problem of finding new antibiotics is urgent.

The need for such a search is even more obvious if we take into account the problem of resistance of microorganisms. With the widespread use of antibiotics as therapeutic drugs, there is a rapid accumulation of forms of microorganisms resistant to these compounds, clinically significant resistance occurs over a period of several months to several years. The vast majority of antibiotics used belong to one of the structural classes of compounds, among which only one is relatively new, and the rest were introduced into clinical practice more than 40 years ago, i.e. there is an “innovative vacuum” in the field of invention of fundamentally new antimicrobial agents [Walsh, S. 2003. Where will new antibiotics come from? Nat. Rev. Microbiol. 1: 65-70]. It should be noted that cases of resistance of common human pathogens are known, such as Enterococcus faecalis, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, Streptococcus pneumoniae. Vibrio cholerae and many others, to almost any of the drugs used [Walsh, S. 2000. Molecular mechanisms that confer antibacterial drug resistance. Nature 406: 775-781].

In the course of studies of the mechanisms underlying the protective reactions of the host organism to the appearance of a pathogen, a new class of natural antibiotics produced by animals and higher plants was discovered. It turned out that a large role in repelling the attack of pathogens by the host immune system is played by the so-called antimicrobial peptides. Today, thanks to the accumulated experimental data, an idea has been formed about these molecules as a universal and evolutionarily ancient mechanism for protecting plant and animal organisms from pathogens [Finlay, B.B., Hancock, R.E.W. 2004. Can innate immunity be enhanced to treat microbial infections? Nat. Rev. Microbiol. 2: 497-504].

Despite the existence of stronger antibiotics, antimicrobial peptides have several advantages. The ability to quickly kill target cells, an unusually broad spectrum of activity, activity against strains resistant to other antibiotics, and the relative difficulty in breeding resistant mutants in vitro allow us to consider these substances as the basis for creating effective drugs, especially against the background of a decrease in the effectiveness of conventional antibiotics in view of the increasing number of resistant strains [Hancock, REW, Lehrer, R. 1998. Cationic peptides: a new source of antibiotics. Trends Biotechnol. 16: 82-88].

To date, several hundred antimicrobial peptides are known [Brahmachary, M. et al. 2004. ANTIMIC: a database of antimicrobial sequences. Nucleic Acids Res. 32: D586-D589], among which several structural classes are distinguished [Brogden, K.A. 2005. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3: 238-250]. However, for most antimicrobial peptides, the target of the action is, apparently, the cell membranes of pathogenic organisms, and the mechanism of action is a violation of the normal permeability of these membranes. The vast majority of antimicrobial peptides are characterized by the following structural and functional features: the general positive charge of the molecule and the tendency to form amphipathic structures, which is believed to be the basis for their interaction with membranes, as well as the ability to inhibit the growth of a number of microorganisms in concentrations of the order of 1-10 μm.

Several models have been proposed to explain the increase in the permeability of the membranes of target cells under the action of antimicrobial peptides. The most valid model for today is the formation of toroidal lipid-peptide pores due to the active incorporation of peptide molecules into the lipid bilayer [Yang, L. et al. 2001. Barrel-stave model or toroidal model? A case study on melittin pores. Biophys. J. 81: 1475-1485].

Antimicrobial peptides that do not contain cysteine residues occupy a special place among others, since such a structural feature greatly facilitates their production by chemical or biotechnological means and sharply reduces production costs. Among the latter, much attention is paid to peptides prone to organization into an amphipathic α-helix upon contact with membranes; approaches to optimizing their properties in order to increase the therapeutic potential are discussed [Tossi, A., Sandri, L., Giangaspero, A. 2000. Amphipathic, α-helical antimicrobial peptides. Biopolymers 55: 4-30].

Among the properties that must be controlled in potential antibiotics, the cytotoxic effect on the cells of the macroorganism, more often evaluated in standard tests for hemolytic activity. The presence of similar activity in a number of antimicrobial peptides is a serious drawback and limits the possibility of their use as medicines.

Closest to the claimed peptides in structure and antimicrobial properties is a fragment of a lipopolysaccharide-binding peptide from rabbit granulocytes [Tossi, A. et al. 1994. Identification and characterization of a primary antibacterial domain in CAP 18, a lipopolysaccharide binding protein from rabbit leukocytes. FEBS Lett. 339: 108-112]. The fragment consists of 21 amino acid residues, completely inhibits the growth of certain bacteria at concentrations of 0.5-4 μM and does not exhibit hemolytic activity at 50 μM. A full-sized lipopolysaccharide-binding peptide from rabbit granulocytes [Larrick, J.W. et al. 2000. U.S. patent No. 6103888] contains 37 amino acid residues, belongs to the group of cathelicidins and is also active against a number of bacteria in concentrations of 0.5-4 μM.

The invention solves the problem of expanding the range of antimicrobial peptides.

The problem is solved due to the structure of the peptide having the following amino acid sequence:

H ₂ N-Ser ¹ -Met ² -Trp ³ -Ser ⁴ -Gly ⁵ -Met ⁶ -Trp ⁷ -Arg ⁸ -Arg ⁹ -Lys ¹⁰ -Leu ¹¹ -Lys ¹² -Lys ¹³ -Leu ¹⁴ -Arg ¹⁵ -Asn ¹⁶ -Ala ¹⁷ -Leu ¹⁸ -Lys ¹⁹ -Lys ²⁰ -Lys ²¹ -Leu ²² -Lys ²³ -Gly ²⁴ -Glu ²⁵ -X,

where X is the free carboxyl group (latarcin-1, LtAMP-1) or the lysine residue Lys ²⁶ (latarcin-1a, LtAMP-1a).

The name of the latarcin peptide comes from the initial syllables of the Latin name of the producer spider Lachesana tarabaevi and the ending -qin, generally accepted to indicate the bactericidal properties of a substance.

The claimed latarcin peptides exhibit antimicrobial activity against a number of gram-positive (Arthrobacter globiformis, Bacillus subtilis, Micrococcus luteus) and gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria, as well as yeast (Pichia pastoris). The minimum concentrations of peptides necessary for complete inhibition of the growth of these organisms in vitro (minimum inhibitory concentrations) are 0.4-17 μM. Moreover, LtAMP-1 and LtAMP1a latarcins are characterized by low hemolytic activity (20% erythrocyte lysis at a peptide concentration of 100 μM). In addition, the claimed peptides have antitumor activity: at a concentration of 6.6 μM cause the death of 50% of human lung adenocarcinoma cells. Thus, the technical result of the present invention is the high antimicrobial and antitumor activity of the peptides LtAMP-1 and LtAMP1a, combined with a low hemolytic, and hence the high therapeutic potential of the proposed antibiotics, which allows us to consider them as the basis for creating drugs with a wide profile for the fight against pathogens of infectious diseases of various origins, as well as malignant tumor formations.

The peptides LtAMP-1 and LtAMP1a consist of 25 and 26 amino acid residues, respectively, i.e. are relatively small molecules, which simplifies their production by chemical synthesis. In addition, the peptides are linear and do not contain cysteine residues, which also simplifies their production by chemical synthesis or biotechnology, since it eliminates the risk of formation of inactive forms due to incorrect closure of disulfides.

The latarcin-1 peptide (LtAMP-1) is obtained from a natural source - the poison of the Central Asian spider Lachesana tarabaevi Zonstein et Ovtchinnikov, 1999, which belongs to the family Zodariidae, order Araneae, class Arachnida, type Arthropoda.

The latarcin-1a peptide (LtAMP-1a) is obtained by chemical synthesis, it is an analog of latarcin-1, differs from the latter by the C-terminal lysine residue and exhibits biological properties identical to latarcin-1.

The inventive peptides are new compounds and are not close homologues of already known proteins or peptides.

The invention is illustrated by examples.

Example 1

Isolation of the antimicrobial peptide Latarcin-1 (LtAMP-1) from the spider venom Lachesana tarabaevi.

The whole venom of the spider Lachesana tarabaevi is subjected to HPLC separation on a Jupiter C ₅ reverse phase column (2 × 150 mm, pore size 300 Å, particle diameter 5 μm, Phenomenex, USA). Fractionation is carried out in a linear gradient of the concentration of acetonitrile from 0 to 60% (v / v) in 0.1% (v / v) trifluoroacetic acid for 60 minutes with an elution rate of 0.3 ml / min. Detection is carried out by optical absorption at 210 nm. The resulting fractions are tested for their ability to inhibit the growth of Escherichia coli DH5α. The LtAMP-1 peptide elutes from the column as a fraction with a retention time of 37 minutes (FIG. 1).

The isolated fraction was then separated by HPLC on an Ascentis RP-Amide column (2.1 × 100 mm, particle diameter 3 μm, Supelco, USA). Fractionation is carried out in a linear gradient of a concentration of acetonitrile from 10 to 60% (v / v) in 0.1% (v / v) trifluoroacetic acid for 50 min with an elution rate of 0.3 ml / min. Detection is carried out by optical absorption at 210 nm. The resulting fractions are tested for their ability to inhibit the growth of Escherichia coli DH5α. The LtAMP-1 peptide eluted from the column with a retention time of 29.5 minutes (FIG. 2).

The impurity content in the purified fraction corresponding to LtAMP-1 is 5%, as evidenced by HPLC separation on a Luna C ₁₈ analytical column (1 × 150 mm, pore size 100 Å, particle diameter 3 μm, Phenomenex, USA) in a linear gradient acetonitrile concentration from 20 to 50% (v / v) in 0.1% (v / v) trifluoroacetic acid for 40 min with an elution rate of 50 μl / min, detection is carried out by optical absorption at 210 nm.

Example 2

Determination of the relative molecular weight of latarcin LtAMP-1 and LtAMP-1a.

The identity of the purified substances is confirmed by mass spectrometric analysis. Mass spectra were obtained on an ultraflex II TOF / TOF MALDI time-of-flight mass spectrometer (Bruker Daltonik, Germany) with identification of positive ions in a reflex mode. The matrix used is α-cyano-4-hydroxycinnamic acid (10 mg / ml) in 50% (v / v) acetonitrile containing 0.1% (v / v) trifluoroacetic acid. To calibrate the instrument, a standard mixture of peptides with a molecular weight range of 700-3500 Da is used (Bruker Daltonik, Germany).

The measured monoisotopic molecular weight of the natural peptide LtAMP-1 is 3072.1 Da (the calculated value is 3071.8 Da), the synthetic LtAMP-1 and LtAMP-1a are 3071.5 Da and 3199.7 Da, respectively (calculated values: 3071.8 Yes and 3199.9 Yes, respectively).

Example 3

Establishment of the amino acid sequence of latarcin-1.

The N-terminal amino acid sequence of the purified LtAMP-1 peptide was determined by Edman stepwise degradation using a Precise 492 automated sequencer (Applied Biosystems, USA). As a result, the complete amino acid sequence of latarcin-1 is established, consisting of 25 amino acid residues:

H ₂ N-Ser ¹ -Met ² -Trp ³ -Ser ⁴ -Gly ⁵ -Met ⁶ -Trp ⁷ -Arg ⁸ -Arg ⁹ -Lys ¹⁰ -Leu ¹¹ -Lys ¹² -Lys ¹³ -Leu ¹⁴ -Arg ¹⁵ -Asn ¹⁶ -Ala ¹⁷ -Leu ¹⁸ -Lys ¹⁹ -Lys ²⁰ -Lys ²¹ -Leu ²² -Lys ²³ -Gly ²⁴ -Glu ²⁵

The calculated monoisotopic molecular weight of the LtAMP-1 peptide is 3071.8 Da, i.e. differs from the measured by 0.3 Yes. Thus, the antimicrobial peptide Latarcin-1 (LtAMP-1) is a linear peptide and does not contain any chemical modifications. Analysis of the amino acid sequence of latarcin-1 (distribution of hydrophobic and hydrophilic residues, the total charge of the molecule) suggests the tendency of this peptide to form an amphipathic α-helix when interacting with membranes.

Example 4

Chemical synthesis of latarcin-1 (LtAMP-1) and its analogue-latarcin-1a (LtAMP-1a).

Peptides are synthesized by the solid-state method in the manual version by chain extension from the C-terminal residue to a 2-chloro-trityl-chloride (with a load of 0.8-1.44 mmol / g) resin (PepChem, Germany), for temporary protection of α-NH ₂ groups use a fluorenylmethoxycarbonyl group (Fmoc) [Chan, WC, White, PD 2000. Fmoc Solid Phase Peptide Synthesis. University Press, Oxford]. The protection of the side groups of amino acid residues is chosen with the expectation of a final release of trifluoroacetic acid: to protect the side groups of the residues, Glu and Asp use the O-tert-butyl group, for Ser and Thr, the tert-butyl group, for Trp, the tert-butyloxycarbonyl group, for Gln and Asn, the trityl group.

To attach the first amino acid to the resin, 1.5 equiv (relative to the number of active groups on the resin) of the Fmoc-protected amino acid is dissolved in dichloromethane (at the rate of 10 ml of dichloromethane per 1 g of resin) with 1.5 equiv of diisopropylethylamine. An additional 1.5 eq of diisopropylethylamine is added to the resulting solution and poured onto a dry resin, gently mixed for 45 minutes. Unreacted active groups on the resin are blocked with methanol (based on 1 ml of methanol per 1 g of resin) for 20 minutes, after which the resin is washed with three volumes of dichloromethane, three volumes of N, N-dimethylformamide and three volumes of ethanol, and dried under vacuum. Amino acid residues are attached to the peptidyl polymer to form a hydroxybenzotriazole ester in the presence of 1,3-diisopropylcarbodiimide, 1.2 equivalents of N-hydroxybenzotriazole and 1 equivalent of 1,3-diisopropylcarbodiimide are used to activate 1 equivalent of amino acid. To build the polypeptide chain during each synthetic cycle, the peptidyl polymer is condensed with 5 eq of activated Fmoc amino acid (1-12 h at 37 ° C), the content of unreacted amino groups is visually controlled by staining the pH-dependent indicator of bromphenol blue.

Cleavage of the product from a 2-chloro-trityl chloride polymer with simultaneous release of the side groups is carried out with a mixture of trifluoroacetic acid, water and ethanedithiol (95: 2.5: 2.5, v / v / v) based on 1 ml of the mixture per 50 mg of resin . The suspension is stirred for 2 hours, then a solution of the peptide in trifluoroacetic acid is filtered off from the polymer. The product is planted with a 10-fold excess (by volume) of chilled diethyl ether, filtered off, washed twice with ether, and then freeze-dried. The reduction of oxidized methionine residues is carried out by treating a solution of the peptide (2 mg / ml) in 10% (v / v) acetic acid with methyl mercaptoacetamide (10 equiv per 1 equivalent of methionine residues) for 24-48 hours at 37 ° C.

The synthesized peptides were purified by HPLC on a Luna C ₁₈ reverse phase column (10 × 250 mm, pore size 100 Å, particle diameter 10 μm, Phenomenex, USA). Fractionation is carried out in a linear gradient of a concentration of acetonitrile from 0 to 50% (v / v) in 0.1% (v / v) trifluoroacetic acid for 60 min with an elution rate of 2 ml / min. Detection is carried out by optical absorption at 210 nm.

The impurity content in the synthetic preparations LtAMP-1 and LtAMP-1a is 1%, which is confirmed by separation by HPLC on a Luna C ₁₈ analytical column (1 × 150 mm, pore size 100 Å, particle diameter 3 μm, Phenomenex, USA) in linear a gradient of acetonitrile concentration from 20 to 50% (v / v) in 0.1% (v / v) trifluoroacetic acid for 40 min with an elution rate of 50 μl / min, detection is carried out by optical absorption at 210 im.

The identity of the synthesized LtAMP-1 to the natural is proved by mass spectrometric analysis, comparison of chromatographic mobilities when separated on a Luna C ₁₈ analytical column (1 × 150 mm, Phenomenex, USA), as well as a comparison of their biological properties.

Example 5

Obtaining absorption spectra of the peptides LtAMP-1 and LtAMP-1a in the UV region.

To obtain absorption spectra in the UV region of latarcin-1 isolated by chromatographic methods, as well as chemically synthesized latarcin LtAMP-1 and LtAMP-1a, some of them (weighed) are dissolved in 200 μl of pure water. Absorption spectra were obtained on a U-3210 spectrophotometer (Hitachi, Japan). The length of the optical path (the thickness of the cuvette) is 1 cm. Pure water serves as a comparison solution.

The concentration of the peptide is determined by the spectrum (figure 3), using the calculated molar absorption coefficient at 280 nm, which is 11380 l / (mol × cm), according to the formula

C = (A ₂₈₀ -A ₃₂₀ ) / ε, where

C is the concentration of the peptide in mol / l,

And ₂₈₀ - the optical density of the solution at 280 nm,

And ₃₂₀ is the optical density of the solution at 320 nm,

ε is the molar absorption coefficient at 280 nm.

Example 6

Obtaining spectra of circular dichroism of the peptides LtAMP-1 and LtAMP-1a.

CD spectra are measured using a J-810 spectropolarimeter (Jasco, Japan) in a cuvette with an optical path length of 0.01 cm in the wavelength range of 190-250 nm in increments of 1 nm. The conformation of peptides LtAMP-1 and LtAMP-1a in water (pH ~ 5), in a mixture of water (pH ~ 5) with trifluoroethanol in a volume ratio of 1: 1, and in a suspension of liposomes formed by dioleylphosphatidylcholine (DOPC) with a diameter of 100 nm is studied. 40 mM sodium phosphate buffer (pH 7.5) and 83 mM NaCl are present in the liposome suspension.

Monolamellar DOPC liposomes are prepared by extrusion. 40 mM sodium phosphate buffer (pH 7.5, 1 ml) was added to dry lipid (10 mg) and the suspension was incubated with non-vigorous stirring for 40-60 minutes at room temperature. Then spend 10 cycles of freezing and thawing the suspension using a refrigerator based on liquid nitrogen. The resulting lipid suspension was forced through an extruder (Avanti, USA) 20 times through a polycarbonate membrane with a pore size of 100 nm at room temperature. The result is a suspension of large (diameter 100 nm) monolamellar liposomes at a lipid concentration of 10 mg / ml.

The concentration of peptides in water is 0.16 mm, in a solution of water-trifluoroethanol - 0.08 mm, in a suspension of liposomes - 0.1 mm. The lipid / peptide molar ratio is 50: 1. CD spectra are given (Fig. 4) in units of molar ellipticity [Θ] calculated by the formula

[Θ] = 115 × Θ _expert / (10 × C × l), where

Θ _expert - observed ellipticity in _{milli degrees}

C is the concentration of the peptide in mg / ml,

l is the optical path length (cell thickness) in cm,

115 is the average molecular weight of one amino acid residue.

The content of secondary structure elements (Table 1) is estimated using the CONTINLL program [Provencher, S.W., Glockner, J., 1982. Estimation of globular protein secondary structure from circular dichroism. Biochemistry. 20, 33-37] for globular proteins.

Table 1 The content of secondary structure elements for the peptides LtAMP-1 and LtAMP-1a according to CD peptide solution α-helix β structure β-bend disordered structure LtAMP-1 water, pH 5.0 8% 28% 23% 42% trifluoroethanol: water, 1: 1 74% 2% 2% 22% DOPC liposomes 42% 22% 0 36% LtAMP-1a water, pH 5.0 7% 28% 22% 43% trifluoroethanol: water, 1: 1 67% four% 7% 22% DOPC liposomes 25% 2% 22% 34%

An analysis of the obtained spectra shows that (1) in the aqueous medium, the latarcins LtAMP-1 and LtAMP-1a are not prone to the formation of an ordered structure, (2) in an environment imitating membranes (trifluoroethanol), latarcins accept the α-helical conformation, (3) LtAMP -1 and LtAMP-1a interact with membranes, at least consisting of dioleylphosphatidylcholine, and (4) when interacting with membranes, they partially form an ordered structure like an α-helix.

Example 7

Antimicrobial properties of latarcin LtAMP-1 and LtAMP-1a.

To determine the antimicrobial activity of the fractions obtained during the chromatographic separation of the components of the spider venom Lachesana tarabaevi, purified latarcin-1, as well as synthetic LtAMP-1 and LtAMP-1a, the following microorganism strains are used: Escherichia coli MH1, Escherichia coli DH5α, Pseudomonas aerugthrob PAO globiformis BKM 348-10, Bacillus subtilis BKM B-504, Micrococcus luteus NCIMB 13267, Pichia pastoris GS115, Institute of Bioorganic Chemistry, Russian Academy of Sciences, antimicrobial activity was evaluated by the method of inhibiting culture growth in a liquid medium. The determination of the minimum concentrations of peptides necessary for complete inhibition of the growth of microorganisms (minimum inhibitory concentrations) is carried out by the method of serial dilutions using a modified method [Amsterdam, D. 1996. Susceptibility testing of antimicrobials in liquid media, pp.52-111. In Loman, V., ed. Antibiotics in laboratory medicine, 4th ed. Williams and Wilkins, Baltimore].

Bacteria are cultured in liquid low-salt medium LB (1% bacto-tryptone, 0.5% bacto-yeast extract, 0.5% NaCl, pH 7) at 37 ° C and stirring at a speed of 220 rpm, yeast in a liquid medium YPD (1% bacto-yeast extract, 2% bacto-peptone, 2% glucose, pH 5.3) at 30 ° C and stirring at a speed of 150 rpm for 18 hours. The resulting cultures were diluted 200 times using the same medium, and grown under the same conditions for 3-5 hours until the exponential growth phase is reached (optical density of the culture at 620 nm OD ₆₂₀ ~ 0.3-0.5), and then diluted again using the original medium, to a cell concentration of the order of 10 ⁵ colonies forming units (CFU) in 1 ml The cultures are placed in sterile 96-well plates, 90 μl per well, where then 10 μl of test substance solutions of various concentrations obtained by serial dilutions are added. A negative control is a cell culture with the addition of 10 μl of pure water, and a clean culture medium is a positive control. The plates are incubated under the conditions described above for 24 hours for bacterial cultures and 48 hours for yeast, after which OD _{620 is} measured, the minimum inhibitory concentrations are defined as the lowest concentrations of substances that completely inhibit the growth of microorganisms. The experiment is carried out in three independent repetitions.

The results of determining the minimum inhibitory concentrations for latarcin-1 are presented in table.2. The biological activity of synthetic latarcin-1 is identical to the activity of natural. The values obtained for latarcin-1 and latarcin-1a are the same, i.e. The C-terminal lysine residue does not affect the antimicrobial properties of the claimed peptides.

table 2 Antimicrobial properties of latarcin LtAMP-1 and LtAMP-1a strain minimum inhibitory concentration, μm A. Gram-negative bacteria Escherichia coli MH1 0.7 Escherichia coli DH5α one Pseudomonas aeruginosa PAO1 4.1 B. Gram-positive bacteria Arthrobacter globiformis BKM 348-10 0.5 Bacillus subtilis BKM B-504 one Micrococcus luteus NCIMB 13267 0.4 B. Yeast Mushrooms Pichia pastoris GS 115 IBCh RAS 17

Example 8

Testing of latarcin LtAMP-1 and LtAMP-1a for hemolytic activity. The hemolytic activity of the peptides is determined using rabbit erythrocytes. Fresh blood is obtained from the marginal vein of the rabbit's ear, 2 ml of blood is collected in a test tube with 8 ml of Hanks balanced saline solution containing heparin (10 units / ml). Cells are pelleted by centrifugation for 5 min at 180-220 g, resuspended in Hanks solution to a concentration of 1-3 × 10 ⁷ cells / ml. 10 μl of the desired concentration of the peptide solution are mixed with 1 ml of the resulting suspension. As a negative control, 10 μl of the pure Hanks solution is added to the cell suspension. Incubated at 37 ° C and stirring at a speed of 120 rpm for 3 hours, after which the cells are precipitated by centrifugation for 3-5 minutes at 4000 g. 100 μl of the supernatant was collected in sterile 96-well plates; the hemoglobin yield from erythrocytes was estimated by optical density at 414 nm. The hemolytic activity of the samples is expressed in% of the maximum possible hemolysis, the maximum possible erythrocyte lysis (positive control) is obtained by resuspending the cells in pure water instead of Hanks solution also to a concentration of 1-3 × 10 ⁷ cells / ml.

As a result, it turns out that LtAMP-1 and LtAMP-1a latarcins do not have pronounced hemolytic activity, at a concentration of 40 μM hemolysis is only 10%, at a concentration of 100 μM - 20%.

Example 9

Testing Latarcin LtAMP-1 and LtAMP-1a for antitumor activity.

A549 human lung adenocarcinoma cells were cultured in Eagle MEM medium supplemented with 2 mM L-glutamine and 7% fetal bovine serum at 37 ° C in a humid atmosphere with 5% CO ₂ . The day before the experiment, the cells were seeded in 96-well flat-bottomed culture plates (0.3 × 10 ⁶ cells / ml, 150 μl of MEM Needle medium containing 2 mM L-GIn and 7% EBS per well). After 24 hours, the studied peptides were added to the wells in the desired concentration; pure water was added to the wells as a negative control. Cells are incubated with various concentrations of peptides for 3 hours at 37 ° C, then Hoechst33342 fluorescent dyes (10 μM, stains the nuclei of all cells) and propidium iodide (10 μM, stains dead cell nuclei) are added to the wells and 10 minutes later fluorescence images are recorded cells using an Axiovert 200 M inverted fluorescence microscope (Zeiss, Germany) with an AxioCam MRc digital camera (Zeiss, Germany) in the Hoechst33342 fluorescence region (excitation filters - BP 365/12, emission filter - LP 397) and in the field of fluorescence propidium iodide (filters for cans denie - BP 510-560, on the issue - the LP 590). From these images, for each concentration of the peptide, the total number of cells (from 500 to 1000 cells per measurement) and the number of dead cells among them are calculated, and then the concentration of the peptide that causes the death of 50% of the cells, IC ₅₀ , is calculated. The results are averaged over three independent experiments.

As a result, the latarcins LtAMP-1 and LtAMP-1a have a pronounced antitumor activity against human lung adenocarcinoma, IR ₅₀ = 6.6 μM. Thus, latarcins LtAMP-1 and LtAMP-1a can serve as the basis for the creation of antitumor drugs.

Claims (1)

A peptide exhibiting antimicrobial activity having the following amino acid sequence: H ₂ N-Ser ¹ -Met ² -Trp ³ -Ser ⁴ -Gly ⁵ -Met ⁶ -Trp ⁷ -Arg ⁸ -Arg ⁹ -Lys ¹⁰ -Leu ¹¹ -Lys ¹² -Lys ¹³ -Leu ¹⁴ -Arg ¹⁵ -Asn ¹⁶ -Ala ¹⁷ -Leu ¹⁸ -Lys ¹⁹ -Lys ²⁰ -Lys ²¹ -Leu ²² -Lys ²³ -Gly ²⁴ -Glu ²⁵ -X, where X is the free carboxyl group or lysine residue of Lys ²⁶ .

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