RU2771493C1 - Peptide with antibacterial activity against microorganisms with multidrug resistance - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biochemistry and can be used in medicine and veterinary medicine. Developed is a new RFR-ChBac3.4(1-14)-NH₂ antimicrobial peptide engineered by modifying (introducing deletions and inserts) the amino acid sequence of natural ChBac3.4 bactenecin.

EFFECT: synthetic RFR-ChBac3.4(1-14)-NH₂ peptide has a high antimicrobial activity against gram-positive and gram-negative bacteria, including multidrug-resistant bacteria, inhibits the growth of pathogenic bacteria synergistically with a number of antibiotics, and is also capable of preventing the formation of biofilms from pathogenic bacteria.

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Description

The invention relates to the field of biochemistry and can be used in medicine and veterinary medicine.

Currently, the world is experiencing a wide spread of pathogenic microorganisms that are resistant to drugs, including simultaneously to several drugs of different chemical nature and different mechanism of action. This phenomenon is called “multi-drug resistance”. The growing use of antibiotics in medicine, veterinary medicine, as well as the addition of antibiotics to the feed of farm animals, birds and fish on farms contributes to the emergence and spread of such microorganisms in nature.

Studies of the genomes of microorganisms with multiple drug resistance show that they contain the following determinants: loss of outer membrane porins due to mutations in the corresponding chromosomal genes, activation of efflux membrane pumping systems caused by the same causes, and acquisition of new drug-degrading enzymes and encoded mobile genetic elements, primarily transmissible plasmids [Nikado H. Multidrug Resistance in Bacteria. Annu Rev Biochem. 2009; 78:119-146]. Treatment of bacterial infections caused by these microorganisms is a difficult task for clinical medicine [Gootz T.D. The Global Problem of Antibiotic Resistance. Critical Reviews in Immunology, 2010, 30 (1): 79-93].

A striking example is the strain of Klebsiella pneumoniae, which in 2016 in the United States caused the death of a patient from sepsis and turned out to be resistant to all 26 drugs prescribed by the state regulatory authority (USA Food and Drug Administration) for the treatment of this infection [Chen L., Todd R. , Kiehlbauch J., Walters M., Kallen A. Notes from the Field: Pan-Resistant New Delhi Metallo-Beta-Lactamase-Producing Klebsiella pneumoniae. CDC Morbidity and Mortality Weekly Report, 2017, 66(1): 33. doi: 10.15585/mmwr.mm6601a7].

The problem of the spread of strains of pathogenic microorganisms with multidrug resistance in Russia does not differ from the situation in other countries [Ergeshov A., Andreevskaya S.N., Larionova E.E., reflects the transmissibility of mutant clones. Molecular biology, 2017. V. 51 No. 4 S. 595-602].

Thus it is medical Smirnova T.G., Chernousova L.N. The spectrum of mutations in the genes associated with resistance to rifampicin, isoniazid and fluoroquinolones in clinical strains of Mycobacterium tuberculosis science is losing the fight against pathogenic microorganisms, not having time to discover and introduce into healthcare practice new drugs, the resistance of bacteria to which is still unknown.

An urgent task is to find ways to treat infections caused by multidrug-resistant microorganisms.

Antimicrobial peptides produced by protective cells of higher eukaryotes are part of the innate immune system and can serve as the basis for the development of new antibiotics. It has also been proposed to use antimicrobial peptides in conjunction with known antibiotics to inhibit the growth of multidrug resistant bacteria.

For example, proposed synthetic peptides with a length of 10 to 14 amino acid residues, with a high content of arginine [RU 2590706]. The peptide RRRFRFFFRFRRR disclosed in this patent, which showed the best results in comparison with the other proposed peptides, meanwhile had high minimum inhibitory concentrations for the growth of gram-positive and gram-negative bacteria in the range from 7.8 to 62.5 mg/ml, which is higher than the minimum inhibitory concentrations (MIC) of the peptide of the present invention, calculated in the range of 1-32 μg/ml (0.5-32 μm).

Artificial synthetic peptides lexicin-1 are known, the sequence of which is IGVLKKYFKIGALIKAIIK [RU 2715854] and the peptide GRFKRFRKKLKRLWHKVGPFVGPILHY [RU 2702661], which have an antimicrobial effect, but their activity against bacteria with multidrug resistance is not shown.

[WO 9840401] discloses the use of the indolicidin peptide and its analogs, obtained by partial substitutions, deletions and insertions of individual amino acids, substitutions of individual amino acids with their D-analogues, chemical modification of the N- and C-termini of the molecule, to suppress bacterial growth and overcome resistance bacteria to antibiotics. This patent also claims the use for this purpose, together with antibiotics (ciprofloxacillin, vancomycin, piperacycline, gentamicin, tobramycin), indolicidin peptide and its analogues, and a number of known types of antimicrobial peptides, including some bactenecins.

It is known that bactenecins - highly cationic peptides of neutrophil granules - have high bactericidal activity [Gennaro R, Skerlavaj B, Romeo D. Purification, composition, and activity of two bactenecins, antibacterial peptides of bovine neutrophils. Infect Immun. 1989; 57(10): 3142-3146. doi: 10.1128/IAI.57.10.3142-3146.1989] and have a high (20% to 50%) proline content.

It is believed that the antimicrobial action of proline-rich peptides is the result of blocking the aminoacylating site of bacterial ribosomes, disruption of protein folding, and, at higher concentrations, destruction of bacterial membranes. [Scocchi M., Tossi A., Gennaro R. Proline-rich antimicrobial peptides: converging to a non-lytic mechanism of action. cell. Mol. life sci. 2011. 68: P. 2317-2330 doi: 10.1007/s00018-011-0721-7; 37; Mardirossian M., Grzela R., Giglione C., Meinnel T., Gennaro R., Mergaert P., et al. The host antimicrobial peptide Bac 71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis. Chem. Biol. 2014.21: 1639-1647. doi: 10.1016/j.chembiol.2014.10.009].

Meanwhile, none of the proposed natural or synthetic peptides with antibacterial action is still used in healthcare practice.

The purpose of the invention is to expand the spectrum of peptides with antibacterial action, including against bacteria with multidrug resistance, with a low level of cytotoxic and hemolytic action.

To achieve the goal of the present invention, a modification was made of the natural proline-rich peptide bactenecin ChBac 3.4, found in leukocytes of the domestic goat Capra hircus, which has a wide spectrum of antimicrobial activity against both gram-positive and gram-negative bacteria [Shamova O., Orlov D., Stegemann C. , Czihal P., Hoffmann R., Brogden K., et al. ChBac3.4: A novel proline-rich antimicrobial peptide from goat leukocytes. Int. J. Pept. Res. Therap.2009. 15:1, P. 31-42. doi: 10.1007/s10989-009-9170-7].

The sequence of amino acid residues of the natural bactenecin peptide ChBac 3.4, amidated from the C-terminus, is shown in Fig. one.

To achieve the goal of the invention, analogues of ChBac 3.4 were designed, synthesized and subjected to comparative studies, differing in substitutions of some amino acids, carboxylated C-terminus, deletions and insertions of individual amino acids, as well as shortened variants - N- and C-terminal fragments of the molecule.

The amino acid sequence of the RFR-ChBac3.4(1-14)-NH ₂ peptide is shown in FIG. 2.

The test results showed that the synthetic peptide variant, named RFR-ChBac 3.4(1-14)-NH ₂ , has antimicrobial activity against gram-positive and gram-negative bacteria, including pathogenic bacteria - it is active against both planktonic (free-floating) bacteria and inhibits the development of biofilms by pathogenic bacteria resistant to several drugs. At the same time, the resulting peptide does not have a pronounced toxicity to human cells.

Comparison of amino acid sequences showed the difference between the obtained peptide and known peptides with antibacterial properties. The created peptide can be used to suppress the growth of multidrug-resistant bacteria, including those forming biofilms.

Molecular design of the synthetic peptide RFR-ChBac3.4(1-14)-NH ₂

The RFR-ChBac 3.4(1-14)-NH ₂ peptide contains a ChBac 3.4 fragment containing amino acid residues 1 to 14, and the N-terminal three amino acid sequence (RFR) is doubled. The C-terminus of the peptide is amidated. The amino acid sequence of the synthetic RFR-ChBac 3.4(1-14)-NH ₂ peptide is shown in Seq ID No. 1 and in FIG. 2. comparison of the amino acid sequences of the peptides ChBac 3.4 (upper sequence) and RFR-ChBac3.4(1-14)-NH ₂ (lower sequence) are shown in FIG. 3.

The molecular weight of the obtained peptide is 2319.8 Da, the peptide has a high content of basic amino acids, it contains 7 residues (41.18%) of arginine.

Chemical synthesis of peptides

Peptides ChBac3.4 and ChBac3.4(1-14)-NH ₂ were obtained by solid-phase synthesis from Fmoc-amino acids using an automatic peptide synthesizer Symphony X (Protein Technologies, USA). After completion of the synthesis and removal from the resin, the peptide was purified by reverse-phase high-performance liquid chromatography RP-HPLC on a Beckman Gold System chromatograph, column - Vydac C18 column, 10×250 mm, 5 μm. The purity of the peptides, according to analytical chromatography, was 96-98%. The molecular weight was confirmed by MALDI TOF mass spectrometric analysis.

The properties of the obtained synthetic peptide, which distinguish it from the original natural peptide - ChBac 3.4 bactenecin, are illustrated by examples 1-5, showing that the RFR-ChBac3.4(1-14)-NH ₂ peptide, characterized by the amino acid sequence Seq ID No 1, can be used to inhibit the growth of bacteria, including multidrug resistant bacteria.

Example 1. Study of growth inhibition of gram-positive and gram-negative microorganisms by peptides ChBac3.4 and RFR-ChBac3.4(1-14)-NH ₂

Minimal inhibiting concentrations (MIC) of the peptides were studied using the method of serial dilutions in liquid nutrient medium (Muller-Hinton broth). Terasaki microchambers for cultivating microorganisms were pre-treated with 0.1% bovine serum albumin (BSA) to prevent sorption of peptides on the plate walls. Serial dilutions of the peptides in a sterile 10 mM Na-phosphate buffer solution, pH 7.4, containing 0.1% BSA were used (with a step of 1:2). growth phase and containing 1×10 ⁶ CFU/ml. The final sample volume was 10 μl and all samples were repeated three times. After 20 hours of cultivation at 37°C, the MIC for each strain of bacteria was determined as the lowest concentration of the peptide that inhibits the growth of bacteria, calculated from the results of 3-5 experiments. Also, the geometric mean MIC value was calculated separately for gram-negative and gram-positive bacteria, designated as GMIC _Gr(-) and GMIC _Gr(+) and the geometric mean GMIC _total for all studied bacteria.

The bacterial strains used for testing are shown in Table 1.

The test results are presented in table 2.

The results presented in Table 2 show that both peptides have similar activity in inhibiting the growth of Gram-negative bacteria, while the RFR-ChBac3.4(1-14)-NH ₂ peptide is more active in inhibiting the growth of Gram-positive bacteria. GMIC _total for the synthetic peptide is reduced compared to the natural peptide.

Example 2. Study of the combined action of the peptide RFR-ChBac3.4(1-14)-NH ₂ and antibiotics to suppress the growth of pathogenic bacteria with multidrug resistance

This study was carried out under the conditions described in example 2, as follows:

The peptide, triturated with a dilution step of 1:2, was added to each row of the microchamber plate for culturing bacterial cells, and the antibiotic triturated in the same way was added to each column. Various combinations of peptide and antibiotic concentrations were thus obtained.

Only dilutions of the peptide were added to the first row, and only dilutions of the antibiotic were added to the last column to establish the MIC of each component separately. After overnight incubation with bacteria, based on the results of taking into account the growth of bacteria in the cells of the microchamber, fractional concentration indices of inhibition (fraction inhibitory concentration index - FICI) of bacterial growth were calculated using the formula:

,

,

where [A] and [B] are the concentrations of A and B in combinations that effectively inhibit the growth of bacteria, and [MICA] and [MICB] are the minimum inhibitory concentrations of A and B when used individually.

The combined effect was estimated as follows:

Minimum FICI ≤ 0.5 - synergism of the peptide and antibiotic (the combined action of the peptide and antibiotic is higher than the simple sum of their individual actions).

0.5 < Minimum FICI ≤ 1.0 - additivity (the actions of the peptide and antibiotic add up)

1.0 < Minimum FICI < 2.0 - independent action (one component acts as if the second was absent).

2 < Minimal FICI - antagonism.

[Orhan G, Bayram A, Zer Y, Balci I. Synergy tests by E test and checkerboard methods of antimicrobial combinations against Brucella melitensis. J Clin Microbiol. 2005 Jan; 43(1): 140-3. doi: 10.1128/JCM.43.1.140-143.2005].

Investigated the action of a number of antibiotics, a brief description of which is given in table. 3.

The results are presented in table 4.

The results presented in Table 4 show that both peptides in most cases are characterized by synergism or additivity of their action with the studied antibiotics.

Both peptides are characterized by independent action with oxacillin for all studied bacterial strains and with meropenem for E. coli ESBL 521/17. Also, independent action of the ChBac3.4 peptide with meropenem was shown for K. pneumoniae ESBL 344/17.

However, Table 4 shows that the synthetic peptide RFR-ChBac-3.4(1-14)-NH ₂ has an advantage over the natural peptide ChBac3.4. So, when used together with erythromycin against A. baumannii, with meropenem against P. aeruginosa, with oflokacin against K. pneumoniae and P. aeruginosa, the synthetic peptide RFR-ChBac-3.4(1-14)-NH ₂ showed synergistic action with antibiotics, and the natural peptide ChBac3.4 showed only additivity.

Example 3 Peptide Inhibition of Biofilm Formation by Multidrug Resistant Pathogenic Microorganisms

Microbial cells have the ability to multiply on various surfaces, forming so-called biofilms. Biofilms contain a dense matrix that protects the cells they contain from drugs and effectors of the immune system. In addition, biofilms contain metabolically inactive bacterial cells that are resistant to antibiotics.

Chokr A, Martinez-de-Tejada G. AMPsasAnti-biofilm Agents for Human Therapy and Prophylaxis. Adv Exp Med Biol. 2019; 1117:257-279. doi: 10.1007/978-981-13-3588-4_14].Most human infectious diseases, such as pneumonia, wound infections, cystic fibrosis, dental caries, and others, are caused by film-forming bacteria [Shahrour H, Ferrer-Espada R, Dandache I,

Chokr A, Martinez-de-Tejada G. AMPsas Anti-biofilm Agents for Human Therapy and Prophylaxis. Adv Exp Med Biol. 2019; 1117:257-279. doi: 10.1007/978-981-13-3588-4_14].

To study the effect of peptides on the formation of biofilms, 50 µl of the peptide, serially triturated in a nutrient medium, was introduced into the cells of a 96-well culture plate. Then, an equal volume of a night culture of Pseudomonas aeruginosa MDR522/17 (strain No. 4 in Table 1) or Acinetobacter baumanii 7226/16 (strain No. 2 in Table 1) diluted 50 times was introduced into the cells.

After incubation at 37°C for 24 hours, the plate was carefully washed from non-adherent bacteria by immersion in a vessel with distilled water, after which the cells attached to the walls of the plate and biofilm matrix components were stained with 0.1% crystal violet dye solution for 10 minutes, after which the plate was washed again, dried, the bound dye was dissolved in 30% acetic acid solution and its amount was determined spectrophotometrically.

Each concentration of the peptide was examined in four parallel samples. The results presented in Table 5 are calculated from three independent experiments.

Thus, the synthetic peptide RFR-ChBac3.4(1-14)-NH ₂ has the ability to suppress the formation of biofilms by multidrug-resistant pathogenic bacteria.

Example 4. Hemolytic activity of peptides ChBac3.4 and RFR-ChBac3.4(1-14)-NH ₂

Peripheral blood samples from healthy donors, collected in standard EDTA vacutainers, were subjected to centrifugation at 300 g and +4°C for 10 minutes, after which the cell pellet was resuspended in buffered saline (PBS). This procedure was repeated two more times to remove blood plasma components and traces of anticoagulant.

The final pellet was resuspended in 10 ml saline and 27 µl of this suspension was added to each sample. To the samples, 3 µl of the titrated peptide solution were added. The mixtures were incubated for 30 minutes at +37°C, after which they were centrifuged at 10,000 g for 3 minutes. The release of hemoglobin into the supernatant was measured spectrophotometrically at a wavelength of 540 nm.

Two controls were used: 100% lysis of erythrocytes was determined in samples with the addition of 3 μl of 1% Triton-X-100 solution, 0% lysis was determined in samples with the addition of 3 μl of PBS instead of the peptide solution.

Another variant of the synthetic ChBac3.4 was used as a positive control, which differed by two tryptophan inserts at positions 10 and 22 and the absence of an amide group at the C-terminus. Three parallel samples were placed on each experimental point, and all experiments were performed three times. The percentage of hemolysis was calculated by the formula

Hemolysis (%)=(A sample - A0% lysis) / (A100% lysis - A0% lysis) × 100%,

where A is the optical density of the solution at a wavelength of 540 nm (A540 nm)

As a result, it was shown that both peptides - ChBac3.4 and RFR-ChBac3.4(1-14)-NH ₂ - at concentrations from 0 to 64 μM do not cause erythrocyte lysis that exceeds the background value, while another variant of the synthetic ChBac3.4 (positive control) showed 19% lysis at 32 μM and 25% at 64 μM. Thus, the synthetic peptide RFR-ChBac3.4(1-14)-NH ₂ has no hemolytic activity.

Example 5 Cytotoxicity study of peptides ChBac3.4 and RFR-ChBac3.4(1-14)-NH ₂ against cultured human cells

Mononuclear cells of human peripheral blood, isolated from the blood of healthy donors by the standard Ficoll gradient centrifugation method and resuspended in RPMI-1640 medium, were introduced into the wells of the culture plate, 2×10 ⁴ cells per well. Serial dilutions of the peptide solution were also added to the wells.

To obtain a positive control (100% live cells), culture medium was added to the cell instead of a peptide; in a negative control (0% live cells), culture medium was added instead of cells.

The plates were incubated at 37°C and 5% CO ₂ overnight, after which a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the wells at a rate of 5 mg/ml .

After 4 hours of cultivation, isopropanol acidified with 0.04 M HCl was added and the absorbance was determined at a wavelength of 540 nm (A ₅₄₀ ). A ₆₉₀ values were also determined for each sample, which were subtracted to equalize the background. The content of surviving cells in the samples was calculated by the formula

Survival (%) \ _u003d (A mod - A _{0% live cells} ) / (A _{100% live cells} - A _{0% live cells} ) × 100%

where: A _o6p - A ₅₄₀ - A _{690 nm} of the sample;

A _{100% live cells} - A ₅₄₀ - A _{690 nm} positive control (100% live cells);

A ₀ % live cell - A ₅₄₀ - A _{690 nm} negative control (0% live cells)

All experiments were performed three times, using 3 parallel samples for each experimental point.

The indicator of toxicity in relation to human mononuclear blood cells - 50% inhibitory concentration (inhibitory concentration ₅₀ ). Toxicity values in IC _{pbmc 50% were} calculated by non-linear regression analysis using the Sigma Plot 11 program (Systat Software Inc., USA).

The result of dividing IC _pbmc 50% by _{GMIC total} (the value of GMIС _total is taken from Table 2) is the selectivity index (SI _h/b ), showing the ratio of the average concentration of the peptide that destroys 50% of human mononuclear blood cells to the geometric mean MIC for all tested microbial cells. The results are presented in table 6.

The results presented in table 6 indicate the multiple superiority of the RFR-ChBac3.4(1-14)-NH ₂ peptide over the ChBac3.4 peptide in terms of the selectivity index. With a high and, in some cases, higher ability to suppress bacterial growth than that of ChBac3.4, the RFR-ChBac3.4(1-14)-NH ₂ peptide has a more than 4.3-fold reduced cytotoxic effect in relation to human blood mononuclear cells than the ChBac3.4 peptide.

Conclusion.

Thus, the synthetic peptide RFR-ChBac3.4(1-14)-NH ₂ , which is a modification product of the natural ChBac3.4 peptide, has clear advantages over the natural peptide:

- At lower minimum inhibitory concentrations, it inhibits the growth of pathogenic strains of gram-positive bacteria: Staphylococcus aureus 1399/17, resistant to ampicillin, oxacillin, gentamicin, amikacin, ofloxacin, and Staphylococcus aureus MRSA (ATCC 33591), resistant to methicillin;

- At a lower minimum inhibitory concentration, it inhibits the growth of the pathogenic gram-negative Escherichia coli 521/17 strain resistant to ampicillin, amoxicillin / clavulonic acid, cefotaxime, ceftazidime, cefixime, aztreonam, netilmicin, ciprofloxacin, trimethoprim / sulfamethoxazole;

- Possesses synergistic inhibition of the growth of bacterial strains resistant to antibiotics when used together with antibiotics: with erythromycin against Acinetobacter baumannii 7226/16 (resistant to imipenem, gentamicin, tobramycin, ciprofloxacin, trimethoprim / sulfamethoxazole), with meropenem and oflokacin against Pseudomonas aeruginosa MDR 522 /17 (resistant to meropenem, ceftazidime, cefixime, amikacin, gentamicin, netilmicin, ciprofloxacin, colistin), with oflokacin against Klebsiella pneumoniae ESBL 344/17 (resistant to ampicillin and other penicillins), in contrast to the natural peptide ChBac3.4, which showed only additivity with these antibiotics.

- The cytotoxic effect of the RFR-ChBac3.4(1-14)-NH ₂ peptide on human blood mononuclear cells is reduced by more than 4.3 times compared to the original ChBac3.4 peptide.

Also, the RFR-ChBac3.4(1-14)-NH ₂ peptide is able to suppress the formation of biofilms by pathogenic microorganisms with multidrug resistance and does not have hemolytic activity.

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SEQUENCE LIST

<110> FGBNU “IEM” (FSBSI “IEM”

<120> A peptide having antibacterial activity against

multidrug resistant microorganisms

<140>

<141>

<150>

<151>

<160> 1

<170>

<210> SEQ ID No: 1

<211> 17

<212> Amino acid sequence

<213> Artificial sequence

<220>

<223> Amino acid sequence of RFR-ChBac3.4(1-14)-NH ₂ peptide

<400> 1

Arg Phe Arg Arg Phe Arg Leu Pro Phe Arg Arg Pro Pro Ile Arg Ile His-NH2

1 5 10 15

<---

Claims (2)

1. Peptide of the formula RFRRFRLPFRRPPIRIH-NH ₂ having antibacterial activity against multidrug resistant microorganisms and characterized by the amino acid sequence of SEQ ID No: 1. 2. A method for inhibiting the growth of bacteria, including pathogenic bacteria with multidrug resistance, which consists in the fact that the peptide according to claim 1 is used together with an antibiotic selected from the group: erythromycin, meropenem, ofloxacin, amikacin.

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