MXPA00012070A - Crosslink-stabilized indolicidin analogs - Google Patents

Abstract

The present invention relates to crosslink-stabilized analogs of indolicidin, which is a naturally occurring peptide having the amino acid sequence Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 ("Indol 1-13";SEQ ID NO:1). The cross-linked indolicidin ("X-indolicidin") analogs of the invention include, for example, analogs such as Indol 1-13(W6,9), which has the structure Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO:3), and Indol 1-13/6,9C(C6,9), which has the structure Ile-Leu-Pro-Trp-Lys-Cys-Pro-Trp-Cys-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO:4), where a crosslink formed between the first and last underlined amino acid residues. In addition, the invention provides nucleic acid molecules encoding the X-indolicidin analogs of the invention, particularly precursors of such analogs. The invention also relates to methods of using an X-indolicidin analog to reduce or inhibit microbial growth or survival by contacting an environment capable of sustaining microbial growth with the X-indolicidin analog.

Description

r ~ INDOLICIDINE ANALOGS STABILIZED BY RETICULATION DESCRIPTION OF THE INVENTION This invention was made with the support of the government under grant number AI22931 granted by the National Institutes of Health. The government has certain rights in the invention. The present invention relates generally to antimicrobial agents and, more specifically, to analogs stabilized by crosslinking of indolicidin and methods for using the analogs to reduce or inhibit microbial growth or survival. Infections by microorganisms, which include bacteria, viruses and fungi, are a major cause of human morbidity and mortality. Although anyone can be a victim of such an infection, the sick person and the elderly are particularly susceptible. For example, hospitalized patients often acquire secondary infections due to a combination of their weakened condition and the prevalence of microorganisms in a hospital. Such opportunistic infections result in increased patient suffering, increased length of hospitalization and, consequently, increased costs to the patient and the health care system. Similarly, the elderly, particularly those who live in care homes or retirement communities are susceptible to infections due to their narrow disposition of life and the impaired sensitivity of their immune systems. Numerous drugs are available to treat infections by certain microorganisms. In particular, various bacterial infections have been responsible for antibiotic treatment. However, the prolonged use of antibiotics since their discovery has resulted in the selection of bacteria that are relatively resistant to these drugs. In addition, few drugs, if any, are effective against microorganisms such as viruses. As a result, continuous efforts are made to identify new and effective agents to treat infections by a variety of microorganisms. The identification of naturally occurring compounds that act as antimicrobial agents has provided novel and effective drugs. Many organisms protect themselves by producing natural products that are toxic to other organisms. Frogs, for example, produce a class of peptides, magainins, that are highly toxic if ingested, thus providing a defense mechanism for frogs against potential predators. The magainins have been purified and shown to have antimicrobial activity, thus providing a useful natural product for reducing or inhibiting microbial infections.

Natural products useful as antimicrobial agents have also been purified from mammalian organisms, including humans. For example, defensins are a class of peptides that have been purified from mammalian neutrophils and shown to have antimicrobial activity. Similarly, indolicidin is a peptide that has been isolated from bovine neutrophils and has antimicrobial activity, which includes activity against viruses, bacteria, fungi and protozoan parasites. Thus, naturally occurring compounds provide a source of drugs that are potentially useful in treating microbial infections. By identifying naturally occurring peptides useful as antimicrobial agents, efforts have begun to chemically modify the peptides to obtain analogs having improved properties. Such efforts have resulted, for example, in the identification of indolicidin analogs which, when administered to an individual, have increased selectivity against infectious microorganisms when compared to the individual's own cells. Thus, the availability of naturally occurring antimicrobial agents has provided new drugs for the treatment of microbial infections and has provided an initial material for identifying analogs of the naturally occurring molecule that has desirable characteristics. Although such natural products and their analogs have provided new agents to treat microbial infectionsIt is well known that microorganisms can become resistant to drugs. Thus, there is a need to identify agents that effectively reduce or inhibit the growth or survival of microorganisms. The present invention satisfies this need and provides additional advantages. The present invention relates to analogues stabilized by cross-linking of indolicidin, which is a naturally occurring peptide having the amino acid sequence Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg -Arg-CONH2 ("Indol 1-13"SEQ ID NO: 1) As described herein, the analogues stabilized by crosslinking of indolicidin (" X-indolicidin analogs ") of the invention are characterized, in part, by having an intrapeptide crosslinked formed , for example, between two residues of Trp, to form a cross-linked di-tryptophan.An analog of X-indolicidin has the structure: X1-X2-X3-X4-X5-X6-P-X6-X6-P-X6- X7-X7-X8, (SEQ ID NO: 2), where XI is Lie, Leu, Val, Ala, Gly or absent, X2 is Lie, Leu, Val, Ala, Gly or absent; X3 is Pro or absent; X4 is Trp, Phe, Cys, Glu, Asp, Lys, AlaL or absent; X5 is Arg, Lys or absent; X6 is Trp, Phe, Cys, Glu, Asp, Lys or AlaL; X7 is Arg, Lys or absent, and X8 is homoserine, Met, Met-X9-Met or absent, wherein X9 is at least one amino acid, with the proviso that the analog contains at least two amino acid residues that are capable of forming a lattice; with the additional condition that if X2 is absent, XI is absent, if X3 is absent; absent, XI and X2 are absent; if X4 is absent, XI, X2 and X3 are absent; and if X5 is absent, XI, X2, X3 and X4 are absent. The X-indolicidin analogues of the invention are exemplified by the peptide Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 3) , wherein the underscore indicates a di-tryptophan reticulate formed between the first and last underlined Trp residues; and by the peptide Ile-Leu-Pro-Trp-Lys-Cys-Pro-Trp-Cys-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 4), wherein underlining indicates a cross-linking of Disulfide formed between the first and last residues of Cys underlined. The X-indolicidin analogs have broad spectrum antimicrobial activity. The invention also provides fusion polypeptides comprising an X-indolicidin analog and a peptide of interest, which may be useful, for example, to facilitate the purification of an expressed indolicidin analog. In addition, the invention provides nucleic acid molecules encoding X-indolicidin analogs of the invention, for example, cross-linked disulfide analogs, as well as precursors of such analogs and fusion polypeptides comprising such analogues. The invention also relates to methods for using an X-indolicidin analog to reduce or inhibit microbial growth or survival in an environment capable of maintaining microbial growth or survival upon contacting the environment with the X-analogue. indolicidin As such, the invention provides methods for reducing or inhibiting microbial growth or survival on a solid surface, for example, surgical instruments, hospital surfaces, and the like. In addition, the methods of the invention are useful for reducing or inhibiting microbial growth or survival in an individual, particularly a mammal such as a human. Thus, the invention provides methods for treating an individual suffering from a pathology caused, at least in part, by microbial infection, by administering an X-indolicidin analogue to the individual, thereby reducing the severity of the pathological condition. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the nucleotide sequence encoding poly- (Indole (1-13) -Met-Ala-Arg-Ile-Ala-Met) 3, which encodes three copies of Indole 1-13, each separated by Met-Ala-Arg-Ile-Ala-Met (SEQ ID NO: 11). The coding strand (sense) is shown in uppercase letters, the antisense strand is shown in lowercase letters, and the encoded amino acid sequence is shown using the three letter code ("Stp" indicates STOP codon). The nucleotide and amino acid sequences correspond to SECs. FROM IDENT. NOS: 12 and 13, respectively. The endonuclease restriction sites Eco Rl and Sal I are indicated. The enterokinase recognition site is simply highlighted, indicating with the double arrow the cleavage site. Simple arrows denote cyanogen bromide cleavage sites. The dotted underlined tetranucleotide sequences correspond to overlays in the oligonucleotides used for ligatures. The doubly underlined sequences denote sebators used for PCR amplification (see example I.C.). Figure 2 shows the dose-dependent microbiostatic activity of indolicidin (Indole 1-13; SEC. FROM IDENT. NO: 1) (closed circle) e X-indolicidin (Indole 1-13 (6, 9); SEQ ID NO: 3) (inverted triangles) in the growth of Escherichia coli ML35. Figure 3 shows the dose-dependent microbiostatic activity of indolicidin (Indole 1-13; ID SEQ ID NO: 1) (closed circles) and X-indolicidin (Indole 1-13 (W6, 9); NO: 3) (inverted triangles) in the growth of Cryptococcus neoformans 27 ÍA. Figure 4 shows the dose-dependent microbiostatic activity of indolicidin (Indole 1-13; ID SEQ ID NO: 1) (closed circles) and X-indolicidin (Indole 1-13 (W6, 9); NO: 3) (inverted triangles) in the growth of Staphylococcus aureus 207A. Figure 5 shows the dose-dependent microbiostatic activity of indolicidin (Indole 1-13, ID SEQ ID NO: 1) (closed circles) and X-indolicidin (Indole 1-13 (W6, 9); NO: 3) (inverted triangles) in the growth of Candida albicans 16820. Figure 6 shows the dose-dependent microbicidal activity of indolicidin (Indole 1-13; ID SEQ ID NO: 1) (closed circles) and X -indolicidin (Indole 1-13 (6, 9); SEQ ID NO: 3) (inverted triangles) in the growth of E. coli ML35. Figure 7 shows the dose-dependent microbicidal activity of indolicidin (Indole 1-13, SEQ ID NO: 1) (closed circles) and X-indolicidin (Indole 1-13 (W6, 9); NO: 3) (inverted triangles) in the growth of C. neoformans 271A. Figure 8 shows the dose-dependent microbicidal activity of indolicidin (Indole 1-13, ID SEQ ID NO: 1) (closed circles) and X-indolicidin (Indole 1-13 (6, 9); NO: 3) (inverted triangles) in the growth of S. aureus 207A. Figure 9 shows the dose-dependent microbicidal activity of indolicidin (Indole 1-13, SEQ ID NO: 1) (closed circles) and X-indolicidin (Indole 1-13 (W6, 9); NO: 3) (inverted triangles) in the growth of C. albicans ML35. The invention provides analogues stabilized by crosslinking indolicidin ("analogs of X-indolicidin"), which are peptides that are characterized, in part, by having an intrapeptide crosslinking. As described herein, an X-indolicidin analog has the general structure: X1-X2-X3-X4-X5-X6-P-X6-X6-P-X6-X7-X7-X8, (SEQ. IDENT NO: 2), where XI is lie, Leu, Val, Ala, Gly or absent; X2 is Lie, Leu, Val, Ala, Gly or absent; X3 is Pro or absent; X4 is Trp, Phe, Cys, Glu, Asp, Lys, AlaL or absent; X5 is Arg, Lys or absent; X6 is Trp, Phe, Cys, Glu, Asp, Lys or AlaL; X7 is Arg, Lys or absent; and X8 is homoserine, Met, Met-X9-Met or absent, wherein X9 is at least one amino acid. In addition, an X-indolicidin analog contains at least two amino acid residues that are capable of forming a cross-linking between its side chains, eg, two Trp residues, which can form a cross-linking of di-tryptophan; or at least two Cys residues, which can form a disulfide crosslinking; or a lanthionine residue, which can form a monosulfide crosslinking. A cross-linking of monosulfide between two AlaL residues forms a lanthionine residue. In addition, if, in an X-indolicidin analogue, X2 is absent, XI is absent; if X3 is absent, XI and X2 are absent; if X4 is absent, XI, X2 and X3 are absent; and if X5 is absent, XI, X2, X3 and X4 are absent. A crosslinking in an X-indolicidin analog can be, for example, between X4, when present, and a residue of X6, or they can be between two X6 residues. In addition, an X-indolicidin analog may have more than one crosslinking. As used herein, the term "crosslinking" means a covalent bond formed between the reactive groups of two amino acids in a peptide. As such, a crosslinking present in an X-indolicidin analog is stable under physiological conditions. A crosslinking in an X-indolicidin analog can be formed, for example, between two Trp residues, which form a di-tryptophan crosslinking; or between two Cys residues, which form a disulfide bond. In addition, a crosslinking may be a monosulfide bond formed by a lanthionine residue. A crosslinking can also be formed between other secondary chains of amino acids, for example, a lactam crosslink formed by a transamidation reaction between the secondary chains of acidic amino acid and a basic amino acid, such as between the α-carboxyl group of Glu (or the group β-carboxyl of Asp), and the e-amino group of Lys; or it can be a lactone produced, for example, by a crosslinking between the hydroxy group of Ser and the? -carboxyl group of Glu (or the β-carboxyl group of Asp), or a covalent bond formed, for example, between two amino acids , one or both of which have a modified secondary chain. Indolicidin is a naturally occurring polypeptide, having the amino acid sequence Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 ("Indol 1-13;" ID SECTION NO: 1). Indolicidin (SEQ ID NO: 1) was named based on its tryptophan-rich nature and microbicidal properties (see US Patent No. 5,324,716, issued June 28, 1994, which is incorporated herein) reference). The indolicidin analogs having the general structure H2N-ILPWKWPWWPWX (SEQ ID NO: 9), wherein X designates one or two independently selected amino acids, have been described (see US Patent No. 5,534,939, issued August 20). of 1996). Such indolicidin analogues, such as indolicidin (SEQ ID NO: 1), are peptides rich in tryptophan and are characterized, in part, by having improved selectivity when compared to indolicidin (SEQ ID NO: 1) . Additional analogs of indolicidin have also been described (International Publication No. WO 97/08199, published March 6, 1997). These previously described indolicidin analogs are distinguishable from those of the present invention in that the previously described analogs do not contain an intrapeptide crosslinking. The X-indolicidin analogs of the invention are exemplified by the peptide Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 3); "Indole 1-13 (W6 / 9)"), wherein underlining indicates a cross-linking of di-tryptophan formed between the first and last underlined Trp residues (also indicated by "(W6 / 9)"); and by the peptide Ile-Leu-Pro-Trp-Lys-Cys-Pro-Trp-Cys-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 4; "Indol 1-13 / 6, 9C (C6 / 9) "), wherein underlining indicates a disulfide crosslinking formed between the first and last underlined Cys residues. As described herein, such X-indolicidin analogs may be relatively more stable to enzymatic degradation than native indolicidin (Example I), and have antimicrobial activity equivalent to indolicidin (Example II). An X-indolicidin analog of the invention may be based on a full-length indolicidin peptide, for example, Indole 1-13 (SEQ ID NO: 1), or a truncated amino terminal indolicidin analog such as Indole 2 -13 (SEQ ID NO: 5) or Indole 3-13 (SEQ ID NO: 6) or truncated carboxy terminal indolicidin analog such as Indole 1-12 (SEQ ID NO: 7; Table 1), non-crosslinked forms of which exhibit antimicrobial activity. An X-indolicidin analog may also be a cross-linked indolicidin analog in which one or more Trp residues are replaced by a Phe residue, since such indolicidin analogues have antimicrobial activity.

TABLE I INDOLICIDIN AND INDOLICIDIN ANALOGS NAME AMINO ACID SEQUENCE SEC. FROM IDENT. DO NOT: Indole 1-13 'H2N-ILPWKWPWWPWRR-C0 H2 1 Indole 1-13 (W6 / 9) "H ^ NILPWKWPWWPWRR-CONH? 3 Indole 1-13 / 6, 9C (C6 / 9) H; NILPW- -CPWCPWRR-CONH7 4 Indole 2-13 H2N-LPWKWPWWPWRR-CONH2 5 Indole 3-13 H2N-PWKWPWWP- -RR-CONH2 6 Indole 1-12 H2N-I- -PW- -WPWWP- -R-CONH2 7 Indole 2-13 / 4F H2N -LPPKWPW- -P- -RR-CONH2 8 Indole 2-13 / 4F (W6 / 9) H ^ NhPFKWPWWPWRR-CO H, 10 - ndolicidin (which occurs naturally). '- the underscore in the sequence indicates reticle For example, a peptide Indol 2-13 (SEQ ID NO: 5), in which the Trp in position 4 is replaced by Phe (Indol 2-13 / 4F; SEQ ID NO: 8; see Table 1) can be crosslinked between the Trp residues in positions 6 and 9 to produce 2-1 / 4F indole (W6 / 9) (SEQ ID NO: 10, see Table 1). The reference to an amino acid position in an X-indolicidin analogue is made herein with respect to the position of the amino acid in the naturally occurring indolicidin (SEQ ID NO: 1). As such, the positions refer to positions 1 to 13, starting with the residue in SEC. FROM IDENT. NO: l (position 1) and ending with carboxy terminal arginine (position 13). As a result, although Leu is the first amino acid in Indole 2-13 (SEQ ID NO: 5), this Leu residue is referred to as being located in position 2 because this is the location of the corresponding Leu in the SEC. FROM IDENT. NO: 1. Followed by the SEC. FROM IDENT. NO: 5 is referred to as Indole 2-13 because it starts with an amino acid corresponding to the second amino acid (Leu) of Indole 1-13 (SEQ ID NO: 1, see Table 1). An analog of X-indolicidin or precursor thereof containing a substitution of a naturally occurring indolicidin residue (SEQ ID NO: 1) with a different amino acid is referred to using the position number and the amino acid code of a letter. For example, replacement of the Trp residue at position 4 with indolicidin (Indol 1-13) with a Cys residue results in an indolicidin analog designated Indol 1-13 / 4C. Similarly, Indol 2-13 / 6F indicates an indolicidin analog lacking an amino terminal amino acid compared to the naturally occurring indolicidin (SEQ ID NO: 1) and contains a substitution of Phe for Trp in the 6-position. Where more than one substitution is made, the positions are separated by a comma; thus, Indol 2-13 / 6, 11F indicates an indolicidin analog that lacks an amino terminal amino acid compared to the naturally occurring indolicidin (SEQ ID NO: 1) and contains Phe by Trp substitutions at positions 6 and 11. The position of a cross-linking in an X-indolicidin analog is indicated in parentheses by a designation of amino acids involved in the cross-linking and amino acid positions. For example, Indole 1-13 (W6 / 9) indicates a peptide having the naturally occurring amino acid sequence of indolicidin and containing a di-tryptophan crosslink between the Trp residues at positions 6 and 9 (see Table 1; ID SECTION NO: 3). Indole 1-13 / 6, 9C (C6 / 9) indicates an indolicidin analog containing Cys by substitutions of Trp at positions 6 and 9 and, additionally, contains a disulfide crosslink between these Cys residues (see SEQ ID. NO: 4, Table 1). For an X-indolicidin analog having a cross-linking formed between amino acid residues that do not have a standard one-letter code, for example, a monosulfide cross-linking formed by a lanthionine residue, the nomenclature can be modified for clarity, such as Indole 1-13 / 6, 9-lanthionine (Lan6, 9) or the like. An X-indolicidin analogue of the invention is based on the general native structure of naturally occurring indolicidin, (SEQ ID NO: 1), except that various deletions of defined amino acids, substitutions or additions are made with respect to indolicidin (see SEQ ID NO: 2). As used herein, the term "amino acid" is used in its broadest sense to mean naturally occurring amino acids as well as non-naturally occurring amino acids, including amino acid analogs. Thus, reference herein to an amino acid includes, for example (L) -naturally occurring proteinogenic amino acids, as well as (D) -amino acids, chemically modified amino acids such as amino acid analogs, naturally occurring non-proteogenic amino acids such as norleucine , lanthionine or the like, and chemically synthesized compounds having properties known in the art to be characteristic of an amino acid. As used herein, the term "proteogenic" indicates that the amino acid can be incorporated into a protein in a cell through a metabolic pathway. The amino acid residue at any position in an indolicidin analog having the structure shown as SEQ. FROM IDENT. NO: 2 can be selected independently. As used herein, the term "independently selected" indicates that the selection of an amino acid residue at any position on an indolicidin analog does not depend on or influences the selection of the amino acid residue at any other position on the analogue. Thus, the selection of a residue Trp for X6 shown in position 6 of SEC. FROM IDENT. NO: 2 does not influence if, for example, the amino acid present in the X6 shown at position 8 is a Trp residue or a Phe residue, with the proviso that at least two amino acids forming a crosslinking, for example, two Trp residues or two Cys residues, are present in the analogue. As described herein, the substitution of Cys residues at positions 6 and 9 of Indole 1-13 (SEQ ID NO: 1), and formation of a disulfide crosslink between the two Cys residues, produces Indole 1 -13 (6, 9C) C6 / 9 (SEQ ID NO: 4, which may have antimicrobial activity equivalent to Indole 1-13 (see Example II) Substitution of various Trp residues in Indole 1-13 ( SEQ ID NO: 1) with Phe residues may also result in indolicidin analogues having antimicrobial activity Thus, the skilled artisan would recognize that various amino acid substitutions can be made in Indole 1-13 (SEQ ID NO: 1) to produce an X-indolicidin analog having antimicrobial activity.Also, the amino terminal interruption of indolicidin occurring naturally results in the production of indolicidin analogues having antimicrobial activity.In addition, the indolicidin analogues lacking a Arg waste c arboxi terminal, or containing a Lys substitution for one or both of the carboxy terminal Arg residues, or lacking the carboxy terminal Arg-13 residue and having a Lys substitution for Arg-12 of Indole 1-13 have antimicrobial activity (see patent No. 5, 547, 939, supra, 1996). Accordingly, the skilled artisan will recognize that deletions can be made in Indole 1-13 (SEQ ID NO: 1) to produce truncated X-indolicidin analogs having antimicrobial activity. Furthermore, in view of the permissibility of deletions at the amino or carboxy terminus of Indole 1-13, the skilled artisan will recognize that various amino acid substitutions can be made at the suppressible positions without destroying the antimicrobial activity of an X-analog derivative. indolicidin A) Yes, since Indole 1-13 (SEQ ID NO: 1) contains a residue at position 1, the skilled artisan knowing that this lie can be suppressed without destroying the antimicrobial activity, would recognize that the lie can also be substituted conservatively with an amino acid such as Leu, Val, Ala or Gly without destroying the antimicrobial activity of an X-indolicidin analog produced therefrom. Similarly, conservative amino acid substitutions are permissible for Leu at position 2. In addition, substitution of an Arg residue for Lys at position 5 is allowed, since the presence of a positively charged amino acid at position 5 correlates with the antimicrobial activity. A precursor peptide of an X-indolicidin analog of the invention can be expressed from a nucleic acid molecule encoding in vi tro or in vivo in a host cell or can be chemically synthesized. With respect to the expression of the analogs, the nucleic acid sequences encoding the various indolicidin analogs of the invention can be prepared based, for example, on the description of the nucleic acid sequence for indolicidin (Del Sal et al., Biochem. Biophys, Res. Comm. 187: 467-472 (1992), which is incorporated herein by reference) and in the knowledge of the codon technique for amino acids comprising the various indolicidin analogues described. Such nucleic acids encoding the X-indolicidin analogs can be cloned into an appropriate vector, particularly an expression vector, and the encoded analogue can be expressed using an in vitro transcription / translation reaction. In addition, such nucleic acid sequences can be used to construct a synthetic gene encoding a polypeptide (X-indolicidin analog), which can be cloned into an expression vector and expressed in vivo in a bacterium, insect host cells. or mammal (see Example IC). It should be recognized that, while reference is made to a nucleic acid encoding an X-indolicidin analog, the nucleic acid encodes a linear precursor peptide that can be cross-linked, for example, in an in vi tro reaction, to obtain a Analog of X-indolicidin. An advantage of expressing a polypeptide poly- (analogue X-indolicidin) in vivo is that large amounts can be prepared using, for example, commercial fermentation methods, since the polypeptide form of the analogs does not appear to have substantial antimicrobial activity, then the The polypeptide can be split to produce active X-indolicidin analogues or precursors thereof. An X-indolicidin analog can also be chemically synthesized using well-known methods (see, for example, van Abel et al., Internatl, J. Pept. Prot. Res. 45: 401-409 (1995), which is incorporated herein by reference; see, also, Example I. A.). An analogue of X-indolicidin was obtained during the acidolytic cleavage and deprotection of Indole 1-13 assembled with Fmoc (SEQ ID NO: 1). During such preparation, a strongly absorbent material of A-320 was detected, but is not present in Indole 1-13 prepared from natural sources. The purification and characterization of the absorbent material A-320 revealed that it was 2 units of atomic mass smaller in mass than the naturally occurring indole 1-13 (SEQ ID NO: 1). The absorption material A-320 was determined to be Indole 1-13 (W6, 9) (SEQ ID NO: 3, see Example IB), which contains a di-tryptophan crosslink between the Trp residues in the positions 6 and 9. When tested for antimicrobial activity, the Indole 1-13 (W6, 9) (SEQ ID NO: 3) demonstrated broad spectrum antimicrobial activity equivalent to native Indole 1-13 (SEQ ID NO: 1, see Example II), but was substantially more stable than native Indole 1-13 (SEQ ID NO: 1) to chymotrypsin digestion (Example IB). The role of the crosslinks in the indolicidin analogues was further examined when preparing Indole 1-13 / 6, 9C (C6, 9) (SEQ ID NO: 4). The disulfide crosslinking in this X-indolicidin analog can stabilize the peptide similarly to Indole 1-13 (W6, 9) (SEQ ID NO.

NO: 3). An advantage of using chemical synthesis to prepare an X-indolicidin analog is that the (D) -amino acids can be substituted by (L) -amino acids, if desired. The incorporation of one or more (D) -amino acids within an X-indolicidin analog may confer desirable characteristics on the peptide, for example, increased stability in vi tro or, particularly, in vivo, since the endogenous proteases are generally ineffective against peptides comprising (D) -amino acids. Naturally occurring antimicrobial peptides that have been chemically synthesized to contain (D) -amino acids maintain their antimicrobial activity (Wade et al., Proc. Nati. Acad. Sci., USA 87: 4761-4765 (1990), which is incorporated in the present for reference The X-indolicidin analogs were synthesized using an automated peptide synthesizer such as an Eppendorf Synostat (Madison Wl) or a Milligen 9050 (Milford MA, although manual methods of peptide synthesis in solution can also be used, then the crosslinking was formed as desired (Example IA) The linear precursors of the X-indolicidin analogs were synthesized in a polyethylene glycol-polystyrene graft resin (PEG-PS) using amino acid derivatives Na-Fmoc. suitable linker such as a valeric peptide PAL acid amide linker (5- (4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy); Fmoc is 9-fluorenylmethyloxycarbonyl; Milligen) or varal acid XAL (5- ( 9-Fmoc-aminoxanten-2-oxy)) was used to produce final carboxamide groups. However, the skilled artisan will recognize that other resins, amino acid derivatives and methods for modifying the amino acid reactive groups or the amino terminus, for example, by acetylation, or the carboxy terminus can be used to obtain a desired indolicidin analog (see, for example, Protein Engineering: A Practical oach (IRL Press 1992), Bodanszky, Principies of Peptide Synthesis (Springer-Verlag 1984), each of which is incorporated herein by reference). The synthesized X-indolicidin analogs were purified by reverse phase HPLC and characterized by mass spectroscopy, absorption spectroscopy, acid-urea gel electrophoresis and analytical HPLC (see Example I) or can be purified and characterized using other routine methods of purification and analysis of peptides. The selective modification of a reactive group, different from the production crosslinking, can impart desirable characteristics to an indolicidin analog. The selection to include such modification is determined, in part, by the required characteristics of the peptide. Such modifications may result, for example, in X-indolicidin analogues having a greater antimicrobial selectivity or potency than the naturally occurring indolicidin. As used herein, the term "antimicrobial selectivity" refers to the relative amount of antimicrobial activity of an X-indolicidin analog against a microorganism when compared to its activity against the environment to which it is administered, particularly its activity against normal cells in a treated individual. For example, an X-indolicidin analog that is characterized as having an antimicrobial activity that is equivalent to native indolicidin, but that has decreased emolitic activity when compared to native indolicidin, is considered to have higher antimicrobial selectivity than native indolicidin. . The indolicidin analogues having greater antimicrobial selectivity than the naturally occurring indolicidin have been described. For example, indolicidin analogues truncated at the carboxy terminus or having one or more lysine substitutions for the carboxy terminal arginines in indolicidin that occur naturally have antimicrobial activity similar to indolicidin, but have decreased haemolytic activity (US Patent No. 5,547,939, supra, 1996). Also, indolicidin analogs in which all Trp residues were substituted with Phe but not analogs having Ala by Pro substitutions, had higher antimicrobial selectivity than native indolicidin (Subbalakshmi et al., FEBS Lett, 395: 48- 52 (1996), which is incorporated herein by reference). Indolicidin analogs containing various other amino acid substitutions or modifications, for example, carboxy terminal carboxymethylation, also have desirable properties (Fall and Hancock, Antimicr Agents Chemother, 41: 771-775 (1997), which is incorporated in the present for reference; see, also, WO 97/08199, supra, 1997). None of the indolicidin analogs described previously, however, contains intrachain chain cross-linking. The antimicrobial selectivity of an X-indolicidin analog can be determined using the methods described herein (see Example II) or using routine methods such as those described in the aforementioned references. As described herein, an indolicidin-X analog, Indole 1-13 (W6, 9) (SEQ ID NO: 3), demonstrating broad spectrum antimicrobial activity similar to that of native indolicidin (Indole 1-13; SEC DE IDEN NO: 1, Example II). As used herein, the term "broad spectrum," when used with reference to the antimicrobial activity of an X-indolicidin analog, refers to the ability of the analog to reduce or inhibit the survival or proliferative capacity of various microorganisms. prokaryotic and eukaryotic. For example, the indolicidin analogs of the invention may show antimicrobial activity against protozoa such as Giardia lambia, Chlamydia sp. and Acanthamoeba sp.; viruses, particularly enveloped viruses, such as HIV-I; yeasts and fungi such as Cryptococcus and Candida; various genera of gram negative and gram positive bacteria, including Escherichia, Salmonella and Staphylococcus; and helminths such as liver flukes. Antimicrobial activity can occur through microbicidal inhibition, which refers to the ability of an X-indolicidin analog to reduce or inhibit the survival of a microorganism by irreversibly killing or damaging it, or through microbiostatic inhibition, which refers to the ability of an X-indolicidin analog to reduce or inhibit the growth or proliferative capacity of an objective microorganism without necessarily terminating it. Indolicidin analogs containing a carboxy terminal homoserine residue ("Indol-Hse" analogs) maintain antimicrobial activity. The determination that an Indol-H analog maintains antimicrobial activity is significant because a Hse group remains in the carboxy terminus of a peptide followed by a cleavage of cyanogen bromide from the peptide in a Met residue. Since the native indolicidin does not contain an internal Met residue and since the indolicidin analogs lack internal Met residues can be produced, such analogs are not split when exposed to the cyanogen bromide. The Hse in the carboxy terminus of a peptide typically exists as a state of equilibrium between the lactone and the carboxylate forms. An an { alogo Indol-Hse can be admitted in the terminal carboxy. The described ability of an Indol-Hse analog to maintain antimicrobial activity provides a means to produce substantial amounts of the precursors of the X-indolicidin analog by expressing a poly- (Indol-Met) N polypeptide where "N" is the number of times that the Indol-Met sequence is repeated, and unfold the polypeptide with cyanogen bromide to produce "N" Indol-Hse analog peptides (see Example IC). The crosslinks can be formed in the precursor peptides to produce X-indolicidin analogues. A method for producing idolicidin-X precursors of the peptide can be performed in vivo in a host cell because the poly- (Indol-Met) N polypeptides do not show substantial antimicrobial activity. Such a method is performed, for example, by synthesizing a nucleic acid sequence that encodes the Indol portion of the analog and a carboxy terminal Met; ligand the nucleic acid sequences, such that the encoded peptides are maintained in the same reading structure, to produce a synthetic gene comprising a concatemer having "N" repeats of the sequence encoding Indol-Met; cloning the synthetic gene into an expression vector such that the encoded poly- (Indol-Met) N is expressed from the promoter in the vector; transform a host cell with the vector; expressing the encoded poly- (Indol-Met) N polypeptide; and unfolding the polypeptide with cyanogen bromide to produce "N" -Indol-Hse analogs. The crosslinks can be formed in the peptides to produce X-indolicidin analogues. Thus, the invention provides X-indolicidin analogs that contain a carboxy terminal homerin residue. The purification of the expressed poly- (Indol-Met) N polypeptide is facilitated by further linking the synthetic gene to a nucleic acid sequence encoding a peptide that is capable of being linked by a molecule. Such a peptide can be a ligand or a receptor, which can be specifically linked by an appropriate receptor or ligand, respectively; or a peptide that is specifically linked by an antibody. In addition, a peptide linked to a poly- (Indol-Met) N polypeptide can be any peptide of interest, for example, a peptide such as alkaline phosphatase or green fluorescent protein, which provides a means to detect the presence of the fusion polypeptide .

To facilitate the purification of a poly-indolicidin analog polypeptide, the linked peptide can be, for example, a maltose-binding protein, which binds maltose or an oligosaccharide containing maltose such as amylose; glutathione-S-transferase (GST), which binds glutathione; His-6, which is linked by a metal ion such as nickel ion or cobalt ion; the FLAG epitope which is linked by an anti-FLAG antibody, or any other peptide for which a specific antibody or other ligand or receptor is available. If desired, the molecule, for example glutathione, which binds the peptide (GST), can be attached to a solid support such as a chromatography matrix and a expressed poly- (Indol-Met) N-GST fusion polypeptide can be purified from contaminating proteins of the host cell by passage over the matrix. If desired, the fusion polypeptide can be eluted from the matrix and treated with cyanogen bromide; or the fusion polypeptide, while bound to the matrix can be exposed to cyanogen bromide, thereby releasing only the Indol-Hse analog precursors of the corresponding X-indolicidin analog. Thus, the invention provides fusion polypeptides comprising an X-indolicidin analog linked to a peptide of interest. As used herein, the term "precursor", when used with reference to X-indolicidin, means a linear peptide that can form an intrapeptide crosslinking to produce an X-indolicidin analog. The invention also provides nucleic acid molecules encoding the X-indolicidin analogs of the invention, specifically the linear peptide or the polypeptide precursors of the X-indolicidin analogues. The skilled artisan will know that the nucleotide sequence of the nucleic acid molecules of the invention can be determined based on the amino acid sequence of the X-indolicidin analogue and the knowledge of the codons encoding the various amino acids. Such codons can be selected using computer-assisted methods. One or other degenerate codon, for example, one of the six codons encoding Arg or one of the six codons encoding Leu or the like, may be selected as desired, for example, to avoid (or include) the insertion of a site of endonuclease restriction in the X-indolicidin analog encoding the sequence. The nucleic acid molecules of the invention are useful, for example, to produce X-indolicidin analogs in vi tro using an appropriate transcription / translation system or in vivo using an appropriate expression system, after which intra-chain reticulations can be formed. in the precursors to produce X-indolicidin analogues. The nucleic acid molecules of the invention may be polydeoxyribonucleotide (DNA) sequences or polyribonucleotide (RNA) sequences, as desired, may contain linkers, adapters or the like to facilitate cloning or concatemerization in the appropriate structure. An X-indolicidin analog having antimicrobial activity can be applied to an environment capable of maintaining the survival or growth of a microorganism or an environment at risk of supporting such survival or growth, thus providing a means to reduce or inhibit the growth or microbial survival. Accordingly, the invention relates to methods for using an X-indolicidin analog to reduce or inhibit microbial growth by contacting the environment capable of maintaining microbial growth or survival with the X-indolicidin analogue. As used herein, reference to "an environment capable of maintaining the survival or growth of a microorganism" means a gaseous, liquid or solid material, including a living organism, in or on which a microorganism can live or spread. In view of the wide range of environments that allow the survival or growth of almost as diverse microorganisms, for example, as viruses, bacteria and fungi, and also in view of the described effectiveness of the X-indolicidin analogues claimed against a broad spectrum of such microorganisms, the range of such environments that can be treated using a method of the invention is necessarily broad and includes, for example a tissue or bodily fluid of an organism such as a human; a liquid such as water or an aqueous solution, for example, contact lens solution; a food such as a crop, a food product or a food extract; an object such as the surface of an instrument used, for example, to prepare food or to perform surgery; and a gas such as that used to anesthetize in preparation for surgery. One method of the invention comprises administering to the environment an effective amount of an X-indolicidin analog such that the analogue can contact a microorganism with the environment, thereby reducing or inhibiting the organism's ability to grow or survive. . The X-indolicidin analogs can be used in a variety of methods to reduce or inhibit the survival or growth of microorganisms, which includes the microbicidal inhibition of the survival of a microorganism as well as the microbiostatic inhibition of growth. As such, an X-indolicidin analog may be used, for example, as a therapeutic agent, a food preservative, a disinfectant or a medicament. An X-indolicidin analog can be used as a therapeutic agent to treat a patient suffering from a bacterial, viral, fungal or other infection due to a microorganism susceptible to the antimicrobial activity of the analog. Thus, the invention provides methods for treating an individual suffering from a pathology caused, at least in part, by microbial infection, by administering an X-indolicidin analogue to the individual under conditions that allow the analog to contact the infectious microorganisms, reducing or inhibiting with that the survival or growth of the microorganisms and alleviating the severity of the infection. For use as a therapeutic agent, the analog of X-indolicidin can be formulated with a pharmaceutically acceptable carrier to produce a pharmaceutical composition, which can be administered to the individual, which can be a human or other mammal. A pharmaceutically acceptable carrier can be, for example, water, sodium phosphate buffer, sulfate-regulated saline, normal saline or Ringer's solution or other physiologically regulated salt solution, or another solvent or carrier such as a glycol, glycerol, an oil such as olive oil or an injectable organic ester. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds which act, for example, to stabilize or increase the absorption of the X-indolicidin analog. Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextrans; antioxidants such as ascorbic acid or glutathione; chelating agents such as EDTA, which disrupt the microbial membranes; divalent metal ions such as calcium or magnesium; low molecular weight proteins; or other stabilizers and excipients. One skilled in the art will know that the selection of a pharmaceutically acceptable carrier, which includes a physiologically acceptable compound, depends, for example, on the route of administration of the composition. A pharmaceutical composition containing an X-indolicidin analog can be administered to an individual by various routes, including intravenous, subcutaneous, intramuscular, intrathecal or intraperitoneal injection; orally, as an aerosol spray; or by intubation. If desired, the X-indolicidin analog may be incorporated into a liposome, a non-liposome lipid complex, or other polymer matrix, which may additionally be incorporated therein, for example a second drug useful in treating the individual. The use of indolicidin incorporated within liposomes, for example, has been shown to have antifungal activity in vivo (Ahmad et al., Biochim, Biophys, Acta 1237: 109-114 (1995), which is incorporated herein by reference). Liposomes, which consist of phospholipids or other lipids, are non-toxic, physiologically acceptable and metabolizable carriers that are relatively simple to manufacture and administer (Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton FL, 1984), which is incorporated herein by reference). The skilled artisan will select a particular route and method of administration based, for example, on the location of a microorganism in a subject, the particular characteristics of the microorganism, and the specific X-indolicidin analog that is administered. Food and food products can also be treated with X-indolicidin analogues for the purpose of preserving food or eliminating or preventing infection by microorganisms. For example, shellfish and poultry products routinely harbor enteric pathogenic microorganisms. The growth or survival of such microorganisms can be reduced or inhibited by contacting the product with an X-indolicidin analogue. Food crops such as fruits, vegetables and grains can be treated with an X-indolicidin analog to reduce or inhibit post-harvest damage caused by microorganisms, for example, by administering the analog locally using an aerosol form of the analogue. In addition, transgenic plants or animals useful in the food industry can be produced by introducing a nucleic acid molecule encoding an analogous precursor of X-indolicidin of the invention into the embryonic cell line of such organisms, particularly a precursor of an analog. of X-indolicidin containing disulfide crosslinks, since the disulfide crosslinks can be established spontaneously in cells in vivo. Methods for producing transgenic plants and animals are well known and routine in the art. An X-indolicidin analog can also be used as a disinfectant to reduce or inhibit the survival or growth of microorganisms on an object, in a solution. An X-indolicidin analog can be used to treat essentially any object or solution that can maintain microbial growth, wherein the survival or growth of the microorganism is undesirable. In particular, an object or solution that comes into contact with a mammal such as a human, for example, wet baby towels, diapers, relief bands, towels, makeup products and solutions for eye washing and contact lenses they can be treated with an X-indolicidin analogue. In such methods, the X-indolicidin analog may be applied locally to the object or it may be added to the solution or it may be in an aerosol form in a gas. To exhibit antimicrobial activity in an environment, an effective amount of an X-indolicidin analog is administered to the environment. As used herein, the term "effective amount" refers to the amount of an X-indolicidin analog that reduces or inhibits the survival or growth of a microorganism in an environment. In particular, an effective amount of an indolicidin analogue produces only minimal effects against the environment although the level of an acceptable detrimental effect is weighed against the benefit caused by the antimicrobial effect. An X-indolicidin analog can be administered to a subject such as a human systematically at a dose ranging from 1 to 100 mg / kg of body weight, for example, at a dose of about 10 to 80 mg / kg, in particular about 10 to 50 mg / kg, and the X-indolicidin analog can be incorporated into liposomes, if desired. In addition, an X-indolicidin analog can be administered locally or in the environment, which can be a human subject, or it can be placed in a solution, at a concentration of about 0.1 to 10 mg / ml, for example at a concentration of approximately 0.5 to 5 mg / ml. Although X-indolicidin analogs are generally effective in microgram amounts, an amount effective to administer to a particular environment will depend, in part, on the environment. For example, when administered to a mammal such as a human, an X-indolicidin analog, in addition to having antimicrobial activity, may have hemolytic activity as a side effect. The skilled artisan will recognize that the level of such effects ^^ Secondary can be considered when prescribing a treatment and should be conserved during the treatment period, and will adjust the amount of the analog that is administered accordingly. An effective amount will also vary, depending, for example, on the characteristics of the target microorganism, the degree of infection or previous growth and the specific X-indolicidin analogue administered: In addition, an effective amount depends on the form in which the Analog of X-indolicidin is administered. For example, the incorporation of native indolicidin into liposomes allows the administration of a higher amount than "free" indolicidin, without producing unacceptable side effects such that fungal infection in mice can be cured (Ahmad et al., Supra, 1995). The following examples are intended to illustrate but not limit the present invention. EXAMPLE I PREPARATION AND CHARACTERIZATION OF INDOLICIDIN ANALOGS This example provides methods for preparing and characterizing X-indolicidin analogues. A. Chemical Synthesis of Indole 1-13 25 Indole (1-13) was assembled on a Fmoc-PAL-PEG-PS resin at a 0.2-mm scale on a Millipore 9050 Plus continuous-flow peptide synthesizer. The resin was swelled for 30 minutes in DMF before starting the synthesis. Fmoc chemistry was completely used. The Fmoc splitting was done with a solution to the 2% DBU-2% piperidine / DMF for 1 to 5 minutes. The following protection groups were used: Arg (Pbf), Lys (tBoc), Trp (tBoc), Glu (OtBu), Ser (tBu), Cys (Trt). All amino acids were coupled by BOP / HOBt / NMM activation, using 5 minutes of preactivation, 60 minutes of coupling time, and 3 times the molar excess of amino acids in each coupling reaction. The coupling of Lie, Leu, Trp (6) and Tpr (9) was repeated (double coupling) for 40 minutes. After the last coupling, the Fmoc group was unfolded from the peptide and the peptidyl resin was washed with DCM and ethanol, then dried for 24 hours in vacuo. For cleavage and deprotection, the peptidyl resin was swollen in DCM in a manual reaction vessel, the excess DCM was red by filtration, and the resin was cooled to 0 ° C. The protecting groups were red and the peptide was cleaved from the resin with K-reagent (TFA-phenol-water-thioanisole-1,2-ethanedithiol; 82. 5: 5: 5: 5: 2.5) using a ratio of 1.5 ml of K reagent per gram of peptidyl resin. The reaction vessel was stirred for 4 hours, then the resin was filtered, washed with freshly prepared Reagent K (1 ml / g of resin), followed by DCM (3 x 10 ml / g of resin) and finally 50 ml of resin. % acetic acid / water (3 x 10 ml / g resin). The combined filtrates were placed in a separatory funnel and the aqueous phase was extracted twice more with DCM. The aqueous peptide solution was diluted with distilled water to a final concentration of 10% acetic acid and then dried by freezing. The lyophilization was repeated with the solution of 5% acetic acid / water of the peptide. The unpurified product was isolated as a white fluffy powder. The unpurified synthetic peptide was dissolved in 5% acetic acid / water (peptide concentration 0.5 mg / ml) and subjected to purification with RP-HPLC (van Abel et al., Supra 1995). A Vydac preparative C-18 reverse phase column (25 x 100 mm) was used for purification and a C-18 Vydac analytical column (0.46 x 25 mm) for purity titration. In both cases, acetonitrile gradients were used (with 0.1% TFA) and water (0.1% TFA) was used for chromatographic fractionation. The elution of the elution of the peptide was observed at 220 nm and 280 nm. The appropriate HPLC fractions were combined, concentrated by centrifugation, and lyophilized. B. Characterization of Indole 1-13 (W6, 9) During the acidolitic cleavage and deprotection of Indole 1-13 assembled with Fmoc (SEQ ID NO: 1), a strongly absorbent material A-320 was detected in the synthetic product that is absent from indolicidin prepared from natural sources. The absorbent material A-320 was purified by cation exchange HPLC, using a sulfoethyl cation exchange column (0.45 x 20 cm). The column was loaded with 1.5 mg of sample dissolved in 100 mM NaOAc containing 25% acetonitrile and eluted in the same solvent isocratically. The absorbent material A-320 was collected and purified by RP-HPLC using the conditions described above and, as discussed below, it was determined to be X-indolicidin, Indole 1-13 (W6, 9). The purified A-320 absorbent material was characterized by electrospray mass spectroscopy. Material A-320 was two units of atomic mass smaller in mass than indole 1-13 that occurs naturally (SEC DE IDENT NO: 1). The monoisotopic mass of indolicidin was 1905.88 (theoretical 1906.05) compared to 1904.13 (theoretical 1904.05) for the absorbent material A-320. UV spectroscopy revealed, in addition to absorption to A-320, which is absent for native indolicidin, absorbance to A-218 and A-280; native indolicidin also shows absorbance at A-218 and A-280. The A-320 material was also highly fluorescent (emission at 400 nm) determined by spectrofluorimetry with an excitation of 325 nm.

The Edman sequence analysis of the A-320 material revealed a dramatic drop in the production of Trp-6 and Trp-9. In relation to the very high fluorescence emission, these results indicate the presence of an extended ring system. Additional analysis for the Trp-Trp connection was carried out by digesting the absorbent material A-320 with trypsin and chymotrypsin and characterizing the resulting fragments by mass spectrometry. The masses of the products confirmed that Trp-6 and Trp-9 were crosslinked through the carbon d of the respective indole rings, generating a di-tryptophan crosslinking. Accordingly, material A-320 was designated Indole 1-13 (W6, 9) (SEQ ID NO: 3). Indole 1-13 (SEQ ID NO: 1) and Indole 1-13 (W6, 9) (SEQ ID NO: 3) were subjected to enzymatic degradation for 6, 24, 48 and 96 hours with a-chymotrypsin (1% by weight) in 0.1 M Tris buffer (pH 7.7) at 37 ° C, then analyzed by RP-HPLC. All native indolicidin (SEQ ID NO: 1) was digested within 6 hours of incubation. Approximately 85% of Indole 1-13 (W6, 9) (SEQ ID NO: 3) was digested after 3 hours, but the remaining undigested material was stable for at least the 18 hour incubation period. These results indicate that an X-indolicidin analog, which contains intrachain chain cross-linking, is stabilized with respect to proteolytic degradation when compared to linear native indolicidin. Di-tryptophan crosslinks were formed in an indolicidin peptide or indolicidin analog using a modification of the method of Stachel et al. J. Amer. Chem. Soc. 118: 1225 (1996), which is incorporated herein by reference). Approximately 1 mg of purified peptide was dissolved in approximately 100 μl of trifluoroacetic acid and incubated under nitrogen at room temperature for 0.5 to 18 hours. The sample was dried in vacuo, washed with 100 μl of chloroform and re-dried in vacuo. The dried peptide was dissolved in approximately 1.0 ml of 1,4-dioxane containing 0.6 μmol of dichloro, dicyanoquinone and stirred for 0.1 to 2 hours at room temperature. The products of the optimized incubation were purified by RP-HPLC, observing the elution at 320 nm. Indole 1-13 / 6, 9C (see SEQ ID NO: 4), which contains Cys for Trp substitutions at positions 6 and 9, was prepared using the method described above. The secondary chain protecting groups were removed by acidolysis and the disulfide bond was formed allowing oxidation with air at pH 7-9 to produce Indole 1-13 / 6, 9C (C6, 9) (SEQ ID NO: 4) ). The air oxidation of reduced Indole / 6,9C was carried out at a peptide concentration of 100 μg / ml in 0.1 M ammonium bicarbonate (pH 8) or in 0.1 M Tris-HLC (pH 8) or other aqueous solvents at pH 8. The solution was stirred at room temperature, in ambient air, for 2 to 72 hours, ^^ with intermittent test of the solution using Ellman's reagent, to determine when free sulfhydryl groups do not remain. The oxidized peptide was purified by RP-HPLC and the disulfide formation was confirmed by MALDI-TOF mass spectrometry or electroaspersion. C. Expression of Indol-Hse analogues: The Indol-Hse was expressed from a construction ^ fc 10 recombinant encoding three replications of the mature peptide, each separated by a hexapeptide spacer sequence; poly- (Indole (1-13) -Met-Ala-Arg-Ile-Ala-Met) 3 (SEQ ID NO: 11). Recombinant indolicidin was produced as a fusion polypeptide with a binding protein to maltose (MBP) and recovered by cleavage with cyanogen bromide. The multicopy indolicidin encoding the DNA sequence was assembled from six synthetic oligonucleotides. The oligonucleotides were phosphorylated and assembled by tuning and ligating each fragment (Ikehara et al., Proc. Nati, Acad. Sci., USA 81: 2956-5960 (1984), which is incorporated herein by reference). Oligonucleotides (2.5 nmol each) were phosphorylated by treatment with 10 mmol of ATP at pH 8.0, heated for 2 minutes in boiling water, then 9.5 units of polynucleotide kinase were added and the samples were incubated at 37 ° C for 120 minutes. The reaction was stopped by incubating the samples for 15 minutes at 70 ° C. Phosphorylated fragments and non-phosphorylated ends were mixed, heated for 2 minutes in boiling water, and quenching of the pairs was completed after cooling slowly to 15 ° C and incubating overnight. The samples were purified with phenol / chloroform and precipitated with EtOH. The hardened DNA mixtures were mixed together and treated with 1.2 units of T4 ligase for 15 hours at 15 ° C. The mixture was heated for 2 minutes at 70 ° C to inactivate the ligase. A ligation product of base pair 211 (bp) was isolated from an agarose gel after electrophoresis using the WIZARD PCR purification kit (Promega, Madison Wl); PCR was performed using the primers as shown in Figure 6 (double underlined sequence). The purified 211 bp PCR product was digested with Sal I and Eco Rl, and then ligated into the Sal I and Eco Rl sites of the precut vector pMAL-c2 (New England BioLabs; Beverly MA). The transformation of INVaF 'E. coli was carried out with the TA cloning equipment according to the instructions of the manufacturers (Invitrogen, La Jolla CA). The DNA sequence shown in Figure 1 was formed by dideoxy sequencing.

INVaF 'cells containing the poly-indolicidin analogue pMAL-c2 fusion polypeptide were grown overnight in 15 ml of LB medium containing 100 μg / ml ampicillin at 37 ° C with constant agitation. Ten ml of the overnight culture was transferred into one liter of fresh medium of LB / ampicillin containing 0.2% glucose and incubated with constant agitation for 4 hours until an OD62O = 0.500. IPTG was added to a final concentration of 0.3 mM and the culture was incubated for an additional 4 hr, then the cells were harvested by centrifugation at 4 ° C. The cell pellet was suspended in 20 ml of ice-cold lysis buffer (0.01 M Tris-HCl, pH 8.0, 1 mM of each of PMSF, DTT and EDTA, 2 mg / ml of lysozyme), then mixed slowly for 30 minutes on ice. 1.6 ml of 10% sodium deoxycholate and 63 μl of a 2 mg / ml DNase I solution were added and the mixture was incubated for an additional 30 minutes on ice. 3.2 ml of 2% protamine sulfate was added and the mixture was mixed for 20 minutes on ice. The soluble fusion polypeptide was obtained in the supernatant after centrifugation for 30 minutes at 12,000 rpm. The fusion polypeptide was purified using an amylose affinity resin (New England BioLabs). The supernatant of the lysis was diluted 10 to 25 times with column regulator (0.2 M NaCl, 0.02 M Tris-HCl pH 8.0, 1 mM each DTT and EDTA) before applying it to the column. From 2 liters of bacterial culture, approximately 80 mg of maltose binding protein (MBP) -indolicidin fusion polypeptide was purified by amylose affinity chromatography. The purified fusion polypeptide (80 mg) was dialyzed against 1% acetic acid, lyophilized, and dissolved in 4 ml of 80% formic acid containing 160 mg of CNBr. The solution was purged with nitrogen, and incubated at room temperature for 5 hours. The solution was diluted 10 times with water, lyophilized, then the digested was purified by RP-HPLC. The recovery of Indol (1-13) -H was approximately 50% of the theoretical yield. Alternatively, a MBP-indolicidin fusion polypeptide can be prepared, having the sequence Met-Ala-Arg-Ile-Ala-Met (SEQ ID NO: 11) in place of the first Met residue in the poly-indolicidin and then in an enterokinase cleavage site. Such a MBP-indolicidin fusion polypeptide can first be cleaved with enterokinase, to release the MBP portion of the fusion polypeptide. The poly-indolicidin moiety can then be treated with CNBr, to release the Indol-Hse analogs, which can be purified as above. The results discussed above indicate that poly-indolicidin analog polypeptides can be produced in vivo in a bacterial expression system, without killing the host microorganism and, therefore, provide a means to produce substantial amounts of Indol-Hse analogs, and, therefore, thus, analogs of X-indolicidin effecting crosslinks as described in the above. EXAMPLE II ANTIMICROBIAL ACTIVITY OF X-INDOLICIDINE ANALOGS This example demonstrates that Indole 1-13 (W6, 9) (SEQ ID NO: 3) exhibits broad spectrum antimicrobial activity similar to the activity of native indolicidin (SEC DE IDENT NO .: 1). The antimicrobial activity was characterized using a method of microbial inhibition, including a modified plaque diffusion test (Hultmark et al., EMBO J. 2: 571-573 (1983); Lehrer et al., J. Immunol. Meth. 137: 167-173 (1991), each of which is incorporated herein by reference). Plates containing nutrient agar (or agarose) were seeded with E. Coli ML35, C. neoformans 271A, S. Aureus 207A or C. Albicans 16820. Five to ten μl Indol 1-13 (SEQ ID NO: 1) or Indole 1-13 (W6, 9) in 10 mM PIPES, pH 7. 4 (final concentration of 10, 30, 100 or 300 μg / ml) were placed into small wells formed in the solid agarose plates. After an initial incubation interval of 1 to 4 hours, the well-containing layer was covered with solid medium (2X normal) enriched to support microbial growth outside the perimeter of inhibition. After incubation overnight at 30 ° C to 37 ° C, the antimicrobial activity was quantified by measuring the light zones around each well (zone of inhibition). Indole 1-13 (SEQ ID NO: 1) and Indole 1-13 (W6, 9) (SEQ ID NO: 3) inhibited the growth of each of the microorganisms tested in a dependent manner of the dose and the zones of inhibition for each peptide were approximately the same for a given microorganism (see Figures 2 to 5). These results indicate that an X-indolicidin analog has essentially the same microbiostatic activity as native indolicidin. The microbicidal activity of Indol 1-13 (SEC.

IDENT. NO: 1) and Indole 1-13 (W6, 9) (SEQ ID NO: 3) for the same four microorganisms (see above) was also examined. The microbicidal activity was measured by first incubating the target organism with the peptide in 10 Mm regulator PIPES (pH 7.4), then the suspension was plated to quantify the surviving microorganisms. Cultures were grown to the log log phase in an appropriate medium, harvested, washed, and resuspended to 1-2 x 10 7 colony forming units (CFU) per ml. To conserve the peptide, the incubation volume was usually 0.05 ml, with the final cell concentration being 1-2 x 10 6 CFU / ml. The solutions • Peptide stock, were usually made in 0.01% acetic acid, diluted in the incubation buffer to a final concentration of 1 μg / ml to 30 μg / ml, and incubation was initiated by the addition of an appropriate volume of the fungal or bacterial stock suspension to the preheated mixture (37 ° C) of regulator-peptide. 10 • At time intervals, samples of 50 μl or 100 μl were removed and serially diluted, then seeded onto plates containing nutrient agar. The destructive activity was quantified determined the reduction in CFU regarding appropriate incubation control. E were incubated. Coli and S a ureus with peptide for 30 minutes; C. Albicans was incubated for 60 minutes; and C. Neoformans was incubated for 4 hours. Following the incubation, the 50 μl or 100 μl samples were removed and serially diluted, then were plated on agar plates containing nutrient. The activid ^^ determining appropriate control incubations. Indole 1-13 (SEQ ID NO: 1) and Indole 1-13 (W6, 9) (SEQ ID NO: 3) had very similar microbiocidal activity (see Figure 6 to 9). At 10 μg / ml, both peptides reduced the survival of E. Coli, S a ureus and C. Neoformans by more than three orders of magnitude, with minimal additional destruction observed at 30 μg / ml of peptide. Both peptides also reduced the survival of C. Albicans by approximately two orders of magnitude at 10 μg / ml, and by more than three orders of magnitude at 30 μg / ml. These results demonstrated that an X-indolicidin analog has microbicidal activity against a variety of different microorganisms and, with the results of the microbiostatic tests discussed above, demonstrate that an X-indolicidin analog such as indolicidin has broad antimicrobial activity. spectrum. Although the invention has been described with reference to the examples given above, it should be understood that various modifications may be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.

Claims (40)

1. CLAIMS 1. An analog of crosslinked indolicidin (X-indolicidin) having the amino acid sequence: Xaal-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Pro-Xaa6-Xaa6-Pro-Xaa6-Xaa7-Xaa7-Xaa8, (SEQ. DE IDENT NO: 2) characterized in that: Xaal is lie, Leu, Val, Ala, Gly- or absent; Xaa2 is Lie, Leu, Val, Ala, Gly or absent; Xaa3 is Pro or absent; Xaa4 is Trp, Phe, Cys, Glu, Asp, Lys, AlaL, or absent; Xaa5 is Arg, Lys or absent; Xaa6 is Trp, Phe, Cys, Glu, Asp, Lys, or AlaL; Xaa7 is Arg, Lys or absent; and Xaa8 is homoserine (Hse), Met, Met-Xaa9-Met or absent; Where Xaa9 is at least one amino acid; with the proviso that a cross-linking can be formed between two amino acids selected from the group consisting of: a) Xaa4, when present, and a Xaa6; and b) a first Xaa6 and a second Xaa6; and with the additional condition that if Xaa2 is absent, Xaal is absent; if Xaa3 is absent, Xaal and Xaa2 are absent; if Xaa4 is absent, Xaal, Xaa2 and Xaa3 are absent and if Xaa5 is absent, Xaal, Xaa2, Xaa3 and Xaa4 are absent.
2. 2. The X-indolicidin analogue according to claim 1, characterized in that it additionally comprises a C-terminal amide.
3. 3. The X-indolicidin analog according to claim 1, characterized in that it has the amino acid sequence: H2N-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg -CONH2 (SEQ ID NO: 3); and
4. 4. The X-indolicidin analogue according to claim 1, characterized in that it has the amino acid sequence: H2N-Ile-Leu-Pro-Trp-Lys-Cys-Pro-Trp-Cys-Pro-Trp-Arg- Arg-CONH2 (SEQ ID NO: 4).
5. 5. The X-indolicidin analogue according to claim 1, characterized in that the cross-linking is a di-tryptophan crosslinking.
6. 6. The X-indolicidin analogue according to claim 1, characterized in that the crosslinking is a disulfide crosslinking.
7. The X-indolicidin analogue according to claim 1, characterized in that the cross-linking is selected from the group consisting of a monosulfide, a lactam and a lactone crosslinking.
8. 8. The fusion polypeptide, characterized in that it comprises the X-indolicidin analogue according to claim 1 linked to a peptide.
9. 9. The fusion polypeptide according to claim 8, characterized in that the peptide is capable of specifically binding by a molecule.
10. 10. The fusion polypeptide according to claim 9, characterized in that the molecule is an antibody that binds specifically to the peptide.
11. The fusion polypeptide according to claim 9, characterized in that the peptide and the molecule, respectively, are selected from the group consisting of: glutathione-S-transferase and glutathione; maltose and maltose binding protein; and His-6 and a metallic ion.
12. 12. The indolicidin analogue according to claim 1, characterized in that it has antimicrobial activity against a microorganism selected from the group consisting of a gram-positive bacterium, a gram-negative bacterium, a yeast and a fungus.
13. 13. The indolicidin analogue according to claim 12, characterized in that the microorganism is selected from the group consisting of Staphylococcus aureus, Escherichia coli, Candida albicans, Salmonella typhimurium and Cryptococcus neoformans.
14. 14. The indolicidin analogue according to claim 1, characterized in that it has antimicrobial activity against a protozoan.
15. 15. The indolicidin analogue according to claim 14, characterized in that the protozoan is selected from the group consisting of Giardia sp. and ñcanthamoeba sp.
16. 16. The indolicidin analogue according to claim 1, characterized in that it has antimicrobial activity against a virus.
17. 17. The indolicidin analogue according to claim 16, characterized in that the virus is human immunodeficiency virus-1.
18. 18. A pharmaceutical composition, characterized in that it comprises the X-indolicidin analogue according to claim 1, and a pharmaceutically acceptable carrier.
19. 19. The pharmaceutical composition according to claim 18, characterized in that it is associated with a liposome.
20. 20. The pharmaceutical composition according to claim 18, characterized in that it is associated with a non-liposome lipid complex.
21. 21. A method for reducing or inhibiting the growth or survival of a microorganism in an environment capable of maintaining the growth or survival of the microorganism, characterized in that it comprises administering an effective amount of an X-indolicidin analog to the environment, thereby reducing or inhibiting the growth or survival of the microorganism.
22. 22. The method of compliance with the claim 21, characterized in that it has antimicrobial activity against a microorganism selected from the group consisting of a gram-positive bacteria, gram-negative bacteria, a yeast and a fungus.
23. 23. The method according to the claim 22, characterized in that the microorganism is selected from the group consisting of Staphylococcus aureus, Escherichia coli, Candida albicans, Salmonella typhimurium and Cryptococcus neoformans.
24. 24. The method according to claim 21, characterized in that it has antimicrobial activity against a protozoan.
25. 25. The method according to claim 24, characterized in that the protozoan is selected from the group consisting of Giardia sp. and Acanthamoeba sp.
26. 26. The method according to claim 21, characterized in that it has antimicrobial activity against a virus.
27. 27. The method according to claim 26, characterized in that the virus is the virus of • human immunodeficiency-1. 5
28. 28. The method of compliance with the claim 21, characterized in that the environment is a food or a food product.
29. 29. The method according to claim 21, characterized in that the environment is a solution. 10
30. 30. The method of compliance with the claim 21, characterized in that the environment is an inanimate object comprising a surface.
31. 31. The method according to claim 21, characterized in that the environment is a mammal. 15
32. 32. The method of compliance with the claim 21, characterized in that the administration is local.
33. 33. The method according to claim 21, characterized in that the administration is by injection.
34. 34. The method according to claim 20 21, characterized in that the administration is oral.
35. 35. A nucleic acid molecule encoding an X-indolicidin analog having the amino acid sequence: Xaal-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Pro-Xaa6-Xaa6- 25 Pro-Xaa6-Xaa7-Xaa7-Xaa8 , (SEQ ID NO: 2) characterized by: Xaal is lie, Leu, Val, Ala, Gly or absent; Xaa2 is Lie, Leu, Val, Ala, Gly or absent; Xaa3 is Pro or absent; Xaa4 is Trp, Phe, Cys, Glu, Asp, Lys, AlaL or absent; Xaa5 is Arg, Lys or absent; Xaa6 is Trp, Phe, Cys, Glu, Asp or Lys; Xaa7 is Arg, Lys or absent; and Xaa8 is Met, Met-Xaa9-Met or absent; wherein Xaa9 is at least one amino acid; with the proviso that a cross-linking can be formed between two amino acids selected from the group consisting of: a) Xaa4, when present, and a Xaa6; and b) a first Xaad and a second Xaa6; and with the additional condition that if Xaa2 is absent, Xaal is absent; if Xaa3 is absent, Xaal and Xaa2 are absent; if Xaa4 is absent, Xaal, Xaa2, and Xaa3 are absent and if Xaa5 is absent, Xaal, Xaa2, Xaa3 and Xaa4 are absent.
36. 36. The nucleic acid molecule according to claim 35, characterized in that the X-indolicidin analog has the amino acid sequence: H2N-Ile-Leu-Pro-Trp-Lys-Cys-Pro-Trp-Cys-Pro-Trp-Arg-Arg-CONH2 (SEQ ID NO: 4);
37. 37. The nucleic acid molecule according to claim 35, characterized in that it additionally comprises a nucleotide sequence encoding a peptide of interest.
38. 38. The nucleic acid molecule according to claim 37, characterized in that the peptide of interest is capable of specifically binding by a molecule.
39. 39. The nucleic acid molecule according to claim 38, characterized in that the molecule is an antibody that binds specifically to the peptide.
40. 40. The nucleic acid molecule according to claim 38, characterized in that the peptide and the molecule, respectively, are selected from the group consisting of: glutathione-S-transferase and glutathione; maltose and maltose binding protein; and His-6 and a metallic ion.