NEW ANTIMICROBIAL PEPTIDES ¹

Technical field

The invention involves novel peptides isolated from natural sources of the following formulas I - IV

H-Gly-Phe-Leu-Ser-IIe-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Al a-His-Met-Lys-NH ₂ (I), H-VaI-ASn-TrP-LyS-LyS-VaI-LeU-GIy-LyS-IIe-IIe-LyS-VaI-AIa-Ly S-NH ₂ (II),

H-VaI-ASn-TrP-LyS-LyS-IIe-LeU-GIy-LyS-IIe-IIe-LyS-VaI-AIa-Ly S-NH ₂ (III),

H-VaI-ASn-TrP-LyS-LyS-IIe-LeU-GIy-LyS-IIe-IIe-LyS-VaI-VaI-Ly S-NH ₂ (IV),

and their new synthetic analogs of general formulas (V) and (VI)

R'-tXaa^ _m -Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala -His-Met-Lys-

R ² (V), and

R'-rXaa^ _m -Val-Asn-Tφ-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys -R ² (VI),

wherein R ¹ represents acyl, alkyl or unprotected amino group, R ² represents amid, substituted amid, unprotected carboxylic group or ester, Xaa represents, independently from each other, proteinogenic amino acid, D-amino acid, non-standard amino acid or amino acid with the side chain modification, n means position of the amino acid replacement and m means the number of the replacements in the peptide chain, and their applications as antimicrobial, antiviral, antifungal, antiparasitic and anticancer compounds.

Background arts

Antimicrobial peptides are evolutionary conserved components of the host's innate immunity system that form the first line of defense against infections [1-3]. They have been identified in almost all classes of life. Among antimicrobial peptides, those from insects constitute a remarkable group [4]. Since insects are uniquely adapted to a variety of natural environments, often considered rather unhealthy by human standards, they have developed amazing resistance to bacterial infection, in which antimicrobial peptides play a major role.

The significant advantage of antimicrobial peptides resides in their mechanism of action, which is markedly different from that of conventional antibiotics. Although the precise mechanism of the broad spectrum of antimicrobial activity of these peptides is not yet fully understood, they appear to act via a specific, but not receptor-mediated, formation of transmembrane pores or ion channels on cellular membrane. This causes leakage of essential metabolites that results in the disruption of microbial cell structure and leads to cell death [5, 6]. In contrast to conventional antibiotics, they do not appear to induce microbial resistance and require only short time to induce killing. Quite peculiar groups of peptides with antimicrobial properties were identified in the venom of stinging insects such as hymenopterans [7]. The main function of hymenoptera venom is the protection against predators or nest defense against intruders causing them pain and inflammation. Antimicrobial effect of peptides isolated from the venoms of wasps, bees, bumblebees and ants is rather secondary as in the case of peptides belonging to the group of mastoparans [8]. Nevertheless, antimicrobial effect of peptides isolated for example from the venom of wild bees is quite significant. As the growing resistance of bacterial pathogens to conventional antibiotics has become serious global health problem, this alarming situation resulted in a search for novel alternative to traditional antibiotics such as antimicrobial peptides. As regards of the synthetic availability of peptides isolated from the venom of wild bees, these peptides may found application in practical medicine.

Disclosure of the invention

The invention involves novel peptides isolated from natural sources of the following formulas I - IV

H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Al a-His-Met-Lys-NH ₂ (I), H-VaI-ASn-TiP-LyS-LyS-VaI-LeU-GIy-LyS-IIe-IIe-LyS-VaI-AIa-Ly S-NH ₂ (II),

H-Val-Asn-Tφ-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Ly s-NH ₂ (III),

H-VaI-ASn-TrP-LyS-LyS-IIe-LeU-GIy-LyS-IIe-IIe-LyS-VaI-VaI-Ly S-NH ₂ (IV),

and their new synthetic analogs of general formulas (V) and (VI)

R'-[Xaa ⁿ ] _m -Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala -His-Met-Lys-

R ² (V), and

R'-tXaa^ _m -Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys -R ² (VI),

where R ¹ represents acyl, alkyl or unprotected amino group,

R ² represents amid, substituted amid, unprotected carboxylic group or ester,

Xaa represents, independently from each other, proteinogenic amino acid, D-amino acid, non-standard amino acid or amino acid with the side chain modification, n means position of the amino acid replacement and m means the number of the replacements in the peptide chain.

It means that n can be 1-18 in general formula V or 1-15 in general formula VI. Several amino acid residues can be replaced simultaneously in this manner in the general formula V or VI, the order of amino acid residues can be altered, or some amino acids in the peptide chain may be omitted or inserted.

This invention further involves application of listed peptides of formulas I - IV and analogs of general formulas V and VI as antimicrobial, antiviral, antifungal, antiparasitic and anticancer compounds.

Detailed description of the invention

We isolated the peptide of the formula (I) from the venom reservoirs of cleptoparasitic bees Melecta albifrons and named it melectin. From the venom reservoirs of primitively eusocial bees Lasioglossum laticeps we isolated the peptides of the formulas (II, III and IV) and named them lasioglossin I, lasioglossin II, and lasioglossin III, respectively. The peptides of general formula (V) are analogs of peptide (I) and the peptides of general formula (VI) are analogs of peptide (IV). The peptides I, II, III and IV possess strong antimicrobial effect and low hemolytic activity, but their primary function in the nature is unknown.

Even if this primary role of melectin and lasioglossins in the nature is not known, we deal with novel unique, so far not published, peptides with significant antimicrobial properties. These peptides possess antimicrobial properties thanks to the shape of their secondary structure, which depends on their distinctive primary peptide sequence. Secondary structure of melectin and lasioglossins is unordered in aqueous media, but it easily undergoes

to the α-helical structure in the presence of α-helix inducing compounds such as trifluoroethanol or SDS or in an anisotropic environment of the bacterial cell membrane [9].

In the so called Edmundson wheel [10] projection (Fig. 1), melectin [9] as well as lasioglossins have well-defined hydrophobic sectors with large aliphatic residues (red) on one side of the helix, and hydrophilic sector (black) dominated by cationic Lys residues (blue) on the opposite side of the helix. Their ability of those peptides to adopt such an amphipathic structure [11, 12] within bacterial cell membrane is one of the most important factors participating in the disruption of bacterial cell membrane finally resulting in the bacteria death. In addition, the cationic character [11] of those peptides ensured by the presence of four lysine residues in melectin [9] and five lysine residues in lasioglossins located in hydrophilic sectors of the helices, plays also important role in the killing process. Due to this space arrangement, the positively charged faces of the peptides are attracted by strong electrostatic interaction to negatively charged phospholipids of bacterial cell membrane. The peptide accumulation on the membrane surface causes tension between the two leaflets of the lipidic membrane bilayer, which above a threshold peptide concentration leads to the disintegration and rupture of the membrane.

In anisotropic environment melectin and lasioglossins may theoretically form the optimal α-helical structures of five and four turns respectively. These helices are stabilized by " hydrogen bonds in which every backbone NH group donates a hydrogen bond to the backbone C=O group of the preceding turn. In the case of melectin, due to the presence of proline residue (Pro) in position 11 , the possibility of making hydrogen bond to the preceding turn is lost since proline lacks the hydrogen on its amide and because of a steric effect of its ring structure. As a result a kink [9, 13, 14] is introduced to the helical structure [9] causing slight bending of α-helix. This conformational element, imposed by proline on a melectin peptide chain, appears to govern its biological functions such as high antimicrobial activity and low hemolytic effect. The replacement of Pro by other amino acid residue in the synthetic peptide results in the increase of undesirable hemolytic activity (Tables 2 and 5). Such effect has been already described in the case of other antimicrobial peptides [13, 14]. The entire cationicity, hydrophobicity or amphipathicity of melectin or lasioglossins can be modified by the replacement of the original amino acid residues in their sequences by other amino acids. This is done in an effort to increase their antimicrobial activity and reduce hemolytic activity [15]. Other different chemical modifications as demonstrated by general formulas V and VI influence the biological properties of melectin and lasioglossins. Tables 1 - 7 show the

examples of chemical modifications of peptides and their consequences on antimicrobial and hemolytic activity.

Examples

Example 1

Isolation from natural sources

Bee specimens of Melee la albifrons were collected in urban area of north-west quarter of Prague, Czech Republic, and kept frozen at -2O ⁰ C for several days. The venom reservoirs of four individuals were removed by dissection and their contents was extracted with 25 μl of a mixture of acetonitrile-water (1 : 1) containing 0.1% TFA. The extract was centrifuged, and the supernatant was fractionated by RP-HPLC. The chromatography was carried out on the Thermo Separation Product instrument with a Vydac C- 18 column (250 x 4.6 mm; 5 μm) at a flow rate 1.0 ml/min using the gradient of solvents from 5% to 70% acetonitrile/water/0.1% TFA over 60 min. The major peptide fractions detected by the UV absorption at 222 run were collected, the solvent was evaporated in a Speed- vac and the material was subjected to mass spectrometry and Edman degradation to determine its primary structure. Melectin represented the main peptide component of the venom extract.

Bee specimens of Lasioglossum laticeps were collected in urban area of the town Hofice in the northeast area of the Czech Republic. Lasioglossins, which represent the main peptide components of the venom extract, were isolated from the venom reservoirs as described above; likewise the primary structure determination.

Example 2

Synthesis of peptides of the general formula I - VI

These peptides may be synthesized according to following procedure: peptides were synthesized manually by using a solid-phase method in 5 mL polypropylene syringes with a bottom Teflon filter. Synthesis was done by using a Fmoc chemistry protocol on a Rink Amide MBHA resin (100 mg) with 0.7 mmol/g substitution. Protected amino acids were coupled in fourfold excess in dimethylformamide as solvent and N,N'- diisopropylcarbodiimide (7 equivalents)/ 1 -hydroxybenzotriazole (5 equivalents) as coupling reagents. Crude peptides of the general formula I and V were deprotected and cleaved from

the resin with a mixture of trifluoroacetic acid/1, 2- ethanedithiol/voda/thioanisol/triisopropylsilane (90 : 2.5 : 2.5 : 3 : 2) for 3.5 h and precipitated with tert-butyl methyl ether. The mixture of trifluoroacetic acid/voda/triisopropylsilane (95 : 2.5 : 2.5) was used for the deprotection and cleavage of peptides of the general formula II, III, IV and VI under the same conditions. Usually, 100 - 150 mg of crude peptides was obtained by this manner. Crude peptides (15 mg) were purified by preparative RP-HPLC using a Vydac C- 18 column (250 x 10 mm) at a flow rate 3.0 ml/min using the gradient of solvents from 5% to 70% acetonitrile/water/0.1% TFA over 60 min yielding 7 - 9 mg of HPLC pure peptides. Their identities were verified by mass spectrometry as given in Tables 1 - 3.

The peptides VI/8 and VI/9 were synthesized similarly on 2-chlorotrityl chloride resin. The peptides were cleaved from the resin in their protected form with a mixture of dichloromethane/trifluoroethanol/acetic acid (7 : 2 : 1) for 1 h. The solvent was concentrated under the vacuum and the residue was triturated with a mixture of tert-butyl methyl ether/n- hexane. The VI/8 peptide was prepared from the obtained protected peptide by the deprotection with a mixture of trifluoroacetic acid/voda/triisopropylsilane (95 : 2.5 : 2.5) for 2 h and then precipitated with tert-butyl methyl ether. The VI/9 peptide was obtained by the following manner: protected peptide was suspended in methanol and esterified with a solution of diazomethane in diethyl ether. The solvent was evaporated, the residue triturated with n- hexane. Then the methyl ester of protected peptide was deprotected as in the case of VI/8 peptide.

Table 1. Amino acid sequences and MS data of melectin (I) and lasioglossins (II, III, IV)

Peptide Peptide sequence Monoisotopic molecular mass (Da)

calculated found

(I) H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Al a-His-Met-Lys-NH ₂ 2038.23 2038.2

(II) H-Val-Asn-Tφ-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Ly s-NH ₂ 1722.14 1721.8

(III) H-VaI-ASn-TrP-LyS-LyS-IIe-LeU-GIy-LyS-IIe-IIe-LyS-VaI-AIa-Ly S-NH ₂ 1736.16 1736.0

(IV) H-Val-Asn-Tφ-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Ly s-NH ₂ 1764.19 1764.9

Table 2. Amino acid sequences and MS data of the synthetic analogs of the formula V peptide

Peptide Peptide sequence Monoisotopic molecular mass (Da) calculated found

V/l H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Lys-Lys-Val-Met-Al a-His-Met-Lys-N^ 2069,27 2069,4

V/2 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Ala-Lys-Val-Met-Al a-His-Met-Lys-NH ₂ 2012,22 2012,3

V/3 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Gly-Lys-Val-Met-Al a-His-Met-Lys-N^ 1998,20 1998,2

V/4 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Al a-Lys-Met-Lys-NH ₂ 2029,27 2029,4

V/5 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Leu-Al a-His-Leu-Lys-NH ₂ 2002,32 2002,4

V/6 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Hi s-Ala-Met-Lys-NH? 2038.23 2038.3

V/7 H-Glv-Phe-Leu-Ser-Ile-Leu-Lvs-Lvs-Val-Leu-Pro-Lvs-Val-Met-Lv s-Ala-Met-Lvs-NH? 2029,27 2029,4

V/8 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-... -Lys-Val-Met-Ala-His-Met-Lys-NH ₂ 1941,18 1940,9 V/9 H-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Ala-Hi s-Met-Lys-NH ₂ 1981,21 1980,7

V/ 10 H-Leu-Ser-Ile-Leu-Lys-Lys- Val-Leu-Pro-Lys- Val-Met-Ala-His-Met-Lys-NH ₂ 1834,14 1833,8

V/l l H-Ser-Ile-Leu-Lys-Lys-Val-Leu-PiO-Lys-Val-Met-Ala-His-Met-Ly s-NH ₂ 1721,06 1720,7

V/l 2 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-PiO-NH2 1212,80 1212,7

V/l 3 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys- VaI-LeU-NH ₂ 1115,74 1115,6

V/14 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-GIy-Pro-Lys-Val-Met-Al a-His-Met-Lys-NH ₂ 1982,17 1982,0

V/15 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-Al a-His-Met-Lys-NH ₂ 2038,23 2038,0

V/16 H-Gly-lNal-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys-Val-Met-A la-His-Met-Lys-NH ₂ 2088,25 2088,0

V/l 7 H-Gly-...-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Lys-Lys-Val-Met-Al a-His-Met-Lys-NH2 1923,15 1923,6

V/l 8 H-Gly-Phe-Leu-Ser-Ile-Leu-Lys-Lys-Val-Leu-Pro-Lys- Val-Nle-Ala-His-Nle-Lys-NH ₂ 2002,32 2002,5

Pro: D-prolin; INaI: 3-(l-naftyl)alanin; NIe: norleucine; bold letters: amino acid residue replacement; underlined: interchanged amino acid residues; -...- the residue was omitted

Table 3. Amino acid sequences and MS data of the synthetic analogs of the formula VI peptide

Peptide Peptide sequence Monoisotopic molecular mass (Da)

calculated found

VI/1 H-Val-Asn-Tφ-Lys-Lys-Ile-Leu-Ala-Lys-Ile-Ile-Lys-Val-Val-Ly s-NH ₂ 1778,20 Mil,9

VI/2 H-Val-Asn-Tφ-Lys-Lys-Ile-Leu-Lys-Lys-Ile-Ile-Lys-Val-Val-Ly s-NH ₂ 1835,26 1834,9

VI/3 H-Asn-Val-Trp-Lvs-Lvs-Ile-Leu-Gly-Lvs-Ile-Ile-Lvs-Val- Val-Lys-NH? 1764,19 1764,9

VI/4 H-VaI-ASn-TrP-LyS-LyS-IIe-LeU-GIy-LyS-IIe-LyS-LyS-VaI-VaI-Ly S-NH ₂ 1779,20 1779,4

VI/5 H-VaI-ASn-TrP-LyS-LyS-IIe-LeU-PrO-LyS-IIe-IIe-LyS-VaI-VaI-Ly S-NH ₂ 1804,22 1804,7

VI/6 H-Val-Asn-Trp-Lys-Lys-Ile-Leu-P/O-Lys-Ile-Ile-Lys-Val-Val-Ly s-N^ 1804,22 1803,8 VI/7 PaImItOyI-VaI-ASn-TrP-LyS-LyS-IIe-LeU-GIy-LyS-IIe-IIe-LyS-Va I-VaI-LyS-NH ₂ 2002,42 2002,3

VI/8 H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Ly s-OH 1765,17 1765,5

VI/9 H-Val-Asn-Tφ-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Ly s-OMe 1779,19 1779,3

VI/10 H-Lys-Asn-Tφ-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys- VaI- VaI-LyS-NH ₂ 1793,21 1793,8

VI/11 H- Val-Asn-Tφ-Lys-Lys-Ile-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val- VaI-LyS-NH ₂ 1877,7 1877,2

VI/12 H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Ly s-Nfy 1764,19 1764,0

Bold letters: amino acid residue replacement; underlined: interchanged amino acid residues; in italics: D-amino acid residues; He: inserted residue; -OH: free carboxylic group; OMe: methyl ester

Example 3

Determination of antimicrobial activity

Antimicrobial activity was determined as a quantitative minimum inhibitory concentration (MIC) by observing bacterial growth in the presence of different concentrations of tested peptides. Mid-exponential phase bacteria were added to the solutions of the tested antimicrobial peptide at different concentrations (0.5 to 100 μM) in LB broth in multi-well plates. The plates were incubated at 37°C for 20 h while being continuously shaken in a Bioscreen C instrument (Finland). The absorbance was measured at 540 run every 15 min and each peptide was tested at least 3 times in duplicate. 1.2.10 ³ - 7.5.10 ³ CFU of bacteria was routinely used in the experiments. Tetracycline in a concentration range of 0.1 - 50 μM was tested as a standard. The results are shown in Tables 4 - 6.

Example 4

Determination of hemolytic activity

The peptides in the concentration (1-200 μM) were incubated with rat red blood cells (5%) for 1 h at 37°C in a physiological solution at a final volume of 0.2 ml. The samples were then centrifuged for 5 min at 250 g, and the absorbance of the supernatant was determined at 540 nm. Supernatants of red blood cells suspended in physiological solution and in 0.2% TRITON XlOO in physiological solution served as controls for zero hemolysis (blank) and 100% hemolysis, respectively. The results are shown in Tables 4 - 6.

Table 4. Antimicrobial and hemolytic activity of melectin (I) and lasioglossins (II, III, IV)

Peptide Antimicrobial activity Hemolytic activity

MIC [μM] IC ₅₀ [μM]

B.s. S.a. E.c. P. a.

I 0,8 6,8 2,0 18,5 >100

II 1,0 20 2,4 22 >200 III 1,0 12,5 2,0 20 >200 IV 1,0 5,4 2.0 23 >200

B. s,. Bacilus subtilis; S.a., Staphylococcus aureus ; E.c, Escherichia coli; P. a., Pseudomonnas aeruginosa

Table 5. Antimicrobial and hemolytic activity of the synthetic analogs of the formula V peptide

Peptide Antimicrobial activity Hemolytic activity

MIC [μM] IC ₅₀ [μM]

B. s. SM. Ex. P.a.

V/l 1,4 13,3 1,8 25,3 27,3

V/2 1,3 4,7 1,6 18,3 29,7

V/3 1,3 4,3 1,8 14,4 41,4

V/4 0,7 16,8 2,1 33,6 >100

V/5 0,8 5,0 1,2 20 43,7

V/6 1,1 9,1 2,1 35,1 150

V/7 0,8 7,7 1,5 31,7 70

V/8 0,9 5,0 1,8 27,5 22,9

V/9 1,2 38,8 9,08 62,1 >200

V/10 7,9 >100 76,2 >100 »200

V/l l 21,8 >100 >100 >100 »200

V/l 2 13,0 >100 78,0 >100 »200

V/l 3 2,9 10,9 9,1 58,7 >100

V/14 1,4 80,0 22,5 80,0 »200

V/l 5 1,7 >100 4,2 100 »200

V/l 6 0,4 2,5 0,4 17,3 25

V/l 7 0,95 40 3,8 10 »200

V/l 8 0.2 2.5 0,75 17,3 50

Table 6. Antimicrobial and hemolytic activity of the synthetic analogs of the formula VI peptide

Peptide Antimicrobial activity Hemolytic activity

MIC [μM] IC ₅₀ [μM]

B. s. S.a. Ex. PM.

VI/1 0,6 2,2 1,4 27,1 55,1

VI/2 0,7 9,6 0,7 26,1 72,2

VI/3 0,7 9,0 1,5 23,1 >200

VI/4 1,0 41,4 3,7 38,0 »200

VI/5 0,8 32,5 1,2 100 »200

VI/6 10,8 >100 85 >100 »200

VI/7 73 >100 100 - 8,8

VI/8 1,2 10 3,5 10 >200

VI/9 0,5 2,5 1,13 10 79,3

VI/10 0,25 12,5 0,25 22,5 »200

VI/11 0,6 >40 3,8 >40 >200

VI/12 0.25 3,0 0,5 12,5 105

Example 5

Cytostatic effect on cancer cells

Tumor cells in suspension or adherent cells were incubated under standard conditions in appropriate culture media for 24-72 h with the peptides in 2-40 micromolar concentration. After this time, the number of living cells was determined using routine tests, e.g. MTT test. The IC ₅₀ was determined as concentration of peptides, which caused 50% decrease in cell viability in comparison to controls (no peptide present). In preliminary tests mouse lymphocytic leukemia L1210 (CCL 219), CCRF-CEM T lymphoblastoid (human acute lymphoblastic leukemia, CCL 119), human promyelocyte leukemia HL-60 (CCL 240), human cervix carcinoma HeLa S3, rat pheochromocytoma PC 12, human colon adenocarcinoma SW480 (CCL-228), and normal rat epithelial cells (IEC-6, CRL-1592) from

ATCC (Manassas, VA, USA), were used. Peptides melectin and lasioglossins and some of their analogues showed IC ₅₀ in the range of 4-30 mM (Table 6).

Table 6. Cytostatic effect of selected peptides on cancer cells

Peptide IC ₅₀ [μM]

L 1210 CCRF-CEM T HL-60 HeLaS3 PC 12 SW480

I >10 >10 >10 18.1 10 25

1/7 6,9 8,8 9,1 9,6 12,5

IV 4,7 5,0 4,4 9,5 10 15

VI/1 3,1 4,6 2,9 7,4 15

VI/2 - - - - 11

VI/3 30

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