PL187999B1 - Method of obtaining peptidic bactericides and fungicides as well as peptides exhibiting bactericidal and fungicidal properties - Google Patents

Abstract

1 The use of peptides prepared on the basis of the sequences of the poison of: these haem-containing proteins (haemoproteins) and their natural and / or synthetic modifications with the formulas ............ e) Human hemoglobin CNBr and / or Arg C peptides, alpha chain with amino acid positions 1-32; 33-76; 77-141; 1-31; 32-92, 93-141, f) Human hemoglobin CNBr and / or Arg C peptides, beta chain having amino acid positions' 1-55, 56-146; 1-30; 31-40, 41-104; 105-146, g) Sperm whale apomyoglobin CNBr peptides with amino acid positions 1-55, 56-131, 132-146, h) a synthetic peptide analogous in sequence to the peptide 59-78 beta globin of human hemoglobin, 1) synthetic peptide analogous in sequence to sperm whale apomyoglobin peptide 59-78, where in the above sequences the corresponding symbols mean 'A, Ala, Alanine, V, Val, Valine, L, Leu, Leucine, I, Ile, Isoleucine; P, Pro, proline, F, Phe, phenylalanma, W, Trp, tryptophan, M, Met, methionine, G, Gly, glycine, S, Ser, serine. T, Thr, threomna, C, Cys, cysteine, Y, Tyr, tyrosin, N, Asn, asparagine, Q Gln, glutamine; D, Asp, Aspartic Acid, E, Glu, Glutamic Acid, K Lys, Lysine; R, Arg, arginine, H, His, histidine, CNBr, cyanogen bromide, which are heme free to produce a pharmaceutical agent with activity bacteriocidal and fungicidal

Description

The subject of the invention is the use of peptide bactericidal and fungicidal agents based on human and / or animal proteins containing heme (haemoproteins).

The invention relates to the biotechnology and pharmaceutical industry, namely the field of obtaining biologically active agents that react with the membranes of Gram (+), Gram (-) bacteria and fungi, causing their perforation and, consequently, the death of microorganisms.

Until now, relatively few clinically useful agents acting directly on the cell membrane of microorganisms have been known and, through its perforation, lead to leakage of the contents and death of the cell. An example of such compounds with limited therapeutic utility are peptide polymyxins which act as cationic detergents.

Extensive research is carried out on endogenous, natural antimicrobial peptides of animals and plants, such as: a and β defensins of mammals, crab protegrin and tachyplesins, plant thionines, frog magainins, oxbactenecins and indolicidin, or silkworm moth cecropins. These compounds are typically cationic amphipathic peptides with a large number of lysine and arginine residues, containing 12 to 45 amino acid residues. Characteristically, many of them assume the alpha-helix conformation if they are in a hydrophobic environment that mimics, for example, the interior of a bacterial cell membrane. Other peptides, in turn, contain a beta structure, often stabilized by the presence of disulfide bridges, as in the case of defensins, and still others are linear peptides rich in proline, tryptophan or histidine residues. As can be seen from this cursory enumeration, natural antibacterial peptides constitute a very diverse group [TIBTECH, 16 (1998) 82-87].

Research on them is widely described in the patent literature, e.g. cecropins: W08604356, W08805826, W08900199; defensins: EP85259, EP162161, EP193351, US 4,659,692; magainines: US-5,221,732, US-5,635,479, US-5,643,876, US-5,686,563). These studies are not only the response of pharmaceutical and biotechnology companies to the increasing demand for effective antibiotics, but also due to the fact that these newly developed peptide compounds act in a way that prevents microorganisms from easily developing resistance to them. So far, the mechanism of action of cationic antibacterial peptides such as melittin, magainin, gramicidin, cecropin and defensins has been studied in detail. The main site of action of these peptides are the cytoplasmic membranes of microorganisms. These compounds have the ability to create channels, perforate the cell membrane or act on the basis of the so-called envelope effect. It is believed that the selectivity of their actions

187,999 per microorganism cells compared to eukaryotic cells is that the surface of the cytoplasmic membranes of the bacteria has a high content of negatively charged lipids, a relatively high electrical gradient potential across the membrane and is cholesterol-free. Additionally, Gram (-) bacteria contain a negatively charged glycolipid - lipopolysaccharide (LPS) in the cell wall.

Based on the antibacterial properties of the bactericidal / permability increasing protein (BPI) protein contained in the intravenous human gastric cells known since 1978, a method of recombinant protein production and its DNA sequence were developed and patented (examples of patents in this field are: US-5,384,942; US-5,488,034; US-5,523,288; US-5,639,727; US-5,652,332; US-5,703,038; US-5,763,567; US-5,770,561; US-5,733,872).

In addition, there are known methods of producing recombinant magainin derivatives (including peptide 21-27 from Xenopus laecis skin) (exemplary patents in this field are: US-5,221,732; US-5,635,479; US-5,643,876; US-5,686,563). The latter compounds may be useful in the treatment of widespread bacterial infections in foot ulcers in advanced diabetic patients and in the treatment of gynecological neoplasms.

Defensins, netrophil peptides and mammalian epithelium have been used in the treatment of bacterial epithelial infection (US Pat. No. 5,656,738).

Antibacterial and antifungal hybrid peptides from naturally occurring peptides: cecropin, defensin, magainin and toxins such as streptolysin, melittin and others are described in the following patents: US-5,519,115; US 5,593,866; US 5,593,866; US-5,714,467, and the production of recombinant antibacterial peptides is described in US-5,593,866; US 5,670,340; US-5,688,767.

A potential limitation to the widespread use of antibacterial peptides is their high production cost. This is due to the fact that natural sources of their occurrence are not efficient enough and the method of chemical synthesis or their production by recombinant technology is still too expensive (in the order of 100 and 20 US dollars per gram, respectively, and the typical daily requirement for the drug is about 0.5- 1 g per day).

The analysis of known and tested antibacterial peptides and the methods of their preparation, all of which are determined by the technical level existing in this field, allows to formulate the task of developing, first of all, a cheap method of isolating a peptide substance with the following properties:

- a wide spectrum of activity from Gram (+) bacteria through Gram (-) to fungi,

- relatively low concentrations at which bacteria are killed [half-lethal dose (LD50) of a few pM]

- uncomplicated production process.

All these properties seem to be fulfilled in the case of the heme-containing peptides described according to the invention and in the method of their production.

The best known hemoglobin binding proteins are hemoglobin and myoglobin. These proteins are known primarily as oxygen transport agents in vertebrates. Hemoglobin in red blood cells carries oxygen, carbon dioxide and hydrogen ions in the blood, and myoglobin carries and stores oxygen in the muscles.

Myoglobin is a single chain polypeptide and hemoglobin is made up of four subunits (2 α chains and 2 β chains). Approximately 75% of the polypeptide chains of both proteins are folded into an alpha helix in the form of eight helical regions (denoted by the letters A-H), and their very compact internal structure is filled almost exclusively with nonpolar amino acid residues. The tertiary structure of myoglobin and the α and β chains of hemoglobin are very similar, despite significant differences in the amino acid sequence of both proteins.

It turned out that hemoglobin and proteins homologous to it are precursors of biologically active peptides formed during physiological and pathophysiological processes as a result of limited proteolysis [Biopolymers 43 (1997) 171-188]. Such peptide degradation products have been found, for example, in the cytosol of human erythrocytes and in the large intestine of pigs. Some peptides obtained from hemoglobin by enzymatic digestion in vitro show biological activity as opioid peptides - hemorphins, others stimulate

187,999 bacterial growth or inhibit the physiological pathways of bacterial killing [Surgery 91 (1982) 75-80; AppliedMicrob. Biotech. 45 (1996) 778-784).

The essence of the invention is based on the fact that the removal of heme from hemoproteins associated with the conformational change of these proteins leads to the activation of "dormant" bactericidal and fungicidal properties, thanks to which they can be precursors of bactericidal peptides and as such can find practical application in medicine and veterinary medicine and in food preservation as peptide "antibiotics".

The method of producing peptide bactericidal and fungicidal agents based on human and / or animal proteins containing heme, is characterized by the following:

- heme is separated from hemoproteins, such as hemoglobin, myoglobin, cytochrome c, isolated by known methods, by precipitating the protein with an acid solution of acetone

- drying or lyophilizing the obtained apoprotein mixture, and preferably the peptide hemoglobin subunits are separated into alpha and beta fractions before drying or lyophilization.

In order to obtain fragments of the polypeptide chain with modified bactericidal and fungicidal activity, the method is used to digest the obtained apoproteins chemically, especially with cyanogen bromide (CNBr), or with proteolytic enzymes, preferably with Arg C proteinase, or chemically synthesize peptide fragments with a sequence analogous to the globins 59 - 78 human or animal.

Peptides with bactericidal and fungicidal activity, produced on the basis of hemoproteins and their natural and / or synthetic modifications, are characterized by the fact that they contain at least one of the peptides of the formula:

a) Human hemoglobin, alpha chain: thirty 40 50 10 twenty NH2-VLSPADKTNV KAAWGKVGAH AGEYGAEALE RMFLSFPTTK TYFPHFDLSH 60 70 80 90 100 GSAQVKGHGK KVADALTNAV AHVDDMPNAL SALSDLHAHK LRVDPVNFKL 110 120 130 140 LSHCLLVTLA AHLPAEFTPA YHASLDKFLA SVSTVLTSKY R-COOH

b) Human hemoglobini, human beta:

10 twenty thirty 40 50 NH2-VHLTPEEKSA VTALWGKVNV DEVGGEALGR LLWYPWTQR FFESFGDLST 60 70 80 90 100 PDAVMGNPKV KAHGKKVLGA FSDGLAHLDN LKGTFATLSE LHCDKLHVDP 110 120 130 140 ENFRLLGNVL VCVLAHHFGK EFTPPVQAAY QKW / AGVANA LAHKYH-COO

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c) Sperm whale skeletal muscle myoglobin:

20 30 40 50

NH2-VLSEGEWQLV LHVWCKVECD VAGHGQDILI RLFKSHPETL EKFDRFKHLK

70 80 90 100

TEAEMKASED LKKHGVTVLT ALGAILKKKG HHEAELKPLA QSHATKHKIP

110 120 130 140 150

IKYLEFISEA IIHVLHSRHP GDFGCDCQGC MNIKALELFRK DIAAKYKELG

YQG-COOH

d) Cytochrome C from the horse's heart:

20 33 44 50

NH2-GDVEKGKKIF VQVQKQCQTV VKGGGHKTTP NLHGLFGRKT GQAPGFTYTD

00 80 90 100

CNKNKGITWK EETLMEYLYN PKKHIPGTHM IFCGIKKKTE REDLICYLKK

CTNE-COOH

e) human hemoglobin CNBr and / or Crg Q peptides, alpha chain with amino acid positions 1-32, 33-76; 77-141; 1-31; 32-92; 93-141;

f) human hemoglobin CNBr and / or Crg C peptides, beta chain with amino acid positions 1-55; 56-146; 1-30; 31-40; 41-104; 105-146;

g) sperm whale apomyoglobin CNBr peptides with amino acid residue positions 1-55; 56-131; 132-146;

h) a synthetic peptide analogous in sequence to the human hemoglobin beta globin 59-78 peptide;

i) a synthetic peptide analogous in sequence to sperm whale apomyoglobin peptide 59-78.

In the above sequences the appropriate symbols are: C, Cla, alanine; V, Val, valine; L, Leu, Leucine; I, Ile, isoleucine; P, Pro, proline; F, Phe, phenylalanine; W, Trp, tryptophan; M, Met, methionine; G, Gly, glycine; S, Cheese, Serine; T, Thr, threonine; Q, Cys, Cysteine; Y, Tyr, Tyrosine ,; N, Csn, asparagine; Q Glut, Glutamine; D, Csp, aspartic acid; E, Glu, Glutamic Acid; K. Lys, Lysine; R, Crg, arginine; H, His, histidine; CNBr, cyan bromide.

The composition of peptide bactericidal and fungicidal agents prepared according to the invention, containing as active ingredient one or any combination of peptide substances derived from human or animal proteins containing heme, exhibits bactericidal and fungicidal parameters comparable to other substances of this type known so far, i.e. peptide "antibiotics" disclosed for example in the patents US-5,221,732; US 5,384,942; US 5,488,034; US-5,519,115; US-5,523,288; US 5,593,866; US 5,635,479; US 5,639,727; US 5,643,876; US 5,652,332; US 5,656,738; US 5,670,340; US 5,686,563; US 5,688,767; US 5,691,301; US 5,703,038; US 5,714,467; US 5,733,872; US 5,753,614; US 5,763,567; US-5,770,561) and with conventional antibiotics [Antibiotics and Chemotherapy Red H.P. Lambert and F.W. O'Grady, Medical Publishing House 1994]

The properties of the prpteud factors according to the invention have been verified experimentally in in vitro tests (Table I - III). The in vitro tests also show that the peptide bactericidal and fungicidal agents obtained according to the invention do not cause haemolysis of human and animal blood cells and have only a slight cytotoxic effect on

187,999 human fibroblasts or HepG2 cells compared, for example, to nonspecifically acting melittin.

Raw material for the production described by The invention of peptides with bactericidal and fungicidal properties, i.e. animal blood, erythrocyte mass or purified proteins, is easy to obtain in sufficient quantities for production, and the production process consisting of simple activities is extremely easy and cheap.

All these advantageous features outlined above mean that the peptide agents of the invention can be used in the preservation of food and feed and in medicine and / or veterinary medicine as a basic bactericidal and fungicidal ingredient in a composition or as an adjunct to treatment with traditional antibiotics.

Embodiments of the invention, as well as the methods used to characterize the physicochemical and biological properties of the substances according to the invention are presented below and illustrated in the drawings, in which Fig. 1 shows a diagram of the separation of cyanogen fragments of myoglobin by RP-HPLC, Fig. 2 - a diagram of the separation of digestion products alpha globin and beta proteinase Arg C by RP-HPLC method, Fig. 3a to 3c - graphs of the effect of pH, divalent ions and salt on E. coli membrane perforation on the peptide concentration in complete nutrient medium, Fig. 4 - graph of the effect of cyanogen bromide peptide 56-131 of myoglobin on the cell lines, and Figure 5 is a graph depicting the circular dichroism spectrum of the myoglobin 46-131 peptide cyanide.

Example I

The starting material for the preparation of the bactericidal and fungicidal substances according to the invention is: human or animal blood; human or animal erythrocyte mass; human or animal hemoglobin; sperm whale or horse muscle myoglobin; horse cytochrome c. 200 ml of blood collected for any anti-clotting substance (heparin, EDTA, sodium citrate, etc.) is centrifuged for 15 minutes with an acceleration of 2,000 g at 4 ° C, the plasma layer with the white "scum" of leukocytes is removed, and the erythrocyte sedimentation is diluted to 1 liter with 0.9% NaCl solution and centrifuged again as above. The washing operation is carried out at least three times. The thus obtained packed erythrocytes devoid of serum proteins are lysed by dilution with distilled water to a volume of 500 ml. The erythrocyte shadows are centrifuged for 30 minutes at 16,000 g at 4 ° C and discarded, and the hemoglobin aqueous solution is concentrated by ultrafiltration on a PM 30 membrane (Amicon) and lyophilized. About 10 g of purified hemoglobin are obtained. The preparation prepared in this way is storage-stable at 4 ° C.

In order to activate the bactericidal and fungicidal properties of the preparation, it is subjected to further treatment consisting in separation of heme from protein by precipitation with an acid solution of acetone. 10 g of the preparation are dissolved in 500 ml of distilled water and 4500 ml of ice-cold acetone containing 0.02 N HCl are added. The precipitate of the resulting protein is centrifuged for 15 minutes at 1,000 g at 4 ° C and the precipitation and dissolution procedure is repeated until the supernatant is colorless after centrifugation, ie until the heme is completely removed.

The apoglobin precipitate consisting of a mixture of alpha and beta globins is air dried or dissolved in 100 ml of distilled water and lyophilized. About 5 g of a globin mixture is recovered. The preparation is storage stable at 4 ° C. The mixture of alpha and beta globins thus obtained can be separated into individual globins by reverse phase high performance liquid chromatography (RP-HPLC) on a C-18 column or by ion exchange chromatography on CM-52 gel [Biol. Chem. Hoppe-Seylers 368 (1987) 681-690].

The same is done in the case of obtaining apomyoglobin from commercial preparations of whale skeletal muscle myoglobin (Sigma) or horse muscle myoglobin (Sigma), while the silver sulfate method is used to separate the heme from cytochrome c from the horse heart (Sigma) [J Biol. Chem. 148 (1973) 3188-3195]. The concentration of native proteins and apoglobins is determined spectrophotometrically using known absorbance coefficients.

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Example II

The alpha and beta hemoglobin subunits, apomyoglobin, apocytochrome c obtained as described in Example 1 were tested in an in vitro test for antibacterial and antifungal activity analogously as described in the Infect publication. Immun. 64 (1996) 4444-4449 except that: Enterococcus faecalis was grown in TSB (trypticase soy broth) medium and Candida albicans in Sabouraud medium (Difco). The following bacterial strains were tested: Staphylococcus aureus ATCC 8325-4 (American Type Cell Culture), Escherichia coli ATCC 33694, Enterococcus faecalis ATCC 29212, Salmonella serotype Krefeld KOS 1497, Klebsiella oxytoca CBM21, Pseudomonas aeruginosa and Candida albicans strain (both clinical). The protein solutions were pre-incubated in 10-fold diluted with HBSS (ang. Hank's Balanced Salt Solution) of 10 ⁵ colony-forming units (CFU) of bacteria in a semi-logarithmic phase of growth for 1 hour at 37 ° C.

After dilution, the pre-incubated bacteria were plated on the appropriate agar medium and left at 37 ° C overnight to obtain macroscopic colonies. The half-lethal dose (LD50) was calculated as the micromolar concentration needed to reduce the number of grown colonies by 50% relative to the control not incubated with the peptide. The results presented in Table I prove that the native proteins hemoglobin and myoglobin did not affect the viability of the tested microorganisms even at the concentration of 330 pM, while the heme-depleted alpha and beta globins and apomyoglobin reduced the number of colonies by 50% at concentrations of the order of micromoles per liter, similar to apocytochrome C. Thus, removal of the heme and the conformational change of the polypeptides under study induced by the described procedure leads to the production of peptide "antibiotics" with a broad spectrum of activity, from Gram (+) bacteria, through Gram (-) to fungi.

Example III

Peptide fragments from a mixture of globins or apomyoglobin (preparations obtained according to Example 1) were obtained by chemical digestion thereof for 24 hours at room temperature in 10% cyanogen bromide (CNBr) solution in 70% formic acid. After incubation, the mixture was diluted with 5 volumes of distilled water and lyophilized. The cyanogen bromide peptides of the globin mixture were separated by molecular filtration on Sephadex G-50 Pharmacia gel equilibrated with 1% actic acid and then on a Waters pBondapak C-18 column in the RP-HPLC water-TFA-acetonitrile system. The peptide fragments of cyanogen bromoglobin were split directly on the C-18 column, and an exemplary separation was obtained from 1 mg of protein in the system: water-TFA-acetonitrile (buffer A-0.1% TFA; buffer B-80% acetonitrile in 0.07% TFA) is shown in Fig. 1. Peptide globin fragments identified by mass spectroscopy (MS) were marked with a pair of numbers defining their location in the corresponding polypeptide chain. The obtained and purified apoglobin peptide fragments were tested for antibacterial activity against E. coli similarly as shown in Example 2 (the results are presented in Table II) and additionally against other microorganisms in the case of apomyoglobin bromide peptides (results are presented in Table III).

Example IV

Peptide fragments were also obtained by enzymatic digestion of the mixture of hemoglobin alpha and beta globin obtained according to Example 1 with the specific Arg-C proteinase (proteinase described in US Pat. No. 5,523,390). A sample of the globin mixture denatured in 6 M guanidine hydrochloride is reduced with dithiothreitol and the cysteine residue is carbamidomethylated with iodoacetamide. The modified protein is desalted on an Amicon PM-10 membrane or on a C-8 column. The enzymatic digestion reaction was then performed for 12 hours at 37 ° C with a 1: 150 weight ratio of the enzyme to the digested globin mixture in an incubation mixture containing: 0.2 M Tris-HCl, pH 7.4, 4 M urea, 10 mM L-cysteine and 1 mM CaCl ₂ .

The peptides were then separated on a Waters C-18 column in the RP-HPLC system in the system: water-TFA-acetonitrile (buffer A-0.1% TFA; buffer B-80% acetonitrile in 0.07% TFA) and an exemplary separation of 1 mg The peptide fragments are shown in Fig. 2. Based on the results, the localization of the obtained peptide fragments in the sequence of the digested protein

187,999 by mass spectroscopy (MS) using the MsFit program available on the internet at http://falcon.ludwig.ucl.ac.uk/msfit.html. The obtained apoglobin peptide fragments were tested for antibacterial activity against E. coli similar to that shown in Example II and the results are shown in Table II. Most of the obtained peptides retain the bactericidal properties of apoglobins, while the attempts to obtain short peptides, e.g. by digestion with trypsin, unfortunately led to the obtaining of products with minimal antibacterial activity, and therefore most of the subsequent studies were performed on the most active of the obtained peptides, i.e. the cyan bromide peptide 56-131 from apomyoglobin ( Table III).

Example V

Synthetic peptides identical in sequence to the hemoprotein fragments were obtained by using the standard Fmoc procedure in an Advanced ChemTech MPS350 automated synthesizer. The peptides were blocked at the N-terminus with an acetyl group and at the C-terminus with an amide group. Purity of all obtained peptides was determined by RP-HPLC and ESI-MS (Electrospray Ionization Mass Spectrometry), and their amount was determined by amino acid analysis. The purified chemically synthesized peptides were tested for their antibacterial activity against E. coli similar to that shown in Example II, and Table II lists the results obtained with the two most active synthetic peptides.

Example Vi

The influence of pH, calcium and magnesium ions and ionic strength on the bactericidal properties of the cyanide peptide 56-131 apomyoglobin (the preparation of which is described in Example III, Fig. 1) was indirectly determined by examining the perforation of the bacterial membrane of E. coli strain JM83. The strain containing the pCH11O (Pharmacia) plasmid with an embedded gene encoding the enzyme continuously, β-galactosidase and ampicillin resistance, was grown in Lennox L Broth Base (Sigma) medium supplemented with 2 µg / ml ampicillin. Measurement was performed in 96-well plates using fresh log phase growth bacteria suspended in an appropriate 10 mM buffer (phosphate pH 6-8 or acetate pH 4-5) so as to obtain 5x10 ⁵ CFU in 50 μΐ. The bacteria were preincubated for 0.5 hour at 37 ° C in the appropriate medium containing 1.5 μΜ of the cyanogen peptide 56-131 apomyoglobin (except for the assay shown in Fig. 3d, which was additionally run in full nutrient medium) and thereafter 200 (ii 0.2 M Tris-HCl pH 8.0 and 5 μ1 12 mM p-nitrophenyl-PD-glucopyranoside substrate solution and after 0.5 hour incubation at 37 ° C, the released P-galactosidase enzyme from the cells was determined spectrophotometrically by measuring the absorbance at 405 nm The total bacterial lysis obtained by using an excess of the lytic peptide of melittin (Boehringer) was taken as 100% perforation of the bacterial membrane. The obtained results are shown in Fig. 3. The peptide shows the maximum activity in the range of pH 6-7.5 (Fig. 3a) the perforation process of the bacterial membrane depends on the concentration of monovalent and divalent ions (Fig. 3b). Even the concentration of 1 mM of calcium or magnesium ions (at a peptide concentration of about 1000 times lower, i.e. 1.5 μΜ) causes perforation rates of up to 50 and 15 percent respectively. A similar effect is observed in the presence of 150 mM NaCl (Fig. 3c), but the perforation inhibition process reflecting in some sense the minimum concentration necessary to kill 99% of bacteria (MIC) is reversible using a higher peptide concentration (approx. 75 pM) (Fig. 3d).

Example VII

The effect of different concentrations of alpha and beta globins, apomyoglobin and peptides obtained by the action of cyanogen bromide (CNBr) and Arg-C enzyme on these proteins on the lysis of human blood erythrocytes was determined by a 1-hour incubation of erythrocytes at 37 ° C in phosphate-buffered saline (PBS) , centrifugation and spectrophotometric measurement of released hemoglobin at 541 nm. No haemolytic activity of the tested compounds was observed even at concentrations 50 times higher than the LD50 doses, ie at peptide concentrations of 100 pM.

The cytotoxic activity of the bactericidal peptide cyanogen 56-131 apomyoglobin was determined on human fibroblasts and HepG2 (ATCC) cells. Cells

187,999 were grown in DMEM (Dulbecco's Modified Eagle Medium) (Gibco) supplemented with 5% fetal bovine serum. 50 µl of medium containing 10 ⁵ cells was incubated in 96-well culture plates with 2 µl of aqueous cyanogen bromide peptide 56-131 apomyoglobin for 1 hour at 37 ° C. 100% of the lysis obtained with an excess of lytic melittin peptide (Boehringer) was used as a control. After incubation with various concentrations of the test peptide, cell viability was determined by the MTT [3- (4.5 dimethylthiosol-2-yl) -2,5-diphenyltetrazolium bromide] assay according to the procedure described by the compound manufacturer (Sigma). The results shown in Figure 4 show little effect of the test peptide on cell viability, and the course of the curve indicates that this effect is due to suppression of mitochondrial metabolism rather than a cytotoxic effect.

Example VIII

The binding capacity of bacterial lipopolysaccharide (E. coli 0111: B4 LPS - Sigma) by the cyan bromide peptide 56-131 from apomyoglobin was determined by two methods: by the displacement of dansylated polymyxin described in "Antb / acterial Peptide Protocols ed. W.E. Shafer, Humana Press, Totowa, 1997, and a method of measuring the neutralization of the LPS effect on ex vivo TNF-a production ("Acute Phase Proleins, ed. A. Mackiewicz et al., CRS Press :, Boca Raton, 1993). In both cases, the interaction of the tested peptide with LPS was negative.

Example IX

The behavior of CNBr-bactericidal peptide 56-131 from apomyoglobin in aqueous solution and in trifluoroethanol is shown in Fig. 5. It can be clearly seen that the peptide shows only 20% of alpha-helical structure in the aqueous environment, and in the environment of the hydrophobic substance this value increases to 60%. This observation suggests the possibility of initiating the formation of such a structure also in the environment of negatively charged bacterial membrane phospholipids, which may be the basis for the disturbance of the structure of bacterial membranes on the basis of the mechanism proposed for other antibacterial peptides: cecropin P1, cathelicidin CAP 18 or melittin [Eur. J. Biochem. 209 (1992) 163-169; FEBSLett. 370 (1995) 46-52; J Am. Chem. Soc. 118 (1996) 8989-8997]. The conducted attempts to find synthetic amphipathic peptides with the alpha-helix structure and antibacterial properties with sequences deduced from the hemoprotein sequences have in two cases (peptide 59-78 beta globin and 59-78 myoglobin) partially confirmed this theory, showing, however, a rather weak antibacterial effect of these peptides (Table II).

Table I.

Bactericidal and fungicidal properties of native hemoglobin, myoglobin and cytochrome c and their heme-free and partially denatured forms expressed as LD50

Microorganism LD50 [pM] 1 2 3 4 5 6 7 8 Hemoglobin Myoglobin Cytochrome c Globina and Globina P. Apomioglobin Apocytochrome c Escherichia coli on on. on. 3.6 4.2 3.1 2.6 Salmonella serotype Krefeld on. on. n.b. 5.1 4.4 4.7 n.b Pseudomonas aeruginosa on a on. n.b. 4.4 6.7 7.9 n.b. Klebsiella oxytoca on. on. n b. 5.8 8.3 4.2 n.b

107 999 table 1 continues

1 2 3 4 5 6 7 8 Enlerococcus faecahs on. on. n.b 6.5 8.0 7.9 n.b. Staphylococcus aureus on. on. n.b. 7.3 7.1 2.6 n.b. Candida albicans on. on. n.b. 14.0 7.5 11.0 n.b.

on. - not active even at the maximum applied concentration (approximately 330 μΜ) n.b. - not tested

Table II

The ability to kill E. coli bacteria by peptides obtained from globin a and β and from apomyoglobin expressed as LD50

Source proteins Peptides LD50 [μΜ] Globina and the human CNBr ^a , 1-32 8.2 CNBr, 33-76 6.7 CNBr, 77-141 3.9 Crg-Cb, 1-31 37.0 Crg-C, 32-92 11.0 Crg-C, 93-141 2.2 Human β globin CNBr, 1-55 4.2 CNBr, 56-146 1.2 Crg-C, 1-30 42.0 Crg-C, 31-40 20.0 Crg-C, 41-104 6.5 Crg-C, 105-146 4.3 Synthetic ^c , 59-78 48.0 Sperm whale myoglobin CNBr, 1-55 10.0 CNBr, 56-131 0.9 CNBr, 132-153 27.0 Synthetic, 59-78 33.0

^a CNBr peptides have N-terminal methionine modified to homoserine lactone ^b Crg-C peptides have cisterns modified to carbamidomethyl derivative ^c N- and C-terminal amino acids of synthetic peptides are blocked with acetyl and amide residues, respectively

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Table III

Bacterial and fungicidal properties of myoglobin bromide peptides

Microorganism LD ₅ o [μΜ] 1-55 56-131 132-153 Escherichia coli 10 0.9 27 Salmonella serotype Krefeld 5 2.7 120 Enterococcus faecalis 17 1.6 187 Staphylococcus aureus 44 4.8 175 Candida albicans 29 8.0 187

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Perforation Percent Perforation Percentage

Ion concentration [mM]

Fig.3b

Salt concentration [mM]

Peptide concentration [μΜ]

Fig.3c

Fig.3d

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Ellipticity [milliost degree] ^{Percentage of} cells surviving

Peptide concentration [µM]

Fig.4

Wavelength [nm] Fig.5

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Time (min.)

100 cn § XI g o uo.

Fig. L

A (220 nm)

Percentage of buffer B

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Claims (2)

Patent claims 1. The use of peptides based on human and / or animal heme-containing proteins (hemnprntein) and their natural modifications and / lhs synthetic formulas: a) Human hemoglobin, alpha chain: 10 twenty thirty 40 50 NH2-VLSPADKTNV KAAWGKVGAH AGEYGAEALE RMFLSFPTTK TYFPHFDLSH 60 70 80 90 100 GSAQVKGHGK KVADALTNAV AHVDDMPNAL SALSDLHAHK LRVDPVNFKL 110 120 130 140 LSHCLLVTLA AHLPAEFTPA YHASLDKFLA SVSTVLTSKY R-COOH b) Human hemoglobin, beta chain: 10 twenty thirty 40 50 NH2-VHLTPEEKSA VTALWGKVNV DEVGGEALGR LLWYPWTQR FFESFGDLST 60 70 80 90 100 PDAVMGNPKV KAHGKKVLGA FSDGLAHLDN LKGTFATLSE LHCDKLHVDP 110 120 130 140 ENFRLLGNVL VCVLAHHFGK EFTPPVQAAY QKWAGVANA LAHKYH-COO c) Sperm whale skeletal muscle myoglobin: 10 twenty thirty 40 50 NH2-VLSEGEWQLV LHVWAKVEAD VAGHGQDILI RLFKSHPETL EKFDRFKHLK 60 70 80 90 100 TEAEMKASED LKKHGVTVLT ALGAILKKKG HHEAELKPLA QSHATKHKIP 110 120 130 140 150 IKYLEFISEA IIHVLHSRHP GDFGADAQGA MJfKALELFRK DIAAKYKELG YQG-COOH d) Cytochrome C from the horse's heart: 10 twenty 33 40 50 NV2-GDVEKGKKIF VQVCKQCHTV VEKGGKVKTG NLHGGFGRKT GCAPGFTETD 60 70 88 99 100 ANKNKGITWK EEEEMEYEEL IKKYIPGTIKM IFCGIKKKTE REDLICELKK ATNE-COOH 187 999 e) Human hemoglobin CNBr and / or Arg C peptides, alpha chain with amino acid positions 1-32; 33-76; 77-141; 1-31; 32-92; 93-141, f) Human hemoglobin CNBr and / or Arg C peptides, beta chain with amino acid positions 1-55; 56-146; 1-30; 31-40; 41-104; 105-146, g) Sperm whale apomyoglobin CNBr peptides with amino acid residue positions 1-55; 56-131; 132-146, h) a synthetic peptide analogous in sequence to the peptide 59-78 beta globin of human hemoglobin, i) a synthetic peptide analogous to the sperm whale apomyoglobin peptide 59-78 sequence, wherein in the above sequences the corresponding symbols are: A, Ala, alanine; V, Val, valine; L, Leu, Leucine; I, Ile, isoleucine; P, Pro, proline; F, Phe, phenylalanine; W, Trp, tryptophan; M, Met, methionine; G, Gly, glycine; S, Cheese, Serine; T, Thr, threonine; C, Cys, Cysteine; Y, Tyr, Tyrosine ,; N, Asn, asparagine; Q Gln, glutamine; D, Asp, Aspartic Acid; E, Glu, Glutamic Acid; K. Lys, Lysine; R, Arg, arginine; H, His, histidine; CNBr, cyanogen bromide, which are heme-free for the preparation of a pharmaceutical with bactericidal and fungicidal activity. 2. The use of peptides based on the sequence of animal proteins containing heme (haemoproteins) and their natural and / or synthetic modifications with the following formulas: a) Bovine hemoglobin, alpha chain: 10 20 30 40 50 NH _Z -VLS? ADKGNV KAAWGKVGGH AAEYGAEALE RMFLSFTTTK TYFPHFDLSH 60 70 80 90 100 GSAQVKGHGA KVAAALTKAV EHLDDLPGAL SELSDLHAHK LRVDPVNFKL 110 120 130 140 LSHSLLVTLA SHLPSDFTPA VHASLDKFLA NVSTVLTSKY R-COOH b) Bovine hemoglobin, beta chain: 10 20 30 40 50 NH2-MLTAEEKAAV tafwgkvkvd evggealgrl lwypwtqrf fesfgdlsta 60 70 80 90 100 DAVMNNPKVK AHGKKVLDSF SNGMKHLDDL KGTFAALSEL HCDKLHVDPE 110 120 130 140 NFKLLGNVLV W / IAtRNFGKE FTPVLQADFQ KWAGVANAL AHRYH-COOH c) Sperm whale skeletal muscle myoglobin: 10 20 30 40 50 NH2-VLSEGEWQLV LHVWAKVEAD VAGHGQDILI RLFKSHPETL EKFDRFKHLK 60 70 80 90 100 TEAEMKASED LKKHGVTVLT ALGAILKKKG HHEAELKPLA OSHATKHKIP YQG-COOH d) Cytochrome C from the horse's heart: 10 20 33 40 50 NH2-GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLLHGLGRKT GQQAGFTYTD 60 70 80 90 100 ANKNKGITWK EETLMEYLEN PKKYIPGTKM IFAGIKKKTE REDLIAYLKK ATNE-COOH 187 999 e) Human hemoglobin CNBr and / or Arg C peptides, alpha chain with amino acid positions 1-32; 33-76; 77-141; 1-31; 32-92; 93-141, f) Human hemoglobin CNBr and / or Arg C peptides, beta chain with amino acid positions 1-55; 56-146; 1-30; 31-40; 41-104; 105-146, g) Sperm whale apomyoglobin CNBr peptides with amino acid residue positions 1-55; 56-131; 132-146, h) a synthetic peptide analogous in sequence to the peptide 59-78 beta globin of human hemoglobin, i) a synthetic peptide analogous to the sperm whale apomyoglobin peptide 59-78 sequence, wherein in the above sequences the corresponding symbols are: A, Ala, alanine; V, Val, valine; L, Leu, Leucine; I, Ile, isoleucine; P, Pro, proline; F, Phe, phenylalanine; W, Trp, tryptophan; M, Met, methionine; G, Gly, glycine; S, Cheese, Serine; T, Thr, threonine; C, Cys, Cysteine; Y, Tyr, Tyrosine ,; N, Asn, asparagine; Q Glut, Glutamine; D, Asp, Aspartic Acid; E, Glu, Glutamic Acid; K. Lys, Lysine; R, Arg, arginine; H, His, histidine; CNBr, cyanogen bromide free of heme for the production of food and feed preservative with bactericidal and fungicidal activity.