The peptide having biocidal activity

 

(57) Abstract:

The invention relates to new peptides of General formula 1 (H-a-Lys-d-Trp-Lys-c - Pro-d-Lys-Pro-Trp-e-Arg-NH2where a = -Ile or Lys; b= -Pro - or-Lys-; C, d = -Lys or Trp; e=Arg or Ala, having biocidal activity, which combine higher compared to indolizidines biocidal activity with no damaging effects on blood cells. table 4.

The invention relates to the field of biotechnology, and in particular to a new peptide structures having biocidal, in particular antibacterial properties.

To date, pharmacology and medicine have created a situation which can be described as the accumulation of tolerance to a fairly wide range of pharmacological preparations. This primarily refers to antibiotics. Many of the traditional antibiotics, which in the second half of our century have been the main instrument in the fight against various infections, gradually lose their active quality due to the constant increase in the number of bacterial strains resistant to the drug (Cassell G. H. Emergent antibiotic resistance: health risks and economic impact // FEMS Immunol. Med. Environ. - 1997. - v.18.- 4.-p.271-274).

Development of new active creation of preparations on the basis of antibiotic peptides, currently used in practical medicine or undergoing clinical trials as an antibiotic, anticandidal and antitumor agents (Hancock R. E. V. Peptide antibiotics // Lancet - 1997.-v.349.-N 4.-R. 418-422; Andreu d, Rivas L. Animal antimicrobial peptides: an overview // Biopolymers (Peptide Science) - 1998.- v.47.- 6.-p.415-433).

The mechanism of cell killing microorganisms such peptides linked to the ability of these compounds to form when interacting with a double layer of lipid cell membrane amphipatic spiral, characterized by cation enriched and hydrophobic areas, which leads to lysis of the cell.

Side effects of antibiotic peptides on the background of their high antibiotic effect is cytolytic activity against a number of normal cells / Bikshapathy E., Sitaram N., Nagaraj R. Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences, model membrane binding and biological activities // J. Pept.Res. - 1999.-v.53.- 1.-p. 47-55/.

Closest to the claimed solution on the structure and properties were peptide indolicidin General formula (1):

(1) H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2, (Selsted, M. E. , Novotny, M. J., Morris W. L, Tang Y. Q., W. Smith, J. S. Cullor, Indolicidin, a novel bactericidal tridecapeptide amide from neurophils. // J. Biol. Chemistry. - vol.267. - 7. - 1992. - p.4292-4295), which possess the viruses etc.

Lack of indolizidine is high hemolytic activity.

The problem faced by the authors in creating the present invention was to develop more effective and safer peptides devoid of cytotoxic side effect of red blood cells, that is, not having hemolytic activity.

It was found that facing the authors of the problem can be solved by creating a peptide of General formula (2):

(2) H-a-Lys-d-Trp-Lys-c-Pro-d-Lys-Pro-Trp-e-Arg-NH2,

where a = -Ile or Lys;

b = -Pro - or-Lys -;

c, d = -Lys - or-Thr-;

e = -Arg or Ala-.

The best results were obtained when using peptides (3-7)

(3) H-Ile-Lys-Pro-Trp-Lys-Lys-Pro-Trp-Lys-Pro-Trp-Arg-Arg-NH2,

(4) H-Ile-Lys-Pro-Trp-Lys-Trp-Pro-Trp-Lys-Pro-Trp-Arg-Arg-NH2,

(5) H-Ile-Lys-Pro-Trp-Lys-Trp-Pro-Trp-Lys-Pro-Trp-Ala-Arg-NH2,

(6) H-Lys-Lys-Pro-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Arg-Arg-NH2,

(7) H-Ile-Lys-Lys-Trp-Lys-Lys-Pro-Trp-Lys-Pro-Trp-Arg-Arg-NH2.

Peptides receive solid phase method of Merrifield (Memfield R. B. and G. Barany in Solid Phase peptide synthesis. // The Pepthides. Analysis, Synthesis, Biology. - Eds. Gross K., J. Meinhofer, N. Y. - Academic Press. - 1980. - vol. 2. - p. 3.) on methylbenzhydrylamine using Vos technology.

For temporary protection of amino groups using tert.-Boo is a group of the arginine - the nitro group, the amino group of a lysine - 2-chlorobenzonitrile protection. The extension of the peptide chain is carried out using oxibendazole ethers with in situ neutralization (Schnolzer, M., Alewood, P. , Jones, A., Alewood, D., Kent, S. B. H. In situ neutralization solid phase peptide synthesis. Rapid, high yield assemdly of difficult sequences. // Int.J.Pept.Prot.Res. - 1992.-v.40.- 2-p.180-193).

Completeness of the reaction control ninhydrin test. Cleavage from the resin and removal of protective groups is carried out in two stages. In the first stage, directly on the polymer using piperidine remove the formyl group in the second phase hydrogen fluoride in the presence of anisole to produce the final release and cleavage of the peptide from the resin.

Purification of synthesized peptides is performed using high performance liquid chromatography high pressure (VGH) on reversed phase.

The purity of the resulting preparations was greater than 95%. They all had satisfactory amino acid analysis and were individually according to analytical HPLC.

Industrial applicability of the invention is illustrated by the following examples.

Example 1. Synthesis of peptide H-Ile-Lys-Pro-Trp-Lys-Lys-Pro-Trp-Lys-Pro-Trp-Arg-Arg-NH2(3)

For the synthesis of peptide were loaded into 10 ml of dimethylformamide. The content of arginine 1.0 mmol/g of polymer. The peptide chain was further increased with the end of the program, are given in table. 1.

If the ninhydrin test is positive, repeated condensation, starting with p. 7.

For the synthesis of the peptide (3) to the original N-tert-butyloxycarbonyl-N-nitro-L-arginine-methylbenzhydrylamine program, are given in table. 1, was sequentially added N-tert-butyloxycarbonyl-N-nitro-L-arginine, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyl-oxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyl-oxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine and N-tert-butyloxycarbonyl-L-isoleucine.

Upon completion of the synthesis, the peptidyl-polymer was dried in a vacuum desiccator over pjatiokisi phosphorus and 0.2 g of peptidyl-polymer is th chromatography on a column With 18 Nova Pack 19300 in a gradient of 0-70% acetonitrile in 0.1% triperoxonane acid. The content of the basic substance, according to the optical density not less than 95%. The amino acid composition of the peptide corresponds to theoretical.

Example 2. Synthesis of peptide H-Ile-Lys-Pro-Trp-Lys-Trp-Pro-Trp-Lys-Pro-Trp-Arg-Arg-NH2(4)

Synthesis, release, purification and characterization of peptide (4) was carried out in the same way that the peptide (3) in example 1, except that to get to the source of N-tert-butyloxycarbonyl-N-nitro-arginine-methylbenzhydrylamine-polymer according to the program shown in table 1, was sequentially added N-tert-butyloxycarbonyl-N-nitro-L-arginine, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine and N-tert-butyloxycarbonyl-L-Isola the hydrated peptide was subjected to purification by reversed-phase chromatography on a column With 18 Nova Pack 19300 in a gradient of 0-70% acetonitrile in 0.1% triperoxonane acid.

The content of the basic substance, according to the optical density not less than 95%. The amino acid composition of the peptide corresponds to theoretical.

Example 3. Synthesis of peptide H-Ile-Lys-Pro-Trp-Lys-Trp-Pro-Trp-Lys-Pro-Trp-Ala-Arg-NH2(5)

Synthesis, release, purification and characterization of peptide (5) are carried out in the same way that the peptide (3) in example 1, except that to get to the source of N-tert-butyloxycarbonyl-N-nitro-L-arginine-methylbenzhydrylamine program, are given in table. 1, was sequentially added N-tert-butyloxycarbonyl-L-alanine, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-L-Proline, N-tert-butylcyclopentadienyl-L-lysine, N-tert-Butylochka-carbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine,N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine and N-tert-butyloxycarbonyl-L-isoleucine.

Polucheniya reversed-phase chromatography on a column With 18 Nova Pack 19300 in a gradient of 0-70% acetonitrile in 0.1% triperoxonane acid.

The content of the basic substance, according to the optical density not less than 95%. The amino acid composition of the peptide corresponds to theoretical.

Example 4. Synthesis of peptide H-Lys-Lys-Pro-Trp-Lys-Trp-Pro-Lys-Lys-Pro-Trp-Arg-Arg-NH2= (6)

Synthesis, release, purification and characterization of peptide (6) are carried out in the same way that the peptide (3) in example 1, except that to get to the source of N-tert-butyloxycarbonyl-N-nitro-L-arginine-methylbenzhydrylamine-polymer according to the program shown in the table. 1, was sequentially added N-tert-butyloxycarbonyl-N-nitro-L-arginine, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine and N-tert-bout is about the program, are given in table. 2, lyophilized peptide was subjected to purification by reversed-phase chromatography on a column With 18 Nova Pack 19300 in a gradient of 0-70% acetonitrile in 0.1% triperoxonane acid.

The content of the basic substance, according to the optical density not less than 95%. The amino acid composition of the peptide corresponds to theoretical.

Example 5. Synthesis of peptide H-Ile-Lys-Lys-Trp-Lys-Lys-Pro-Trp-Lys-Pro-Trp-Arg-Arg-NH2(7)

Synthesis, release, purification and characterization of peptide (7) are carried out in the same way that the peptide (3) in example 1, except that to get to the source of N-tert-butyloxycarbonyl-N-nitro-L-arginine-methylbenzhydrylamine program, are given in table. 1, was sequentially added N-tert-butyloxycarbonyl-N-nitro-L-arginine, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyloxycarbonyl-Nin-formyl-L-tryptophan, N-tert-butyloxycarbonyl-L-Proline, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine, N-tert-butyloxycarbonyl-N-chlorobenzylidene biloxicasino-L-lysine, N-tert-butyloxycarbonyl-N-chlorobenzenesulfonyl-L-lysine and N-tert-butyloxycarbonyl-L-isoleucine.

The resulting release program, are given in table. 2, lyophilized peptide was subjected to purification by reversed-phase chromatography on a column With 18 Nova Pack 19300 in a gradient of 0-70% acetonitrile in 0.1% triperoxonane acid.

The content of the basic substance, according to the optical density not less than 95%. The amino acid composition of the peptide corresponds to theoretical.

Example 6. The effect of synthetic peptides on the growth of microorganisms.

Cell culture grown on solid nutrient medium, passed the night in the refrigerator at +6oC. Before measurement, the cells were removed by a loop in a liquid LB and brought a concentration of up to 5106-107cells/ml In sterile tablets "Costar" cell culture (96 wells) were made in 50 μl of peptide solution in water (concentration of 200 μg/ml) and 100 μl of cell suspension in LB medium, and spent the next dilution step 2. Incubated the plates for 16 hours at 37oWith, shook the tablets at room temperature for 20-30 minutes for weighing cells and removed the optical indicators is assessed % inhibition of growth of the organism compared to control.

The results are given in table. 3.

Example 7. Hemolytic activity of the peptides.

The human erythrocytes (blood is taken on heparin) were washed 3 times Veronelli buffer (VBS) and diluted to a concentration of 0.5% (vol.) in VBS. Samples of the peptides were dissolved at a concentration of 200 μg/ml in VBS, while the initial concentration of peptide in the cell was 100 μg.

The peptide solution was introduced into the cells sterile tablets for the cell culture company "Costar", starting with a concentration of 100 μg of drug and 50 μg of human erythrocytes in the cell. Spent rastarivanie with step 2. The plates were incubated 1 hour at 37oC, centrifuged, the supernatant was collected and organized the screening of the optical density at 415 nm.

As control was used: K1- VBS without peptide;2- VBS with 1% Triton X-100. The indicators of hemolysis was treated according to the formula:

< / BR>
The results are given in table. 4.

Our data indicate that the peptides of the claimed structure combine higher compared to indolizidines biocidal activity with no damaging effects on blood cells. Tested peptides with internal introduction to capocasale lack of toxicity for all peptides.

The peptide of General formula

H-a-Lys-d-Trp-Lys-c-Pro-d-Lys-Pro-Trp-e-Arg-NH2,

where a = -Ile or Lys;

b = -Pro - or-Lys-;

C, d = -Lys or Trp;

e = Arg or Ala

having biocidal activity.

 

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