WO2003035677A2 - Antimicrobial peptides from the venom of the spider cupiennius salei - Google Patents

Abstract

The present invention relates to highly cationic peptides from a novel family of antimicrobial compounds designated cupiennins with an unique helical structure isolated from the venom of the hunting spider Cupiennius salei. Said peptides are characterized by having antimicrobial, cytotoxic and/or insecticidal activity. The invention further comprises nucleic acid molecules encoding the peptides, vectors and host cells containing said nucleic acid molecules; methods for producing the peptides; and pharmaceutical compositions comprising them.

Description

Antimicrobial Peptides from Spider Venom

The present invention relates to peptides having antimicrobial, cytotoxic and/or insecticidal activity, nucleic acid molecules encoding them, vectors and host cells containing said nucleic acid molecules; methods for producing the peptides; and pharmaceutical compositions comprising them.

Antimicrobial peptides are ubiquitous in nature as a part of the innate immune system and host defense mechanism. They are produced by various species, both in prokaryotic and eukaryotic cells. Many of these peptides act within minutes through a cell lytic-/ionophoric, non-stereoselective mechanism against a broad spectrum of bacteria, protozoa, filamentous fungi, tumor cells and viruses. Additional mechanisms involving events other than the breakdown of the membrane barrier function and killing bacteria in a longer time period have been described. As a selective response to microbial invasion, several antimicrobial peptides have been identified in the hemolymph of insects, spiders and scorpions. Only a few antimicrobial peptides are exclusively and constitutively present in venom glands of insects: melittin and crabolin, and in scorpions hadrurin and scorpine. In 1989 the first report on the bactericidal peptides in spider venom (Lycosa singoriensis) was published and later lycotoxins were isolated from Lycosa carolinensis.

Spiders are hunting predators and use paralytic venoms to immobilize their prey. Most components in their venoms act on the nervous systems and are enzymatically active causing cell membrane disruption and tissue necrosis. From the venom of Cupiennius salei, a hunting spider found in Central America, neurotoxically acting peptides (named CSTX-1 to CSTX-13) have been isolated (1).

According to a further report (2, 3), an insecticidal and also bactericidal peptide having a molecular mass of 3701.25 daltons has been isolated from the venom of Cupiennius salei. The peptide has been designated CSTX-4. The reports do not disclose any sequence data.

Peptide antibiotics are available from various sources as mentioned above, e.g. from mammalians, insects, plants, bacteria and viruses. Although various peptide antibiotics are known, there still remains a need for peptides having antimicrobial activity which are useful for medical purposes.

One technical problem underlying the present invention therefore represents the provision of novel peptides having antimicrobial, hemolytic and/or insecticidal activity.

The technical problem is solved by the provision of a peptide comprising an amino acid sequence selected from any of SEQ ID NOS. 1 to 4 and variants thereof, wherein the variant exhibits

i) an antimicrobial activity of at least 50% of the peptide;

ii) a hemolytic activity of at least 50% of the peptide; and/or

iii) an insecticidal activity of at least 50% of the peptide.

Further embodiments of the present invention are outlined in the claims which are hereby incorporated. In the following some terms are defined to clarify their meaning in the context of the present application.

The term "variant" designates any modification of a given amino acid sequence. In particular, the term "variant" designates muteins differing by at least one addition, substitution, deletion, insertion and/or inversion of one amino acid from a given amino acid sequence.

"Antimicrobial activity" designates the microbicidal activity of the peptides according to the present invention. The antimicrobial activity may be determined as follows: Bacteria (Escherichia coli ATCC 25922; Staphylococcus aureus ATCC 29213; Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853) were cultured in Mueller Hinton broth. Determination of the minimal inhibitory concentration (MIC) for the cationic antimicrobial peptides is performed using a 2-fold microtiter broth dilution assay (4). Mueller Hinton broth is used to dilute the bacterial inoculum, which is prepared from mid log phase cultures to give a final concentration of 1.7 - 3.8 x 10⁵ cfu/ml in the wells. First, 100 μl of the bacteria dilution is added into the wells, followed by 10 μl of the test peptides in 0.01% acetic acid, 0.2% BSA. Peptides (0.04-100 μM), non-treated growth control and a sterility control are tested in triplicate. The microtiter plates are incubated at 37 °C for 24 hours. The content of the first four wells showing no visible growth of bacteria (measured as an increase of optical density at 630 nm) are plated out on blood agar plates and incubated at 37 °C for 18 hours. Minimal inhibitory concentrations (MIC) are expressed at intervals of concentrations [a]-[b]; where [a] is the highest concentration of peptide at which bacteria still grow and [b] being the lowest concentration causing 100% of growth inhibition (no colony forming bacteria estimated after additional plating out of 91 % of the tested bacteria suspension).

"Hemolytic activity" relates to the hemolytic activity of the peptides of the present invention on human red blood cells (hRBC). Determination of the hemolytic activity may be performed as follows:

1 ml citrated blood is washed 4 times with 6 ml of PBS buffer (50 mM sodium phosphate buffer, 150 mM NaCI, pH 7.2) and centrifuged (900xg) for 6 minutes at room temperature. The pellet is resuspended in 3 ml and further diluted to a concentration of 1 X 10⁹ hRBC/ml in PBS buffer. Lyophilized toxins in various concentrations are resolved in 200 μl PBS buffer and 50 μl of human red blood cells are added following incubation under gentle shaking at 37 °C for 1 hour. The samples are then placed on ice and immediately centrifuged at 4 °C. The supernatant is carefully removed and the pellet is resuspended in 240 μl of water. Release of hemoglobin is monitored by measuring the absorbance of supernatant and water treated pellet at 541 nm in a 0.1 cm cell (Jasco, V- 550, Japan). The negative control (0% hemolysis) is 50 μl human red blood cells in 200 μl PBS buffer and the positive control (100% hemolysis) is 50 μl human red blood cells in 200 μl water. The concentrations of peptide at which 50% hemolysis are observed. (EC₅o) are derived from the dose-response curves (Prism) [Graph Pad Prism, 3.0; Graph Pad Software, Inc.].

"Insecticidal activity" relates to the insecticidal activity of the peptides of the. present invention, in particular, on flies. The determination of insecticidal activity may be performed as follows: bioassays using Drosophila melanogaster according to (5) are performed to estimate the LD₅₀ (24h post injection) of the peptides. For each assay 20 flies are used as control (injecting 0.05 μl of insect ringer) and 20 for each of the 3 peptide concentrations. LD50 stands for the lethal dose (50% of the test flies die of intoxication) and calculations are done as described in (6). The term "homologous" relates to the degree of relationship among two or more polypeptides which can be determined by alignment of amino acid sequences according to known methods, e.g. computer based sequence comparison (basic local alignment search tool, S.F. Altschul et al., J. Mol. Biol. 215 (1990), 403-410). The percentage of "homology" results from the percentage of identical regions in two or more sequences having regard to gaps and other sequence particularities. Generally, certain computer programs using algorithms are used taking into account of the particular requirements.

Preferred methods for determining the homology first generate the maximum identity among the sequences to be investigated. Computer programs for determining homology among two sequences include, however, are not restricted to the GCG program package, including GAP (Devereux J. et al., Nucleic Acids Research 12:387 (1984)); Genetics Computer Group University of Wisconsin, Maddison (WI); BLASTP; BLASTN, and FASTA (Altschul S. et al., J. Mol., Biol., 215:403-410) (1990)). The BLASTX program can also be accessed via the National Center for Biotechnology Information (NCBI) and from further sources (BLAST Manual, Altschul S. et al., NCB NLM NIH Bethesda MD 20894; Altschul S. et al., see above). Also the well-known Smith- Waterman-algorithm may be used for the determination of homologies.

Preferred parameters for amino acid sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-445 (1970)

Comparison matrix: BLOSUM 62 from Henikoff and Henikoff PNAS USA 89 (1992), 10915-10919.

Gap penalty: 12

Gap length: 4

Threshold of Similarity: 0

The GAP program is also suitable for use with the afore-mentioned parameters. The afore-mentioned parameters represent default parameters for amino acid sequence comparison wherein gaps at the end do not reduce the homology value. Regarding very short sequences relative to a reference sequence, this may necessitate to increase the expectation value up to 100,000 and optionally to reduce the word size as low as 2.

Further exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, including the ones indicated in the program manual, Wisconsin package, version 9, September 1997, may be used. The choice will depend on the comparison to be performed and furthermore on the comparison of sequence pairs wherein GAP or BLAST are preferred, or between a sequence and a sequence database containing numerous sequences wherein FASTA or BLAST are preferred.

An agreement of 60% using one of the above algorithms will be called 60% homology according to the present invention. The same is valid for higher degrees of homology.

"Cloning" in the context of the present invention designates all cloning methods known in the art which may be used in the context of this application, which are, however, not further outlined because they are generally known to the person skilled in the art.

"Recombinant expression in a suitable host cell" means all known expression methods in known expression systems which may be used in the present context, which are, however, not further described, since they are part of the general knowledge of the person skilled in the art.

Surprisingly, a novel family of antimicrobial compounds designated cupiennins has been isolated from the venom of the hunting spider Cupiennius salei (SEQ ID NOS. 1-4). Sequence analysis of said highly cationic peptides and helix projection revealed a unique structure distinctly different from that of other potentially helical peptides isolated so far. The high antimicrobial hemolytic and insecticidal activity and the structural properties indicate a membrane-disturbing function of the cupiennins on prokaryotic as well as eukaryotic cells.

Cupiennin 1a has the following amino acid sequence:

GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH₂ (SEQ ID NO: 1) Cupiennin 1b has the following amino acid sequence:

GFGSLFKFLAKKVAKTVAKQAAKQGAKYIANKQME-NH₂ (SEQ ID NO:2)

Cupiennin 1c has the following amino acid sequence:

GFGSLFKFLAKKVAKTVAKQAAKQGAKYIANKQTE-NH₂ (SEQ ID NO:3)

Cupiennin 1d has the following amino acid sequence:

GFGSLFKFLAKKVAKTVAKQAAKQGAKYVANKHME-NH₂ (SEQ ID NO:4)

Cupiennin 1a* represents a variant of Cupiennin 1a, wherein the C-terminus is not amidated.

Cupiennin 1d* represents a variant of Cupiennin 1d, wherein the C-terminus is Gin and not amidated

Cupiennin 1d° represents a truncated variant of Cupiennin 1d (residues 1-26), which is not amidated:

FKFLAKKVAKTVAKQAAKQGA (SEQ ID NO:5)

Cupiennin 1d°° represents a truncated variant of Cupiennin 1d (residues 6-26) which is not amidated:

GFGSLFKFLAKKVAKTVAKQAAKQGA (SEQ ID NO:6)

Regarding the antimicrobial activity as determined against different bacteria species, the peptides of the present invention are significantly more potent than melittin or magainin 2 isolated from different species. Regarding hemolytic and insecticidal activity, the peptides of the present invention exhibit a significantly increased hemolytic and insecticidal activity. Also, having regard to melittin, the peptides of the present invention demonstrate an improvement in view of EC₅₀ and LD₅₀ values; cf. examples. In a preferred embodiment the variant exhibits i) an antimicrobial activity of at least 80%, more preferably, 90% of the peptide; ii) a hemolytic activity of at least 80%, more preferably 90% of the peptide; iii) an insecticidal activity of at least 80%, more preferably, 90% of the peptide.

In a further preferred embodiment, the variant is at least 60%, more preferably 80% and most preferably 90% homologous with the peptide.

In a further embodiment, the variant differs from the peptide by at least one addition, substitution, deletion, insertion and/or inversion of at least one amino acid. The introduction of mutations into a given amino acid sequence is generally well-known in the art. The mutated amino acid sequence may be generated by Merrifield synthesis and/or fragment condensation. On the nucleic acid level a modification may be facilitated by de novo synthesis of the coding nucleic acid sequence or alternatively site-directed mutagenesis may be performed among the numerous methods known in the art for introducing mutations in a given amino acid sequence. It is preferred that the general structural features of the Cupiennins which will be discussed below are maintained when introducing mutations into the peptide.

In a particularly preferred embodiment the substitution represents a conservative substitution. Four different physico-chemical groups are known in which the naturally occurring amino acids are divided into. The amino acids arginine, lysine and histidine belong to the group of basic amino acids. The amino acids glutamic acid and aspartic acid belong to the group of acidic amino acids. The non-charged/polar amino acids include glutamine, cysteine, asparagine, serine, threonine and tyrosine. The non-polar amino acids include methionine, phenylalanine, tryptophan, glycine, alanine, valine, proline, leucine and isoleucine. A conservative substitution in this context means the replacement of a given amino acid by an amino acid from the same physico-chemical group.

In a further preferred embodiment the peptide of the present invention carries a modification at the N-terminus, C-terminus and/or at a side chain of an amino acid. Amino acid modifications are well-known in the art. The modification may e.g. be selected from a covalent linkage of one or more of the following groups selected from: carboxylic acids, amines, polyethylene glycol, biotin and sugars. In a particularly preferred embodiment, the modification is selected from an acetylation, an amidation or an esterification.

Modifications also include di-, oligo- and polymerization of monomeric starting products, e.g. by cross-linkage, by dicyclohexyl carbodiimide or pegylation or association (self- assembly). The thus produced dimers, oligomers and polymers can be separated from each other by gel filtration, cationic exchange chromatography and reverse phase HPLC.

Modifications further include cyclization of the peptide or truncated variants thereof. Additionally, modifications may include the insertion of cysteines and/or pralines and/or other amino acids.

Further modifications include the use of at least one D-amino acid for the synthesis of the peptide. Particularly preferred, the peptide consists of D-amino acids. It is well known in the art that peptides comprising D-amino acids are less susceptible to proteolytic degradation than peptides lacking them. It is assumed that the use of D-amino acids does not interfere with the presumed mechanism, i.e. attack of the membranes.

According to another aspect of the present invention peptides are provided comprising at least one amino acid segment of the formula X^ X₂, X3.X4,

wherein Xi is Lys;

X₂ may be any amino acid;

X₃ may be selected from Leu, Val, Ala or Gly; and

X₄ may be selected from Ala or Val.

According to another embodiment, the present invention provides a construct comprising at least one peptide of the present invention and at least one tag. In this context, the term "tag" means any compound being capable of specifically recognizing a target structure. Preferably, the target structure is selected from cells, tissues or organs. The tag may represent a carbohydrate, the peptide, a protein or a lipid and mixtures thereof. Preferably, the carbohydrate may be selected from sialic acid or a derivative thereof. Particularly preferred are uronic acids. The peptide may preferably be selected from a peptide or polypeptide carrying a negative or positive net charge and being capable of forming charge-charge interactions with a binding partner having an opposite charge. The binding partner may be immobilized. Particularly preferred, the peptide or polypeptide may be selected from a his-tag or heparin.

The tag may also be selected from a sequence facilitating the purification of the produced peptide. Preferably, the tag represents a "his-tag" comprising at least four histidine amino acid residues.

According to a further embodiment, the present invention provides a fusion protein comprising at least one peptide of the present invention and at least one biologically active polypeptide or an active fragment thereof. In this context, the term "biologically active polypeptide" comprises any peptide or protein having biological activity. It is preferred that the biological activity represents an activity involved in the development and regeneration of cells, tissues or organs of the human or animal body. The term "fusion protein" means in this context that at least one peptide of the present invention is added to the amino acid sequence of the biologically active polypeptide or active fragment thereof and/or inserted into the amino acid sequence of the biologically active polypeptide and/or substitutes for an amino acid sequence naturally occurring in the biologically active polypeptide.

Preferably, the biologically active polypeptide is selected from, for example, maltose binding protein, thioredoxin, glutathione S-transferase or protein A.

The use of tags and fusion proteins is done using standard processes (7).

In a preferred embodiment the peptide comprises at least four, preferably at least 6, of said segments.

In a preferred embodiment, the peptide exhibits

i) an antimicrobial activity of at least 50% of any of the peptides having SEQ ID NOS: 1 to 4; ii) a hemolytic activity of at least 50% of any of the peptides having SEQ ID NOS:1 to 4; and/or

iii) an insecticidal activity of at least 50% of any of the peptides having SEQ ID NOS:1-4.

Surprisingly, it has been found that the Cupiennins isolated from spider venom are characterized by repeats of four amino acids to form the central part of the peptide chain. Without being bound by theory, it is assumed that said consensus motif may represent part of a receptor recognition sequence of said peptides.

In a further embodiment the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the peptide according to the present invention.

The nucleic acid molecule containing the nucleotide sequence may be derived from genomic DNA, cDNA or synthetic DNA, wherein synthetic DNA sequences also mean such sequences containing modified internucleoside bonds. Furthermore, the nucleotide sequence may be derived from RNA sequences, which may be necessary for the expression using recombinant RNA-vector systems.

The nucleotide sequence is also obtainable by using a detectably labeled probe corresponding to the nucleotide sequence encoding one peptide according to the present invention, or a fragment or the anti-sense strand thereof for screening of cDNA and genomic DNA libraries, respectively, from organisms, preferably the organism belonging to the genus Cupiennius.

The identification of positive cDNA and genomic DNA clones is done using standard processes (8, 9).

According to a preferred embodiment, a vector is provided comprising the nucleic acid molecule and optionally a promoter, wherein the promoter is operatively linked to the nucleic acid molecule such that the nucleic molecule may be expressed under the control of the promoter. The vector may be preferably selected from bacteriophages, adenoviruses, vaccinia viruses, baculoviruses, SV40 virus, retrovirus, plasmids like Ti- plasmids of Agrobacte um tumefaciens, YAC vectors and BAG vectors. Furthermore, the present invention provides host cells comprising the nucleic acid molecule and/or the vector. In the state of the art, numerous prokaryotic and eukaryotic expression systems are known wherein the host cells may e.g. be selected from prokaryotic cells like E. coli or B. subtilis or from eukaryotic cells like yeast cells, plant cells, insect cells and mammalian cells, e.g. CHO cells, COS cells or HeLa cells, as well as derivatives thereof. The vector may further comprise a nucleotide sequence encoding a signal peptide suitable for secretion of the encoded peptide from the host cell, thereby facilitating the purification.

According to a further embodiment of the present invention, a method for the recombinant production of the peptide is provided comprising the steps of:

i) transfecting a host cell with a vector comprising the nucleic acid molecule;

ii) culturing the transfectant under conditions allowing the expression of the peptide; and

iii) optionally isolating the peptide from the transfectant or the medium.

In a further embodiment the present invention provides a method for purifying a peptide having antimicrobial/hemolytic and/or insecticidal activity and having an amino acid sequence of any of SEQ ID NOS: 1-4 from spider venom comprising:

a) separating a sample from venom of the spider Cupiennius salei by gel filtration chromatography;

b) recovering the active fractions therefrom;

c) contacting the active fractions of step b) with a cationic exchange chromatography material;

d) eluting active fractions at about 30 to 100% 2 M NaCI therefrom.

e) contacting the active fractions of step d) with a reversed phased chromatography material; f) eluting the active fractions at about 30 to 40% acetonitrile; and

g) optionally repeating steps e) and f) to obtain further purification of the active fractions.

Preferably, the separation step a) is performed on a Superdex^® 75HR10/30 in 200 mM ammonium acetate buffer pH 5.5. In a preferred embodiment the cationic exchange chromatography is performed on a MonoS^® HR10/10 column in 200 mM ammonium acetate buffer pH 5.5. The active fractions are preferably collected between about 50 to about 90% 2 M NaCI in 200 mM ammonium acetate buffer pH 5.5.

In a preferred embodiment the reversed phase chromatography material according to step e) represents the Cι_S material. Particularly preferred, the reversed phase chromatography material represents a nucleoside 120-5 Cι₈ column. Preferably, the active fractions are eluted at about 37% acetonitrile. In a preferred embodiment the optional further purification step g) may represent a further reversed phase HPLC chromatography step. Particularly preferred is an isocratic gradient having about 36% (v/v) of a mixture consisting of 0.1% trifluoretic acid (TFA) and 0.1% TFA in acetonitrile.

The present invention further provides antibodies capable of specifically binding the peptides according to the present invention and obtainable by immunization of laboratory animals with the peptides of the present invention. Polyclonal antibodies may be obtained by immunization, e.g., of rabbits, mice or rats and subsequent recovery of antiserum. Monoclonal antibodies may be obtained by standard processes by immunization of e.g. mice, recovery and immortalization of spleen cells and cloning of hybridoma producing the peptide specific antibody.

The present invention further provides pharmaceutical compositions comprising at least one peptide according to the present invention and a pharmacologically acceptable carrier well-known in the art.

Preferably, the pharmaceutical composition may further include an antibiotic agent and/or a cytotoxic compound. The cytotoxic compound may be selected from known cytotoxic compounds used in cancer treatment, preferably antibodies and cell targeting compounds. Due to their high antimicrobial activity (bactericidal concentration: 0.16 to 5 μM) pharmaceutical compositions comprising the peptide are considered to be useful for the treatment of bacterial infections, including some of the most difficult to treat antibiotic- resistant pathogen-mediated diseases. The peptides according to the present invention are considered to be useful for the diseases mediated by gram negative as well as gram positive bacteria.

Experimental data showing a cytotoxic effect on bloodstream forms of Trypanosoma brucei of submicromolar concentrations of the peptide have been obtained. The peptides are therefore indicated for the treatment of parasitic diseases, including malaria and sleeping sickness (African trypanosomiasis).

The peptides of the present invention exhibit a high hemolytic activity. The ECso-value has been determined in the range of 14.5 to 24.4 μM. Such a strong cytolytic effect is considered to be useful for the treatment of diseases characterized by an exceeding growth of cells as e.g. tumors, in particular leukemias. The peptides of the present invention are also considered useful for the treatment of autoimmune diseases in view of their potential to eradicate hyperactive T-cells.

Due to their highly positive charge, the peptides, in particular the cupiennins, act preferably on cells containing a negatively charged surface. The peptides of the present invention are thus indicated for the treatment of diseases mediated by negatively charged cells including, but not limited to erythrocytes, tumor cells, bacteria, protozoa and vertebrate cells containing gangliosides.

Regarding the mode of administration of the pharmaceutical composition to the patient, the pharmaceutical compositions may be administered in any way, e.g. intravenous, intramuscular, intraperitoneal, subcutaneous, or topical. Particularly preferred is the topical administration of the pharmaceutical compositions.

The useful dosages may be routinely determined by the physician and are well-known in the art.

In a further embodiment the present invention provides pharmaceutical compositions comprising at least one nucleic acid molecule encoding a peptide according to the present invention and a pharmacologically acceptable carrier. Preferably, the pharmaceutical compositions are used in the context of a gene therapy, wherein upon transformation with a suitable vector the peptides are expressed and may serve for one of the above indicated medical purposes.

In another embodiment the present invention provides a method for inactivating bacteria comprising: contacting the object to be inactivated with at least one peptide according to the present invention, preferably, the method is an in vitro method.

In a further embodiment the present invention provides a method for inactivating insects comprising: contacting the object to be inactivated with at least one peptide according to the present invention. Preferably, the method is an in vitro method.

In the following, examples showing the invention in practice will be described. The examples are, however, not intended to limit the scope of the invention as will be apparent to the person skilled in the art.

FIG. 1. Isolation of cupiennins from the venom of the spider Cupiennius salei. A, crude venom was first separated by gel filtration on a Superdex 75 column and the obtained antimicrobial fractions were pooled. B, Further separation of the pooled fraction was achieved by cationic exchange on a Mono S column. C, using RP-HPLC on a nucleosil 120-5 Ci₈ column, the cupiennins were isolated as a broad peak. D, in a last purification step using RP-HPLC on a nucleosil 100-5 C₈ HD column cupiennin 1a, b, c and d (1 nmol) were isolated as described in materials and methods. Synthesized cupiennin 1 a* (1 nmol, not amidated) differed only slightly in retention time. Arrows show small peaks of Met34 sulfoxidated cupiennins. Absorbance was measured in milli-absorbance units.

FIG. 2. Amino acid sequences of cupiennins from the venom of the spider Cupiennius salei. A, amino acid sequence of cupiennin 1 a acquired by sequence analysis until position Met34 and the chymotryptic peptide 29-35. B, overview of amino acid sequences of cupiennin 1a*, 1a, 1b, 1c, 1d, 1d*, 1d° and 1d°°, deduced from a combination of tryptic peptide mapping and sequence analysis of non-identical tryptic peptides. FIG. 3. Tryptic peptide mapping of cupiennins from the venom of the spider Cupiennius salei. A, after digestion of cupiennin 1a, 1b, 1c and 1d (5 μg) with trypsin (0.5 μg), peptides were fractionated isocratically (0.1% trifluoroacetic acid in water) on a nucleosil 120-5 Ci₈ column. B, in a second part further separation was obtained using a linear gradient (0.23% acetonitrile/min) as described under materials and methods. The separated peptides (F) were identified by ESI-MS [monoisotopic masses, Da] and compared with peptides of cupiennin 1a. Non-identical peptides (F*) were sequenced by Edman degradation.

Fig. 4. α-Helical wheel projection of the cupiennin sequences and net projection of cupiennin 1a. A, gray circles correspond to residues with positively charged side chains. Polar amino acids are marked with interrupted circles, θ is the denoted angle subtended by the hydrophilic helix face.

FIG. 5. CD spectra of cupiennin 1 a, 1 a*and 1 d*. 5a, CD characteristics of cupiennins 1 a (C = 5 x 10^"5 M) in buffer (5 mM sodiumphosphate, 150 mM sodiumfluoride, pH 7.25) ; and in TFE/buffer mixtures: TFE (30%v), — , TFE (50 %v). 5b, and 5c, CD- spectra of cupiennin 1a 1a* and — 1d* in TFE/buffer mixtures, [θ] is the mean residue ellipticity.

FIG. 6. Sequence alignment of cupiennin 1 a and 1 d compared with amino acid sequences of other antimicrobial peptides. Lysine motifs are boxed and identical residues are shaded in grey. Amino acid differences of cupiennin 1 a to cupiennin 1 d and identities with other peptides are shaded in dark grey. The asterisks (*) labeled C- terminal amidation.

EXPERIMENTAL PROCEDURES

Chemicals— -TFA (for protein sequencing), TFE and acetonitrile (LiChrosolv®, for chromatography) were purchased from Merck. Melittin (amidated) was purchased from Tocris (Anawa Trading SA, Switzerland) and magainin 2 (not amidated) from Sigma (St. Louis, U.S.A). Isolation of Toxins — Cupiennius salei (Ctenidae) spider maintenance, venom collection by an electrical milking procedure, separation of venom by gel filtration, cationic exchange chromatography, and RP-HPLC were performed as previously described (1). Briefly, 450 μl of crude venom was fractionated into nine 50 μl aliquots and diluted with 150 μl of 200 mM ammonium acetate buffer pH 5.5 (buffer A). The diluted venom was separated on a Superdex 75 HR 10/30 column (Amersham Pharmacia Biotech, Sweden) in buffer A and fractions were collected as noted on the chromatogram (see Fig. 1A). Further separation of the pooled fractions was achieved by cationic exchange on a MonoS HR10/10 column (Amersham Pharmacia Biotech, Sweden) in buffer A. Elution was done with a salt gradient (2 M NaCI in buffer A, pH 5.5) as shown in Fig. 1 B. Similar fractions from four chromatographies were combined. The pooled fractions were further desalted and separated by RP-HPLC on a nucleosil 300-5 C₄ column (4.6 x 250 mm, Macherey-Nagel, Germany) using 100% solvent A with a flow rate of 0.5 ml/min for 0-15 min (100% A) followed by a first 10-min gradient of 1% solvent B in A/min and a second 120-min gradient of 0.4% solvent B in A/min. Solvent A: 0.1% TFA in water, solvent B: 0.1% TFA in acetonitrile (not shown). After each step of purification the fractions were tested for their antimicrobial activity by a plate growth assay as described in (3). Further purification was achieved by RP-HPLC on a nucleosil 120-5 C-ι₈ column (4 x 250 mm, Macherey-Nagel, Germany) equilibrated with 22% solvent B in solvent A and a flow rate of 0.5 ml/min. Ten minutes after injection of the sample the first gradient (22-40% solvent B) was started for 115 min followed by a second 10-min gradient (40-100% solvent B). The obtained peptide fractions were pooled (Fig. 1C) and purified in a final RP-HPLC step on a nucleosil 100-5 C₈ HD column (4 x 250 mm, Macherey-Nagel, Germany) with constant 36% solvent B and a flow rate of 0.5 ml/min (Fig. 1 D). This step was repeated three to five times to obtain homogenous peptides. The purity of these peptides was controlled by ESI-MS and amino acid analyses.

Enzymatic cleavages — 10 μg .of cupiennin a were incubated, with 1 μg chymotrypsin . . (sequencing grade, Roche Diagnostics, Switzerland) in 10 mM Tris-HCI, 1 mM CaCI₂, pH 7.5 for 4 h at 25°C. Separation of the chymotryptic peptides was achieved with RP- HPLC on a 120-5 Cι₈ column (2 x 125 mm, Macherey-Nagel, Germany) equilibrated with 100% solvent A. 10 minutes after injection of the sample a 250 min gradient of 0.2% solvent B in A/min was started. Comparative digestions of 5 μg of purified cupiennin 1a, 1a*, 1b, 1c and 1d with 0.5 μg trypsin (sequencing grade, Roche Diagnostics, Switzerland) were carried out in 100 mM ammoniumhydrogencarbonate buffer pH 8.0 for 4 h at 25°C. Separation of the tryptic peptides was obtained under isocratic conditions with RP-HPLC on a 120-5 C₁₈ column (2 x 125 mm, Macherey-Nagel, Germany) equilibrated with 100% solvent A. A second run was started and after 10 minutes followed by a 120-min gradient of 0.23% solvent B in A/min. Solvent A: 0.1% TFA in water, solvent B: 0.1% TFA in acetonitrile (Fig. 3A, B). Mass analysis of the digested samples and of the isolated peptides was performed on ESI-MS.

Amino acid analysis - Samples were hydrolyzed in the gas-phase with 6 M hydrochloric acid containing 0.1 % (by vol.) phenol for 24 h at 115° C under N₂ vacuum (10). The liberated amino acids were labeled with phenylisothiocyanate and the resulting phenylthiocarbamoyl amino acids analyzed by RP-HPLC on a Nova Pak ODS column (3.9 x 150 mm, 4 μm; Waters) in a Hewlett Packard liquid chromatograph 1090 with an automatic injection system (11).

Amino acid sequence analysis — N-terminal sequence analysis was carried out either in a Procise cLC 492 protein sequencer or in a pulsed-liquid-phase sequencer 477A, both from Applied Biosystems. The released amino acids were analyzed on-line by RP-HPLC according to instructions from Applied Biosystems.

ESI-MS — Determination of the molecular mass of isolated cupiennins and proteolytic peptides was done using electrospray ionization mass spectrometry on a single-stage quadrupole instrument (VG Platform, Micromass, Manchester, UK), calibrated with horse myoglobin in a mass range of 600-2000 m/z. Peptides from enzymatic cleavages represent monoisotopic masses, native cupiennins average masses.

Synthesis of peptides— Solid-phase peptide synthesis was performed on a Millipore 9050 continuous flow peptide synthesizer (Millipore Corp., Milford, MA, USA) using Fmoc chemistry. Cleavage and deprotection was carried out in 88% TFA, 5% liquefied phenol, 2% triisopropylsilane, 5% water for 1-2 h at room temperature. The free peptide was then repeatedly precipitated with ice-cold ether and dried under vacuum. After resuspension in 10% acetic acid the peptides were purified by preparative RP-HPLC on a C-ι₈ column (25 x 100 mm, 15 μm, 300A, Delta-Pak, Waters, Millipore Corp., USA) which was eluted at 5 ml/min with a gradient of 0 to 60 % acetonitrile in 0.1 % TFA at an increment of 1.3 % per min. Peak fractions were repurified on a semi-preparative C₁₈ column (10 x 250 mm, 7 μm, 300A, Vydac, Holland, Ml). Purity and protein composition was analyzed by ESI-MS, amino acid analysis and N-terminal sequence analysis.

Circular Dichroism — Stock solutions of the peptides were prepared by dissolving the samples in 5 mM sodium phosphate buffer, pH 7.2 containing 150 mM NaF. For CD measurements aliquots of the solution were diluted with buffer or mixed with TFE to give a final concentration of 50 μM and the desired solvent composition. Measurements were carried out on a J 720 spectrometer in 0.1 cm cells between 195 and 260 nm at room temperature (Jasco, Japan). Each spectra was the average of 5 scans. The baseline was subtracted. The helicity ( ) of cupiennin 1a, 1a* and 1d*, was determined from the mean residue ellipticity at 222 nm (Θ₂₂₂), according to the equation: α (%)= (Θ₂₂₂+2340) x 100%/-30300 (12).

Antimicrobial assays— Bacteria (Escherichia coliATCC 25 922; Staphylococcus aureus ATCC 29213; Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27 853) were cultured in Mueller Hinton broth. Determination of the minimal inhibitory concentration for the cationic antimicrobial peptides was performed using a 2-fold microtiter broth dilution assay (4). Mueller Hinton broth was used to dilute the bacterial inoculum, which was prepared from mid log phase cultures to give a final concentration of 1.7 - 3.8 x 10⁵ cfu/ml in the wells. First, 100 μl of the bacteria dilution was added into the wells, followed by 10 μl of the test peptides in 0.01% acetic acid, 0.2% BSA. Peptides (0.04-100 μM), non-treated growth control and a sterility control were tested in triplicate. The microtiter plates were incubated at 37°C for 24h. The content of the first 4 wells showing no visible growth of bacteria (measured as an increase of optical density at 630 nm) were plated out on blood agar plates and incubated at 37°C for 18h. Minimal inhibitory concentrations (MIC) are expressed as intervals of concentrations: [a]-[b]; where [a] is the highest concentration of peptide at which bacteria still grow and [b] being the lowest concentration causing 100% of growth inhibition (no colony forming bacteria estimated after additional plating out of 91 % of the tested bacteria suspension).

Hemolytic assay— Hemolytic activity of cupiennin 1a, 1a*, 1b, 1d, 1d*, 1d°, 1d°°, magainin 2 and melittin was determined using fresh human red blood cells (hRBC). 1 ml citrated blood was washed 4 times with 6 ml of PBS buffer (50 mM sodium phosphate buffer, 150 mM NaCI, pH 7.2) and centrifuged (900xg) for 6 min at room temperature. The pellet was resuspended in 3 ml and further diluted to a concentration of 1 X 10⁹ hRBC/ml in PBS buffer. Lyophilized toxins in various concentrations were resolved in 200 μl PBS buffer and 50 μl of hRBC were added following incubation under gentle shaking at 37°C for 1 hour. The samples were then placed on ice and immediately centrifuged at 4°C. The supernatant was carefully removed and the pellet was resuspended in 240 μl of water. Release of hemoglobin was monitored by measuring the absorbance of supernatant and water treated pellet at 541 nm in a 0.1 cm cell (JascoN- 550, Japan). The negative control (0% hemolysis) was 50 μl hRBC in 200 μl PBS buffer and the positive control (100% hemolysis) was 50 μl hRBC in 200 μl water. The concentrations of peptide at which 50% hemolysis was observed (EC₅o) were derived using a sigmoidal curve fitting software (Graph Pad Prism, 3.0; Graph Pad Software, Inc. USA).

Bioassays— Bioassays using Drosophila melanogaster according to (5) were performed to estimate the LD₅o (24h post injection) of the peptides. For each assay 20 flies were used as control (injecting 0.05 μl of insect ringer) and 20 for each of the 3 peptide concentrations. LD₅o stands for the lethal dose (50% of the test flies die of intoxication) and calculations were done as described in (6).

RESULTS

Purification of cupiennins— 450 μl of venom were separated in a five step protocol that included gel filtration (Fig. 1A), cationic exchange chromatography (Fig. 1B) and successive RP-HPLC on a nucleosil 300-5 C₄ column (not shown), a nucleosil 120-5 Cι₈ (Fig. 1C) and on a nucleosil 100-5 C₈ column (Fig. 1D). The retention times of the purified antimicrobial peptides went from 18.35 min for cupiennin 1d to 20.12 min for cupiennin 1a. The retention profiles revealed no other impurities. The purity of the obtained peptides were additionally examined by ESI-MS, N-terminal sequence analysis and amino acid composition. The yield of cupiennin 1a (ESI-MS 3798.63 ± 0.51 Da, theoretically 3798.59 Da) was 4.7 μg/μl of fractionated venom. This implies that the toxin concentration is 1.2 mM in crude venom. Cupiennin 1b (ESI-MS 3800.25 ± 0.28 Da, theoretically 3800.57 Da) occurred in the venom at a concentration of 0.4 μg/μl. Due to very similar retention times, the last RP-HPLC purification step had to be repeated several times for cupiennin 1c (ESI-MS 3769.75 + 0.50 Da; theoretically 3770.48 Da) and cupiennin 1d (ESI-MS 3795.13 ± 0.79 Da, theoretically 3795.55 Da). The procedure was accompanied by a substantial loss of peptides. The yielded amounts of cupiennin 1c and 1d were 0.02 and 0.1 μg/μl crude venom, respectively. The purification procedure was impeded by methionine oxidation at position 34 in cupiennin 1a, 1b and 1d. The sulfoxide containing cupiennins eiuted directly in front of the nonoxidated peptides (Fig. 1 D, peaks are labeled with arrows) as small peaks and were identified by ESI-MS (for cupiennin 1a: ESI-MS 3814.50 + 0.35 Da, theoretically 3814.59 Da) and additionally amino acid analyses (not shown).

Amino acid and sequence analysis of the C-terminal tryptic peptide 33-35 of sulfoxidated cupiennin 1a (ESI-MS 421.28 Da, theoretically 421.09 Da) confirmed the presence of methionine sulfoxide at position 34. The oxidation of methionine is a well known artifact during the repeated purification cycles on RP-HPLC.

Sequence analysis of cupiennin 1a — Sequence analysis of cupiennin 1a ceased at position 34. Chymotryptic peptides were separated by RP-HPLC on a nucleosil 120-5 C₁₈ column (not shown). The five obtained peaks were identified by ESI-MS and the C- terminal peptide (residue 29-35; ESI-MS 845.40 Da, theoretically 846.43 Da) was sequenced. Glutamic acid was identified as the C-terminal amino acid (Fig. 2A). The observed molecular mass difference of 1 Da between the theoretical and measured mass of the chymotryptic C-terminal fragment implied an amidation of glutamic acid. Cupiennin 1 a* (*acidic C-terminus) was synthesized to confirm the assumed posttranslational amidation in cupiennin 1a. After purification, cupiennin 1a* (ESI-MS 3799.38 + 0.39 Da, theoretically 3799.58 Da) eiuted as a single sharp peak in RP-HPLC on an analytical nucleosil 120-5 C₈ HD column. The retention time differed slightly from the retention time of cupiennin 1a (Fig. 1 D) indicating the chemical modification. The correct sequence of cupiennin 1a* was confirmed by amino acid analysis, ESI-MS and Edman degradation (not shown). The determined amino acid sequence of cupiennin 1a and 1a* agree well with the results of the amino acid composition analyses (Table I).

Sequence analysis of cupiennin 1b, 1c and 1d— Comparing the results of the amino acid analysis of cupiennin 1a, 1a*, 1b, 1c and 1d (Table I) only slight differences in the content of Ala, Ser, Glx, lie, Val, His, Thr and Met were observed. To determine the amino acid sequences of cupiennin 1b, 1c and 1d, comparative tryptic peptide mapping was performed. Peptide separation was carried out by RP-HPLC (Fig. 3A, B) and the obtained peptides identified by ESI-MS. The tryptic peptides of cupiennin 1a and 1a* are identical (Table II) except the C-terminal peptides, being amidated in the case of cupiennin 1a as revealed by the mass difference of 0.85 Da (ESI-MS). Cupiennin 1b differs from cupiennin 1 a: 1 ) in the N-terminal peptide 1 -7, Ala4 is exchanged by Ser and 2) in the peptide sequence 28-32, Val29 and Val30 are replaced by Ile29 and Ala30. Cupiennin 1c and 1d bear Ser at position 4 in peptide 1-7. The sequence 28-32 of cupiennin 1c is identical with the 28-32 fragment of cupiennin 1b (Val29, Val30 substituted by Ile29 and Ala30) but in addition Met34 is replaced by Thr in the C-terminal peptide 33-35. Cupiennin 1d differs in the C-terminal region from cupiennin 1a in fragment 28-32, where Val30 is replaced by Ala and in fragment 33-35, where Gln33 is exchanged by His (Fig. 2B). The sequences of all tryptic peptides which differed from cupiennin 1a fragments were determined by Edman degradation (Table II). The deduced amino acid sequences of cupiennin 1b, 1c and 1d (Fig. 2B) are in good agreement with the results of the amino acid analyses (Table I). For further studies a cupiennin 1d analogue with Gin as non-amidated C-terminus was synthesized (cupiennin 1d*). The correct sequence of cupiennin 1d* was confirmed by amino acid analysis (Table I) and ESI-MS (ESI-MS 3794.90 ± 0.44 Da, theoretically 3795.55 Da) (not shown).

General structural features— Cupiennin 1a, 1b, 1c and 1d are linear peptides consisting of 35 amino acid residues. The C-terminus is amidated and the net positive charge is at least + 8 at neutral pH (Fig. 2B). The theoretical isoelectric point is for all peptides 11.54. The N-terminal part of the sequences (Gly-Phe-Gly-Ala/Ser-Leu-Phe) is rather hydrophobic whereas polar amino acid residues predominate in the C-terminal region. All cupiennins are characterized by 6 repeats of 4 amino acids which form the central part of the peptide chain. These repeats are defined by the following sequence: position 1 is always lysine; position 2 is variable (hydrophobic, charged or polar amino acid); position 3 is always a hydrophobic amino acid (Leu, Val, Ala) or Gly and in position 4 Ala or Val (Fig.6). Based on the consensus scale of the hydrophobicity for the individual amino acid residues of Eisenberg (13) the mean hydrophobicity (H) of the cupiennins were found to range between -0.138 and -0.168. Assuming an -helical conformation their hydrophobic moment (μ) was determined to vary between 0.0121 to 0.0282 (Fig. 4A, Table III). These parameters distinctly differ from H and μ values characterizing other antimicrobial peptides such as lycotoxins (14) isolated from spider venom, melittin (15) from bee venom, and magainin 2 (16) found in the frog skin. The mean hydrophobicity as well as the hydrophobic moment of the cupiennins are distinctly lower than H and μ of melittin (H = -0.086, μ = 0.224), magainin 2 (H = -0.036, μ = 0.286) and lycotoxin I (H = - 0.083, μ = 0.0681 )(Table III). The angle subtended by polar residues (Φ) describing the hydrophilic helix surface is with 220° unambiguously greater than the polar face of most other helical antimicrobial peptides (Fig. 4A).

Circular dichroism — The CD spectra of the peptides in sodium phosphate buffer characterize an unordered peptide structure (Fig. 5A). Addition of TFE induces pronounced spectral changes (Fig. 5B, 5C). The negative bands at 207 and 222 nm and the positive ellipticity below 200 nm are characteristic of a α-helical conformation. All peptides were found to be completely helical in the TFE/buffer (1/1 v/v) mixture (Fig. 5). Following Lehrman et al. (17), who suggested that TFE induced helicity of peptides is a measure of their helix propensity, we take the high of the investigated cupiennins as an indicator of their very high capacity to assume a helical conformation (Table III).

Antibacterial effects — The cupiennins are highly active against bacteria. All four tested bacteria species were susceptible to cupiennin 1a, 1a*, 1d, 1d°° and 1d* in the nanomolar to the micromolar concentration range (Table IV). Differences in the activity between the amidated natural and C-terminal free synthesized cupiennins against E. coli

(0.31-0.63 μM; cupiennin 1a and 1a*) and E. faecalis (2.5-5.0 μM cupiennin 1a and 1a*;

1.25-2.5 μM cupiennin 1d and 1d*) were not observed. The activity of cupiennin 1a (more active) and cupiennin 1a* against P. aeruginosa and S. aureus and of cupiennin 1d and

1 d* against P. aeruginosa and S. aureus differed by one dilution step. The activity of cupiennin 1d (more active) and 1d* against E. coli differed by two dilution steps.

Pronounced differences in the susceptibility of gram positive and gram negative bacteria could not be found. E. faecalis exhibited the weakest susceptibility against the and Cupiennin Id cupiennins. Here, melittin was one dilution step more active. Magainin 2 αid not snow growth inhibiting activity against the tested bacteria up to a concentration of 60 μM.

Hemolytic effects— The half maximal concentrations (EC₅o) of the tested cupiennins.to ,,. induce hemolysis were found to range between 14.5 and 24.4 μM (Table V). Compared to melittin (EC50 .7 μM) the lytic activity was reduced by a factor of 8.5 for cupiennin 1 d* and a factor of 14.4 and 12.1 for cupiennin 1a and cupiennin 1a*. At a concentration of 8 μM Cupiennin 1b induced 15% hemolysis, thus being about 2 times more active than cupiennin 1a and less effective than cupiennin 1d (30%). Magainin 2 and 1 ° showed no hemolytic effect. Insecticidal effects— We have investigated insecticidal effects in a bioassay with Drosophila melanogaster. Cupiennin 1a, 1a*, 1b, 1 d and 1 d* showed LD₅o concentrations between 4.7 and 7.9 pmol/mg fly measured after 24 h (Table V). Differences in the LD₅o doses of the synthesized cupiennins 1a* and 1d* and the natural forms were marginal. Obviously, the neurotoxic effects is independent of the C-terminal amidation. In comparison to melittin (14.6 pmol/mg fly) the toxicity of the cupiennins was 2.3 - 3.1 fold enhanced and compared to magainin 2 (123.1 pmol/mg fly), it was 19.2 - 26.2 times higher (Table V). Injection of peptides at sublethal doses paralyzed the flies in most cases within the first 3 minutes. Dose-dependent recovery from the paralysis was observed within 1-6 h.

TABLE

Amino acid analyses of natural and synthesized (*) cupiennins.

Amino acid composition of cupiennin 1a, 1b, 1c and 1d from Cupiennius salei venom purified by RP-HPLC and synthesized cupiennin 1a* and 1d*. The values in parentheses are calculated from the amino acid sequence. n.d. not determined.

amino cupiennin 1a cupiennin 1a* cupiennin 1b cupiennin 1c cupienninld cupiennin ld* acid (mol/mol) (mol/mol) (mol/mol) (mol/mol) (mol/mol) (mol/mol)

Asx 0.8 (1) 0.9 (1) 0.9 (1) 0.8 (1) 0.8 (1) 0.9 (1)

Glx 3.8 (4) 4.0 (4) 3.9 (4) 3.7 (4) 2.9 (3) 3.0 (3)

Ser 0.1 (0) 0.0 (0) 1.0 (1) 0.9 (1) 0.9 (1) 0.8 (1)

Gly 2.9 (3) 3.1 (3) 3.0 (3) 2.9 (3) 2.9 (3) 2.9 (3)

His 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.8 (1) 1.0 (1)

Arg 0.0 (0) 0.0 (0) 0.1 (0) 0.0 (0) 0.0 (0) 0.0 (0)

Thr 0.9 (1) 1.0 (1) 1.0 (1) 1.8 (2) 1.0 (1) 1.0 (1)

Ala 7.0 (7) 7.5 (7) 6.5 (7) 6.5 (7) 6.7 (7) 7.4 (7)

Pro 0.0 (0) 0.0 (0) 0.1 (0) 0.0 (0) 0.0 (0) 0.0 (0)

Tyr 1.0 (1) 1.0 (1) 1.0 (1) 1.1 (1) 1.1 (1) 1.0 (1)

Val 3.3 (4) 3.3 (4) 2.0 (2) 2.0 (2) 2.9 (3) 3.0 (3)

Met 0.7 (1) 1.0 (1) 0.6 (1) 0.1 (0) 0.6 (1) 0.9 (1)

Cys 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0)

He 0.0 (0) 0.0 (0) 0.9 (1) 0.9 (1) 0.1 (0) 0.0 (0)

Leu 2.0 (2) 2.1 (2) 2.1 (2) 2.0 (2) 2.0 (2) 2.1 (2)

Phe 3.0 (3) 3.2 (3) 2.8 (3) 2.9 (3) 2.9 (3) 3.0 (3)

Lys 7.6 (8) 7.8 (8) 7.4 (8) 7.4 (8) 7.5 (8) 8.0 (8)

Trp n.d. (0) n.d. (0) n.d. (0) n.d. (0) n.d. (0) n.d. (0)

Total 33.1 (35) 34.9 (35) 33.3 (35) 33.0 (35) 33.1 (35) 35.0 (35)

TABLE II

Tryptic peptide mapping of cupiennin 1a* 1a, lb, 1c, 1d and identification by ESI-MS. Cupiennins were incubated with trypsin (10:1) for 4 h at 25°C. Tryptic peptides were isolated by RP-HPLC (Fig. 3), the molecular mass measured by ESI-MS and the (t)amino acid sequence determined by Edman degradation. Numbers in parenthesis are theoretical values (monoisotopic, Da). The replacement of the amino acids in cupiennin 1b, 1c and 1d is mentioned in Fig. 2. * not amidated.

cupiennin cupiennin cupiennin cupiennin cupiennin

Fragment 1 a* 1a 1 b 1c 1d

1-7 738.30 738.30 1-754.51 754.47 754.47 (738.41) (754.41)

8-11 477.25 477.40 477.65 477.39 477.33 (477.30)

13-15 316.12 316.06 316.05 315.92 (316.22)

12-15 444.46 444.33 444.34 444.4 444.53 (444.31)

16-19 417.20 417.22 417.40 417.29 417.29 (417.27)

20-23 416.39 416.22 416.40 416.41 416.27 (416.25)

24-27 402.18 402.80 402.32 402.32 402.38 (402.22)

28-32 621.31 621.31 T607.30 607.63 1-593.29 (621.36) (607.34) (607.34) (593.33)

33-35 406.09 405.24 405.29 t375.09 t414.11 (406.16) (405.18) (375.18) (414.18)

TABLE III

Structural properties of cupiennins and other antimicrobial peptides.

The net charge of the peptides was calculated under the assumption that under physiological conditions Lys, Arg and the N-terminal NH₂ are positively and Glu and the C-terminal COOH are negatively charged. The His residues were calculated as not charged. H and μ were calculated on the basis of the Eisenberg consensus scale of hydrophobicity (13).

Φ The molecular masses were determined using the ExPASy-PeptideMass program as average [M] (www.expasy.ch).

The percentage of helicity ( ) in sodium phosphate buffer TFE = 1:1 (v/v) of the peptides was determined from the molar ellipticity at 222 nm according to Chen (12).

* not amidated, n.d. not determined.

^A data from (18).

Truncated cupiennin 1d (residue 1-26) not amidated.

"Truncated cupiennin 1d (residue 6-26) not amidated. mean hydro- molecular mean α (%)

Peptide net charge phobic moment mass (Da)Φ hydrophobicity H μ (50% TFE) cupiennin 1a 3798.59 +8 -0.138 0.0226 100 cupiennin 1a* 3799.58 +7 -0.136 0.0226 100 cupiennin 1b 3800.57 +8 -0.155 0.0229 n.d. cupiennin 1c 3770.48 +8 -0.168 0.0121 n.d. cupiennin 1d 3795.55 +8 -0.152 0.0282 n.d. cupiennin 1d* 3795.55 +8 -0.152 0.0282 100 cupiennin 1d° 2233.73 +6 -0.175 0.1160 84 cupiennin 1d°° 2695.25 +6 -0.095 0.0738 100 magainin 2* 2466.93 +3 -0.036 0.2861 ^A57 melittin 2846.51 +6 -0.086 0.2244 n.d. lycotoxin I 2843.50 +6 -0.083 0.0681 n.d. lycotoxin II* 3206.94 +6 -0.219 0.1267 n.d.

TABLE IV

Antimicrobial activity of cupiennins (native and synthetic derived), melittin and magainin 2 against different bacteria species. cfu = colony forming units

"Minimal inhibitory concentration (MIC), determined by a liquid growth assay after 24h, are expressed as intervals of final concentrations [a] - [b]:

[a] the highest concentration, where bacteria are growing, [b] the lowest concentration causing 100% of growth inhibition. ° (-) indicates not performed measurements

Bacteria cfu^a/ml Minimal inhibitory concentration (μM)^D (x10⁵)

cupiennin cupiennin cupiennin cupiennin cupiennin cupiennin melittin magainin2 1a 1a* 1d 1d* 1d° 1d°°

Escherichia coli (K12, strain 5.0 0.45-0.90 0.9-1.8

C600)

Escherichia coli ATCC 25922 3.1 0 0..3311--00..6633 0.31-0.63 0.08-0.16 0.31-0.63 >160.00 1.25-2.50 0.63-1.25 >60.00

Pseudomonas aeruginosa ATCC 3.8 00..3311--00..6633 0.63-1.25 0.16-0.31 0.31-0.63 >160.00 0.63-1.25 2.50-5.00 >60.00

27853

Pseudomonas putida 10.0 1 1..8800--44..5500 " - " _."

(KT3442/RSF 1010)

Staphyloccocus aureus ATCC 2.6 0 0..3311--00..6633 0.63-1.25 0.63-1.25 0.31-0.63 >160.00 10.00- 1.25-2.50 >60.00

29213 20.00

Staphylococcus epidermidis 3.0 0 0..1188--00..4455

Bacillus subtilis 3.0 00..0099--00..1188

Enterococcus faecalis ATCC 1.7 22..5500--55..0000 2.50-5.00 1.25-2.50 1.25-2.50 >160.00 >20.00 0.63-1.25 >60.00

29212

Paracoccus denitrificans TH 7.0 0.09-0.18 0.09-0.18

Delft Paco 0001

TABLE V

Hemolytic and insecticidal activity of cupiennin 1a, 1a* 1b, 1d, 1d*, 1d°, 1d°°, melittin, magainin 2, CSTX-1 and 13peptidef.

Human erythrocytes were incubated with peptides for 1 h at 37°C. Hemolysis (EC₅₀) was determined as percentage of released hemoglobin by measuring the absorbance at 541 nm.

Estimation of the lethal dose (LD₅₀), were 50% of the test flies die of intoxication 24h after injection, were done using bioassays on Drosophila melanogaster. Different amounts of peptides were dissolved in insect ringer and 0.05 μl injected into the flies. n.d. not determined. t the last 13 C-terminal amino acids of CSTX-1

Hemolysis Bioassay

Peptide EC₅₀ (μM) LDso (pmol/mg fly)

(95% confidence limits) (95% confidence limits)

Cupiennin 1a 24.4 (22.9-26.0) 5.9 (4.2-8.3)

Cupiennin 1a* 20.5 (19.4-21.8) 6.2 (5.1-7.5)

Cupiennin 1 b n.d. 4.7 (3.9-5.7)

Cupiennin 1c n.d. n.d.

Cupiennin 1d n.d. 6.4 (5.4-7.6)

Cupiennin 1d* 14.5 (14.4-14.7) 7.9 (6.4-9.9)

Cupiennin 1d° >100 >400

Cupiennin 1d°° 50.4 (49.7-51.1) 30.7 (28.7-33.0)

Melittin 1.7 (1.6-1.9) 14.6 (11.0-18.4)

Magainin 2 >100 123.1 (107.4-147.5)

CSTX-1 0.35 (0.30-0.42)

13-peptidef >1000 6910 (5920-8390)

References

1. Kuhn-Nentwig et al., Toxicon 32 (1994), 287-302

2. Kuhn-Nentwig et al., Toxicon 36 (1998), 1276-1277

3. Hae erli, S. et al., Toxicon 38 (2000), 373-380

4. Wu, M. and Hancock, R.E.W., J. Biol. Chem. 274 (1999), 29-35

5. Escoubas et al., Toxicon 33 (1995), 1549-1555

6. Kuhn-Nentwig et al., Arch. Insect. Biochem. Physiol. 44 (2000), 101-111

7. Current Protocols in Protein Science (1995 - Annual Updates), J. Wiley and Sons

8. Maniatis et al., Molecular Cloning (1989), CSH Press

9. Current Protocols in Molecular Biology (1994 - Annual Updates), J. Wiley and Sons

10. Chang, J.~Y. and Knecht, R. (1991) Anal. Biochem. 197, 52-58

11. Bindlingmeyer, B. A„ Cohen, S. A., and Tarvin, T. L. (1984) J. Chromatogr. 336, 93-104

12. Chen, Y.-H., Yang, J. T., and Martinez, H. M. (1972) Biochemistry 11 , 4120-4131

13. Eisenberg, D. (1984) Ann. Rev. Biochem. 53, 595-623

14. Yan, L. Z. and Adams, M. E. (1998) J. Biol. Chem. 273, 2059-2066

15. Dathe, M. and Wieprecht, T. (1999) Biochim. Biophys. Acta 1462, 71-87

16. Zasloff, M. (1987) Proc. Natl. Acad. Sci. USA 84, 5449-5453

17. Lehrman, S. R„ Tuls, J. L., and Lund, M. (1990) Biochemistry 29, 5590-5596

18. Dathe, M. et al., (1997) FEBS Lett. 403, 208-212

Claims

1. A peptide comprising an amino acid sequence selected from any of SEQ ID Nos. 1 to 4 and variants thereof wherein the variant exhibits

(i) an antimicrobial activity of at least 50 % of the peptide; (ii) a hemolytic activity of at least 50 % of the peptide; and/or (iii) an insecticidal activity of at least 50 % of the peptide.

2. The peptide according to claim 1 , wherein the variant exhibits (i) an antimicrobial activity of at least 80 % of the peptide;

(ii) a hemolytic activity of at least 80 % of the peptide; and/or (iii) an insecticidal activity of at least 80 % of the peptide.

3. The peptide according to claim 1 , wherein the variant exhibits (i) an antimicrobial activity of at least 90 % of the peptide;

(ii) a hemolytic activity of at least 90 % of the peptide; and/or (iii) an insecticidal activity of at least 90 % of the peptide.

4. The peptide according to claim 1 , wherein said variant is at least 60 %, preferably 80 %, and more preferably 90 % homologous with the peptide.

5. The peptide according to any of claims 1 to 4, wherein said variant differs from the peptide by the addition, substitution, deletion, insertion and/or inversion of at least one amino acid.

6. The peptide according to claim 5, wherein the substitution is a conservative substitution.

7. The peptide according to any of claims 1 to 6, wherein the peptide carries a modification at the N-Terminus, C-terminus and/or at a side chain of an amino acid.

8. The peptide according to claim 7, wherein the modification is selected from an acetylation, an amidation or an esterification.

9. A peptide comprising at least one amino acid segment of the formula X_!X₂X₃X , wherein

X₂ may be any amino acid;

X₃ may be selected from Leu, Val, Ala or Gly; and

X may be selected from Ala or Val.

10. The peptide of claim 9, wherein the peptide comprises at least four, preferably at least six segments.

11. The peptide of claim 10, wherein the peptide exhibits

(i) an antimicrobial activity of at least 50 % of any of the peptides having SEQ ID Nos. 1 to 4;

(ii) a hemolytic activity of at least 50 % of any of the peptides having SEQ ID Nos. 1 to 4; and/or

(iii) an insecticidal activity of at least 50 % of any of the peptides having SEQ ID Nos. 1 to 4.

12. The peptide of claim 10, wherein the peptide exhibits

(i) an antimicrobial activity of at least 80 % of any of the peptides having SEQ ID

) Nos. 1 to 4;

(ii) a hemolytic activity of at least 80 % of any of the peptides having SEQ ID Nos. 1 to 4; and/or

(iii) an insecticidal activity of at least 80 % of any of the peptides having SEQ ID Nos. 1 to 4.

13. The peptide of claim 10, wherein the peptide exhibits

(i) an antimicrobial activity of at least 90 % of any of the peptides having SEQ ID Nos. 1 to 4;

(ii) a hemolytic activity of at least 90 % of any of the peptides having SEQ ID Nos. 1 to 4; and/or (iii) an insecticidal activity of at least 90 % of any of the peptides having SEQ ID Nos. 1 to 4.

14. A construct comprising at least one peptide according to any of claims 1 to 13 and at least one tag.

15. A fusion protein comprising at least one peptide according to any of claims 1 to 13 and at least one biologically active polypeptide or active fragment thereof.

16. A nucleic acid molecule comprising a nucleotide sequence encoding a peptide according to any of claim 1 to 13.

17. A vector comprising the nucleic acid molecule according to claim 16 and optionally a promoter wherein the promoter is operatively linked to the nucleic acid molecule such that the nucleic acid molecule may be expressed under the control of the promoter.

18. A host cell comprising the nucleic acid molecule according to claim 16 and/or the vector sequence according to claim 17.

19. A method for the recombinant production of the peptide according to any of claims 1 to 13 comprising:

) (i) transfecting a host cell with a vector according to claim 17;

(ii) culturing the transfectant under conditions allowing the expression of the peptide; and

(iii) optionally isolating the peptide from the transfectant or the medium.

20. A method for purifying a peptide having antimicrobial activity and having an amino acid sequence of any of SEQ ID Nos. 1 to 4 from spider venom comprising:

(a) separating a sample from venom of the spider Cupiennius salei by gel filtration chromatography;

(b) recovering the active fractions therefrom; (c) contacting the active fractions of step (b) with a cation exchange chromatography material;

(d) eluting the active fractions at about 30 % to about 90 % 2 M NaCI therefrom;

(e) contacting the active fractions of step (d) with a reversed phase chromatography material;

(f) eluting the active fractions at about 30% to about 40 % acetonitrile therefrom; and

(g) optionally repeating steps (e) and (f) to obtain further purification of the active fractions.

21. An antibody capable of specifically binding to the peptide according to any of claims 1 to 13.

22. The antibody according to claim 21 , wherein said antibody is monoclonal.

23. The antibody according to claim 21 , wherein said antibody is polyclonal.

24. A pharmaceutical composition comprising at least one peptide according to any of claims 1 to 13 and a pharmacologically acceptable carrier.

25. The pharmaceutical composition according to claim 24 additionally comprising an antibiotic agent and/or a cytotoxic compound.

26. A pharmaceutical composition comprising at least one nucleic acid molecule according to claim 14 and a pharmacologically acceptable carrier.

27. Use of at least one peptide according to any of claims 1 to 13 for the preparation of a medicament for the treatment of diseases mediated by negatively charged cells.

28. Use of at least one peptide according to claims 1 to 13 for the preparation of a medicament for the treatment of bacterial infections.

29. Use of at least one peptide according to claims 1 to 13 for the preparation of a medicament for the treatment of tumors, preferably leukemias.

30. A method for inactivating bacteria comprising the step of contacfing the object to be inactivated with at least one peptide according to any of claims 1 to 13.

31. A method for inactivating insects comprising the step of contacting the object to be inactivated with at least one peptide according to any of claims 1 to 13.